improved approaches to protein-protein coupling and the

Improved Approaches to Protein-Protein Coupling and the Efficient Formation of Hemoglobin bis-Tetramers

Serena Singh

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Department of Chemistry

University of Toronto

© Copyright by Serena Singh 2016

ii

Improved Approaches to Protein-Protein Coupling and the

Efficient Formation of Hemoglobin bis-Tetramers

Serena Singh

Doctor of Philosophy

Department of Chemistry

University of Toronto

2016

Abstract

My research focused on developing new methods for coupling proteins, with an emphasis on

producing hemoglobin (Hb) bis-tetramers. These are predicted to be safe and effective alternatives

to red cells in transfusions. Based on previous work, it was known that stabilized tetrameric Hb

causes increases in blood pressure in circulation, presumably as the result of their extravasation,

which leads to scavenging of nitric oxide, the signal for relaxation of the muscles that surround

blood vessels. Our initial approach involved optimizing solubility-directed click chemistry based

on the copper-catalyzed azide-alkyne cycloaddition (CuAAC). To increase coupling specificity,

we developed a double cross-linking approach in which reactive sites on the α-subunits are blocked

with a fumaryl cross-linker and with azide installed on a linker specifically bridging the β-subunits.

While this approach gives substantial success, the inefficiencies of the CuAAC process with our

protein of interest motivated us to improve the process. Thus, strain-promoted alkyne-azide

cycloaddition (SPAAC) was successfully adapted for coupling proteins. Using SPAAC, we also

developed extended coupling to produce ‘Hb clusters’ with albumin. The resultant Hb-albumin

cluster is a breakthrough product that can be scaled with minimal manipulation. We took the

project in a new direction using biotin-avidin affinity as the basis for protein-protein coupling. The

approach adds a biotin derivative to a cysteine thiol on each β-subunit of Hb. Avidin then

iii

scavenges the cysteine biotinylated Hbs by the high affinity biotin-avidin interaction to give the

fully functional triple protein HbAvHb. We evaluated the physical and circulatory properties of

the products developed here. These approaches not only apply to creating Hb assemblies but also

provide a general route to coupling like proteins.

iv

Acknowledgments

“A sign of a good leader is not how many followers you have, but how many leaders you

create” –Gandhi. I think of Dr. Ronald Kluger when I hear this quote – as a great leader, he inspired

greatness in myself to push the frontier. It is by gifted leaders like Ron that the world is driven

forward.

I am grateful to our collaborator, Dr. Warren Zapol (Massachusetts General Hospital,

Harvard Medical School), for the perspective I gained during my exchange. My special thanks to

Dr. Binglan Yu who assisted me with the animal studies. I must also express my appreciation for

my supervisory committee, consisting of Dr. G. Andrew Woolley and Dr. Xiao-an Zhang, who

encourage me to expand the scope of my abilities.

The influence of my brilliant colleagues thoroughly enriched my experience on this project:

Dr. Ina Dubinsky-Davidchik, Ms. Aizhou Wang, Ms. Erika Siren, Ms. Megan Roberts, Ms.

Yasamin Heidari, Ms. Liliana Opinska, Dr. Adelle Vandersteen, Mr. Michael Bielecki, Mr.

Graeme Howe and Mr. Yuyang Li. The technical assistance of Mr. Chung-Woo Fung was critical

to the timely completion of this work.

This thesis is dedicated to my mother, Kiran Singh, my father, Jag Singh and my brother

Vishu Singh, who I can never repay for the sacrifices they have made for me so that I may pursue

my dreams. My final thoughts are to my fiancé, Matthew Wong-Fung, who provides me with the

unequivocal support I need to reach for those stars.

v

Table of Contents

Acknowledgments.......................................................................................................................... iv

Table of Contents .............................................................................................................................v

List of Tables ................................................................................................................................. ix

List of Schemes ................................................................................................................................x

List of Figures ................................................................................................................................ xi

Abbreviations ............................................................................................................................... xvi

Chapter 1 ..........................................................................................................................................1

Hemoglobin-Based Oxygen Carrier (HBOC) Design ................................................................1

1.1 Hemoglobin allostery ...........................................................................................................1

1.2 Contemporary HBOC candidates ........................................................................................3

1.3 Bioorthogonal protein modification .....................................................................................7

1.3.1 Copper-catalyzed azide-alkyne cycloaddition (CuAAC) ........................................7

1.3.2 Strain-promoted alkyne-azide cycloaddition (SPAAC) .........................................10

1.3.3 The high affinity avidin-biotin interaction .............................................................11

1.4 Purpose of thesis ................................................................................................................12

Chapter 2 ........................................................................................................................................14

Hemoglobin bis-Tetramers by the Copper-Catalyzed Azide-Alkyne Cycloaddition

(CuAAC) ...................................................................................................................................14

2.1 Results and Discussion ......................................................................................................14

2.2 Concluding remarks ...........................................................................................................25

2.3 Experimental ......................................................................................................................26

2.3.1 General ...................................................................................................................26

2.3.2 Sequentially cross-linked Hb (α99-fumaryl-α99, β82-trimesoyl-β82) ..................26

2.3.3 Preparation of a doubly cross-linked Hb bis-tetramer ...........................................27

2.3.4 Circular dichroism (CD) spectroscopy ..................................................................27

vi

2.3.5 Thermal denaturation and turbidity .......................................................................28

2.3.6 Nitrite reductase activity ........................................................................................28

2.4 Supplemental Information .................................................................................................29

Chapter 3 ........................................................................................................................................30

Physiological Responses of a Bioorthogonally Coupled Hemoglobin bis-Tetramer in

Circulation .................................................................................................................................30



3.3 Experimental ......................................................................................................................39

3.3.1 General ...................................................................................................................39

3.3.2 Preparation of protein solutions for transfusion.....................................................39

3.3.3 Preparation of mice ................................................................................................39

3.3.4 Blood pressure measurements................................................................................40

3.3.5 Blood gas and methemoglobin analysis .................................................................40

3.3.6 Nitric oxide consumption assay .............................................................................40

3.3.7 Statistical analysis ..................................................................................................40

Chapter 4 ........................................................................................................................................41

Assembly of Hemoglobin bis-Tetramers and Hemoglobin-Albumin Clusters by Metal-free

Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC) ........................................................41



4.3 Experimental ......................................................................................................................58

4.3.1 General ...................................................................................................................58

4.3.2 Hb bis-tetramers (Hb-azide) ..................................................................................58

4.3.3 Hb bis-tetramers (bis-azide linker) ........................................................................59

4.3.4 Preparation of Hb-DIBO and Hb-exclusive clusters .............................................59

4.3.5 Preparation of albumin-azide .................................................................................60

vii

4.3.6 Preparation of Hb-albumin cluster from albumin-azide ........................................60

4.3.7 Preparation and SPAAC of Hb-PEG-azide and albumin-DIBO............................60


Chapter 5 ........................................................................................................................................72

Self-assembly of Hemoglobin and Avidin ................................................................................72



5.3 Experimental ......................................................................................................................82

5.3.1 General ...................................................................................................................82

5.3.2 Self-assembly of Hb and avidin .............................................................................83

5.3.3 Biotinylation of Hb ................................................................................................83

5.3.4 Hb-avidin conjugation ...........................................................................................83

5.3.5 HABA occupancy assay ........................................................................................84


Chapter 6 ........................................................................................................................................89

General Experimental Methods.................................................................................................89

6.1 Reagents .............................................................................................................................89

6.2 Hb oxygenation/deoxygenation .........................................................................................89

6.3 Reverse-phase HPLC .........................................................................................................89

6.4 Size-exclusion HPLC .........................................................................................................89

6.5 Mass spectrometry analysis ...............................................................................................90

6.6 SDS-PAGE analysis...........................................................................................................90

6.7 Native PAGE analysis........................................................................................................90

6.8 Oxygen binding analysis ....................................................................................................90

Chapter 7 ........................................................................................................................................91

Conclusions and Future Work ...................................................................................................91

viii

References ......................................................................................................................................94

ix

List of Tables

Table 2.1: Oxygen binding properties of doubly cross-linked Hb and the bis-tetramer (compared

to native Hb).

Table 2.2: Minimum temperatures required for the denaturation of native and modified

carbonmonoxyHb species under anaerobic conditions in sodium phosphate buffer (0.01 M, pH

7.4).

Table 2.3: Comparison of oxygen affinity, cooperativity and nitrite reductase activity (NiR) of

doubly cross-linked Hb and its PEG modified derivative with other chemically modified species.

Table 6.1: Reverse-phase HPLC elution gradient.

Table 7.1: Comparative overview of the Hb-based products designed.

x

List of Schemes

Scheme 1.1: Huisgen cycloaddition.

Scheme 1.2: CuAAC mechanism.

Scheme 1.3: The formation of reactive oxygen species in the presence of copper: AH- + Cu2+ →

A∙- + Cu+ + H+.

Scheme 1.4: Phase-directed copper-catalyzed azide-alkyne cycloaddition (PDCuAAC).

Scheme 1.5: Glaser-Hay homocoupling with CuI/TMEDA (tetramethylethylenediamine).

Scheme 1.6: SPAAC reaction.

Scheme 2.1: Doubly cross-linked Hb can be prepared quantitatively if the α-subunits are

modified first.

Scheme 2.2: Synthesis of azide cross-linker 1.

Scheme 2.3: Addition of azide cross-linker 1 to α-subunit protected Hb to give α99-fumaryl-α99,

β82-azido-β82.

Scheme 2.4: CuAAC with bis-alkyne 2 to produce the doubly cross-linked bis-tetramer.

Scheme 3.1: Hb bis-tetramer prepared by the PDCuAAC.

Scheme 4.1: Preparation of Hb-cyclooctyne (top) and Hb-azide (bottom).

Scheme 4.2: Copper-free click of Hb-cyclooctyne with Hb-azide.

Scheme 4.3: CuAAC of Hb-alkyne with Hb-azide.

Scheme 4.4: Preparation of Hb bis-tetramer by copper-free coupling of Hb-cyclooctyne with bis-

azide.

Scheme 4.5: Decoration of native Hb with NHS-DIBO to give Hb-DIBO.

Scheme 4.6: Copper-free click of Hb-DIBO with Hb-azide.

Scheme 4.7: CuAAC of Hb-alkyne(s) with Hb-azide.

Scheme 4.8: SPAAC of Hb-DIBO with albumin-azide.

Scheme 4.9: Preparation of Hb-PEG-azide by treatment of native Hb with NHS-azide.

Scheme 4.10: SPAAC of Hb-PEG-azide with albumin-DIBO to assemble the Hb-albumin

cluster.

Scheme 5.1: Biotinylation of Hb with the biotin-maleimide bifunctional reagent.

Scheme 5.2: Self-assembly of biotinylated Hb to avidin.

xi

List of Figures

Figure 1.1: Oxygen dissociation curve for normal adult Hb (red line).

Figure 1.2: The DPG binding site at the interface between the two β-subunits.

Figure 1.3: Coordinate-difference plot indicating the spatial change in residue position relative

to a native COHb quaternary structure reference point.

Figure 1.4: Hb cross-linkers. Bis(3,5-dibromosalicyl) fumarate (DBSF) (left) and trimesoyl

tris(3,5-dibromosalicylate) (TTDS) (right).

Figure 1.5: Scavenging of nitric oxide from nitric oxide synthase (NOS) following extravasation

of cell-free Hb.

Figure 1.6: Hb bis-tetramer.

Figure 1.7: Hb-albumin cluster HemoAct.

Figure 1.8: Comparing second order rate constants for strained cyclooctynes OCT

(cyclooctyne), DIBO (dibenzocyclooctyne), DIBAC (dibenzoazacyclooctyne) and BARAC

(biarylazacyclooctynone).

Figure 1.9: pH dependent formation of avidin/ovalbumin microspheres as visualized by phase-

contrast micrograhs.

Figure 2.1: Reverse-phase HPLC trace under dissociating conditions of α99-fumaryl-α99, β82-

trimesoyl-β82.

Figure 2.2: Reverse-phase HPLC trace under dissociating conditions of the product of the

reaction of α99-fumaryl-α99, β2 with linker 1.

Figure 2.3: Reverse-phase HPLC trace under dissociating conditions of the product of the

reaction of α99-fumaryl-α99, β2 with linker 1.

Figure 2.4: Size-exclusion HPLC of the CuAAC mixture before purification.

Figure 2.5: SDS-PAGE analysis of the bis-tetramer before purification.

Figure 2.6: Oxygen binding curves of α99-fumaryl-α99, β82-trimesyl-β82 and the bis-tetramer

(compared to native Hb).

Figure 2.7: CD spectra of α99-fumaryl-α99, β82-trimesyl-β82 Hb and the bis-tetramer

(compared to native carbonmonoxyHb) in the far UV.

Figure 2.8: CD spectra of α99-fumaryl-α99, β82-trimesyl-β82 Hb and the bis-tetramer

(compared to native carbonmonoxyHb) in the near UV.

Figure 2.10: Absorbance profile output of the reaction of deoxyHb with nitrite.

Figure 2.9: Turbidity curves for native and modified carbonmonoxyHb species under aerobic

conditions in 0.1 M sodium phosphate buffer (0.1 M, pH 7.4).

Figure 2.11: Initial rate plot for NiR of α99-fumaryl-α99, β82-trimesoyl-β82 Hb.

Figure 2.12: Size-exclusion HPLC traces of the products from PEGylation of α99-fumaryl-α99,

β82-trimesoyl-β82 Hb at room temperature and 37 °C.

Figure 2.13: Oxygen binding curve of PEG modified α99-fumaryl-α99, β82-trimesoyl-β82 Hb.

xii

Figure 2.14: Initial rate plot PEG modified α99-fumaryl-α99, β82-trimesoyl-β82 Hb.

Figure 2.15: Mass spectrum of peak collected from reverse-phase chromatography

corresponding to β82-azido-β82.

Figure 3.1: Measurement of tail systolic blood pressure (SBP) in awake wild-type mice following

tail-vein injection of modified PBS buffer containing N-acetyl cysteine, native Hb or Hb bis-

tetramer.

Figure 3.2: Measurement of tail systolic blood pressure (SBP) in awake db/db mice following tail-

vein injection of modified PBS buffer containing N-acetyl cysteine, native Hb or Hb bis-tetramer.

Figure 3.3: Total plasma free Hb from arterial whole blood of wild-type mice two hours after tail


Figure 3.4: Total plasma free Hb from arterial whole blood of db/db mice two hours after tail vein

injection of modified PBS buffer containing N-acetyl cysteine, native Hb or Hb bis-tetramer.

Figure 3.5: Total plasma metHb from arterial whole blood of wild-type mice two hours after tail

vein injection of the modified PBS buffer containing N-acetyl cysteine, native Hb or Hb bis-

tetramer.

Figure 3.6: Total plasma metHb from arterial whole blood of db/db two hours after tail vein


Figure 3.7: Nitric oxide consumption by native Hb standards and the bis-tetramer as measured

using the chemiluminescent nitric oxide analyzer.

Figure 3.8: Nitric oxide consumption by native Hb standards (3.25, 6.5, 13, 26 and 52 µM heme).

Figure 3.9: Nitric oxide consumption by the bis-tetramer (3.25, 6.5, 13, 26 and 52 µM heme).

Figure 4.1: Reverse-phase HPLC trace of cross-linked Hb-cyclooctyne under dissociating

conditions.

Figure 4.2: Reverse-phase HPLC trace of Hb-azide under dissociating conditions.

Figure 4.3: A) Size-exclusion HPLC trace of the products of the reaction of Hb-cyclooctyne with

Hb-azide. B) Percent yield based on the theoretical maximum. C) Consumption of starting material

fit to second order kinetics. D) Linear inverse plot.

Figure 4.4: Size-exclusion HPLC trace under high salt conditions of the products of CuAAC of

Hb-alkyne with Hb-azide.


bis-azide. B) Percent yield based on the theoretical maximum. C) Consumption of starting material

fit to second order kinetics. D) Linear inverse plot.

Figure 4.6: Reverse-phase HPLC trace of Hb-DIBO under dissociating conditions.

Figure 4.7: Reverse-phase HPLC trace of xlHb-DIBO under dissociating conditions.

Figure 4.8: Size-exclusion HPLC trace under high salt conditions of the products of the copper-

free click of Hb-DIBO with Hb-azide.


free click of xlHb-DIBO with Hb-azide.

xiii

Figure 4.10: Size-exclusion HPLC trace under high salt conditions of the products of the

reaction between albumin-azide and Hb-DIBO (3 equiv albumin to 1 equiv Hb).


reaction between albumin-azide and Hb-DIBO (6 equiv albumin to 1 equiv Hb).

Figure 4.12: Reverse-phase HPLC trace of Hb-PEG-azide under dissociating conditions.


reaction between albumin-DIBO and Hb-PEG-azide after 4 days.

Figure 4.14: Oxygen binding curve of Hb-albumin cluster prepared from combination of Hb-PEG-

azide with albumin-DIBO.

Figure 4.15: Mass spectrum of Hb-cyclooctyne (β-subunits cross-linked).

Figure 4.16: Mass spectrum of Hb-azide (β-subunits cross-linked).

Figure 4.17: Mass spectrum of Hb-cyclooctyne (β-subunits cross-linked) modified with bis-

azide (4,4’-diazidediphenylsulfone) only.

Figure 4.18: Native PAGE analysis of the products of the copper-free click reactions after

approximately half the starting material is consumed.

Figure 4.19: Mass spectra of Hb-DIBO (the pre-deconvolution spectra were too complex to

yield accurate masses).

Figure 4.20: Mass spectrum of TTDS cross-linked Hb-DIBO (the pre-deconvolution spectra

were too complex to yield accurate masses).

Figure 4.21: Native PAGE analysis of the Hb-exclusive clusters after incubation for 1 day at 4

°C.

Figure 4.22: Mass spectrum of native albumin as purchased.

Figure 4.23: Mass spectrum of maleimide-azide reagent.

Figure 4.24: Mass spectrum of albumin-azide (the pre-deconvolution spectrum was too complex

to yield accurate masses).

Figure 4.25: Native PAGE analysis of the Hb-albumin clusters prepared from SPAAC of Hb-

DIBO with Hb-PEG-azide.

Figure 4.26: Native PAGE analysis of the products of the SPAAC coupling of albumin-azide

with Hb-cyclooctyne.

Figure 4.27: Mass spectra of Hb-PEG-azide.

Figure 4.28: Mass spectrum of albumin-DIBO.

Figure 4.29: Native PAGE analysis of the products of SPAAC of Hb-PEG-azide and albumin-

DIBO.

Figure 4.30: Reverse-phase HPLC of Hb-alkyne under dissociating conditions.

Figure 4.31: Mass spectrum of Hb-alkyne (β-subunits cross-linked).

Figure 4.32: Reverse-phase HPLC of Hb-alkyne(s) under dissociating conditions.

Figure 4.33: Mass spectra of Hb-alkyne(s).

xiv

Figure 4.34: Reverse-phase HPLC of TTDS cross-linked Hb-alkyne(s) under dissociating

conditions.

Figure 4.35: Mass spectra of TTDS cross-linked Hb-alkyne(s).

Figure 5.1: Absorbance at 700 nm, from turbidity, resulting from formation one to one mixtures

of native or cross-linked Hb and avidin in buffers of varying pH and ionic strength.

Figure 5.2: Absorbance at 700 nm, associated with solution turbidity, of one to one mixtures of

fumaryl cross-linked Hb and avidin in buffers of varying pH and ionic strength.

Figure 5.3: Turbidity increase associated with increasing the fumaryl cross-linked Hb to avidin

ratio.

Figure 5.4: The turbidity changes associated with adding inositol hexaphosphate (IHP) to

fumaryl cross-linked Hb/avidin mixtures in HEPES, pH 7.2 (I=2 mM).

Figure 5.5: Absorbance at 700 nm, associated with solution turbidity, of protein mixtures of

increasing lysozyme to native/cross-linked Hb ratio in Tris-HCl, pH 9.0 (I=1 mM).

Figure 5.6: Insoluble shards obtained upon glutaraldehyde treatment of a solution of native Hb

and avidin.

Figure 5.7: Spectral changes associated with heating native Hb (top panel) and native

biotinylated Hb (bottom panel) at 60 °C for 10 min.

Figure 5.8: Size-exclusion HPLC traces of Hb-avidin conjugates with Hb bis-tetramer as a

reference.

Figure 5.9: Absorbance changes accompanying addition of HABA to avidin (5 µM avidin).

Figure 5.10: Absorbance changes accompanying addition of HABA to the Hb-avidin conjugate

(1.3 µM avidin).

Figure 5.11: Binding curves for the Hb-avidin conjugate and avidin titrated with HABA.

Figure 5.12: Native PAGE analysis of the Hb-avidin conjugates (4% stacking gel, 6% separating

gel, 1 hour, 200 V).

Figure 5.13: Oxygen binding curve of the Hb-avidin conjugate compared to the curve for native

Hb.

Figure 5.14: Reverse-phase HPLC traces of Hb species after treatment with biotin-maleimide

cross-linker.

Figure 5.15: Reverse-phase HPLC trace of fumaryl cross-linked Hb after treatment with biotin-

maleimide cross-linker.

Figure 5.16: Biotinylated β-subunit (from modification of native Hb).

Figure 5.17. Biotinylated trimesoyl cross-linked β-subunits.

Figure 5.18: Biotinylated β-subunit (from modification of fumaryl Hb).

Figure 5.19: Absorption spectrum of (non-cross-linked) Hb-avidin conjugate with excess

biotinylated Hb.

Figure 5.20: Absorbance changes accompanying addition of HABA to non-conjugated

biotinylated Hb.

xv

Figure 5.21: Native PAGE analysis of the Hb-avidin conjugates with a high percentage

separating gel.

Figure 5.22: Native PAGE analysis of the Hb-avidin conjugates.

Figure 5.23: Absorbance profile of the Hb-avidin conjugate (5 µM avidin) in the presence of

HABA (19 µM) compared to that of native avidin (5 µM) in the presence of the same

concentration of HABA.

xvi

Abbreviations

BARAC biarylazacyclooctynone

COHb carbon monoxide-bound hemoglobin

CuAAC copper-catalyzed azide-alkyne cycloaddition

DBSF bis(3,5-dibromosalicyl) fumarate

deoxyHb deoxygenated hemoglobin

DIBAC dibenzoazacyclooctyne

DIBO dibenzocyclooctyne

DPG 2,3-diphosphoglycerate

HABA hydroxyazobenzene-2-carboxylic acid (HABA)

Hb hemoglobin

HBOC hemoglobin-based oxygen carrier

HSA human serum albumin

IHP inositol hexaphosphate

LUMO lowest unoccupied molecular orbital

L-NAME Nω-nitro-L-arginine

metHb methemoglobin

NiR nitrite reductase activity

OCT cyclooctyne

oxyHb oxygenated hemoglobin

PAGE polyacrylamide gel electrophoresis

PBS phosphate buffered saline

PDCuAAC phase-directed copper-catalyzed azide-alkyne cycloaddition

PEG poly(ethylene) glycol

pI isoelectric point

SDS sodium dodecyl sulfate

SOD superoxide dismutase

SPAAC strain-promoted alkyne-azide cycloaddition

TMEDA tetramethylethylenediamine

TTDS trimesoyl tris(3,5-dibromosalicylate)

1

Chapter 1

Hemoglobin-Based Oxygen Carrier (HBOC) Design

1.1 Hemoglobin allostery

The primary function of hemoglobin (Hb) is to transport oxygen from the lungs to the

tissues.1 Approximately 250 million of 64 kDa Hb tetramers (α2β2) reside within one heathy adult

red blood cell that has a circulatory lifetime of approximately 120 days. Reversible binding of

oxygen at each subunit heme give rise to the archetypal sigmoid-shaped dissociation curve (Figure

1.1).2 The oxygen affinity (P50) is the oxygen pressure (pO2) at half-occupancy, which for normal

adult Hb is approximately 26 torr. Oxygen is loaded near the alveoli (pO2 ≈ 75 torr) and delivered

near resting (pO2 ≈ 30 torr) or hypoxic (pO2 less than 15 torr) tissue with maximum efficiency.

Figure 1.1: Oxygen dissociation curve for normal adult Hb (solid line). Image reproduced with

permission from Ralston et al.3

Crystal structures from Perutz in 1970 of deoxygenated Hb (deoxyHb, T-state) and

oxygenated Hb (oxyHb, R-state) provided initial insights into the structural changes that

accompany the conformational switching responsible for the observed positive cooperativity.4

High-spin iron (as in deoxyHb) is greater in spherical radius than low spin iron (as in oxyHb), such

that T-state heme prefers an out-of-plane geometry assisted by proximal His-94. Binding of

2

oxygen promotes iron to adopt an in-plane geometry and the accompanying structural changes are

transmitted to adjacent subunits via interactions at the interface. Negative anionic allosteric

effectors such as DPG (2,3-diphosphoglycerate) bind to deoxyHb at specific residues within Hb’s

cationic funnel and stabilize this T-state, thereby decreasing the oxygen affinity. The residues that

associate with DPG include β-Val-1 β-His-2, Lys-82 and β-His-143 (Figure 1.2). The oxygen

binding curve is also shifted to the left by carbon dioxide and H+ (the Bohr effect) because the

association of carbon dioxide with the α-subunit N-terminus and H+ with essential histidine

residues results in salt bridge formation. The physiological significance of a lower affinity for CO2

and H+ at high oxygen pressure is the output of CO2 at the lungs (the Haldane effect).

Figure 1.2: The DPG binding site at the interface between the two β-subunits. Image reproduced

with permission from Kluger et al.5

Hb’s quaternary structure is further altered by the chemical installation of covalent cross-

links that bridge adjacent subunits. X-ray crystal structures of three cross-linked Hb derivatives

with varying oxygen affinities were obtained by Schumacher et al.6 These materials were cross-

linked in the deoxyHb state and crystallized in the COHb state. The extent of rotation of the α2β2

dimer about its axis relative to that of native COHb was quantified based on the distances displaced

by each residue (Figure 1.3, ‘quaternary differences’). The structural features of α2β1-stilbene-

β82 Hb, that has an oxygen affinity similar to that of native Hb, is virtually superimposable with

the crystal structure of native COHb. Meanwhile, the low oxygen derivatives α2β1-trimesoyl-β82

Hb and α2β1,82-trimesoyl-β82 Hb are prevented from fully reaching the R-state conformation.

These results provide important insights about that nature of allosteric transitions and the effect

that chemical modification has on both the tertiary and quaternary structure of the protein.

3

Figure 1.3: Coordinate-difference plot indicating the spatial change in residue position relative to

a native COHb quaternary structure reference point. Native deoxyHb (blue), α2β1-trimesoyl-β82

(yellow; P50 = 17.1 mmHg), α2β1,82-trimesoyl-β82 (green; P50 = 18.1 mmHg) and α2β1-stilbene-

β82 (white, P50 = 3.4 mmHg). Image reproduced with permission from Schumacher et al.6

1.2 Contemporary HBOC candidates

Development of a red cell substitute could reduce the limitations of currently used blood

products.7 Circulation of acellular Hb in large quantities results in hemoglobinuria and

vasoconstriction.7, 8 Thus, as a minimum, an acellular oxygen carrier based on Hb requires

modifications to prevent these adverse effects. Cross-linking the ~64 kDa Hb tetramer prevents

dissociation into ineffective ~32 kDa αβ-dimers, which undergo renal filtration.9 It was apparent

during clinical trials of HemAssist, Hb α-subunit cross-linked with the anionic electrophile bis(3,5-

dibromosalicyl) fumarate (DBSF) (Figure 1.4), that stabilization alone is insufficient.7 We now

understand that the propensity for this material to elicit abnormally elevated blood pressure is

related to scavenging of nitric oxide (NO).

4

Figure 1.4: Hb cross-linkers. Bis(3,5-dibromosalicyl) fumarate (DBSF) (left) and trimesoyl

tris(3,5-dibromosalicylate) (TTDS) (right). DBSF (in the presence of inositol hexaphosphate

(IHP)) and TTDS modify the α-Lys99 and the β-Lys82 residues, respectively.

Nitric oxide in the endothelium is produced from L-arginine by nitric oxide synthase and

affects vascular tone by its interaction with guanylyl cyclase.10 By mediating vasodilation, NO is

directly involved in the body’s response to hypoxia. The membrane of the red cell prevents its

extravasation (leakage across the blood vessel wall) and thus the enclosed Hb does not reach

locally produced nitric oxide from nitric oxide synthase.8 In contrast, cell-free Hb can penetrate

the endothelium where it binds NO, thereby inducing vasoconstriction (Figure 1.5). The

hypothesis is supported by experiments with the nitric oxide synthase inhibitor L-NAME (Nω-

nitro-L-arginine).11

Figure 1.5: Scavenging of nitric oxide from nitric oxide synthase (NOS) following extravasation

of cell-free Hb. Image reproduced with permission from Kim-Shapiro et al.8

5

HemAssist’s tendency to extravasate suggests that there is a need to increase the overall

size of the protein to a point where it does not penetrate the endothelium.8 Simply cross-linking

the protein is not enough to differentiate its extent of endothelial permeability from that of native

Hb.12 The diameter of healthy endothelial pores is estimated to be between 5 and 12 nm.13

Therefore, reduced endothelial permeability is exemplified by much larger entities such as Hb-

polyoxyethylene conjugate (average molecular weight of 90 kDa) and the Hb-haptoglobin

complex (~150 kDa).12 Charge and geometric factors may also influence the extent of

extravasation. The reduced permeability of bovine serum albumin (pI = 4.7) is probably a result

of the negative surface charge repelling anionic endothelial cells.12 Geometrical differences in

margination are complex, but, in general, non-spherical particles experience torque and tumble

away from the center of the blood vessel.14

The adverse reactions of glutaraldehyde polymerized PolyHeme (derived from human Hb)

and Hemopure (derived from bovine Hb), with average molecular weights exceeding 100 kDa,

were then attributed to the presence of undermodified vasoactive species.7, 15 Hemospan is another

heterogeneous mixture that is constructed by treating native human Hb tetramer with 2-

iminothiolane (Traut’s reagent) followed by maleimide-poly(ethylene) glycol (PEG). This product

is finding utility as a plasma expander16 and CO-delivery agent17, since carbon monoxide has been

shown to have vasodilatory, anti-inflammatory and cell-protective properties.17, 18 However, it

remains that there is currently no FDA approved blood substitute to date. The urgency to produce

one escalates with the emergence of new viruses (e.g. Zika) threatening to contaminate the blood

supply.19

A well-defined Hb bis-tetramer20 (Figure 1.6) and the Hb-albumin cluster HemoAct21

(Figure 1.7) are examples of recent HBOCs that are well-tolerated in animal studies. Hb-

superoxide dismutase (SOD) conjugates are an extension of Hb bis-tetramers, with the added

capacity to deactivate reactive oxygen species related to reperfusion injury.22

6

Figure 1.6: Hb bis-tetramer. Image reproduced with permission from Lui et al.23

Figure 1.7: Hb-albumin cluster HemoAct. The Hb-albumin bridge is constructed with SMCC

(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Images reproduced with

permission from Hosaka et al.24

The Hb bis-tetramer exhibits enhanced nitrite reductase activity, which may serve to

counteract HBOC-induced vasoconstriction. The one-electron reduction of nitrite to nitric oxide

by deoxyHb was first described by Brooks in 1937.25

NO2- + HbFe2+ + H+ → NO + HbFe3+ + OH-

NO + HbFe2+ → HbFe2+-NO

The process is auto-catalytic, which is apparent from the sigmoidal rather than hyperbolic

reaction profile.26 R-state shifted deoxy hemes catalyze the reduction about 60 times faster than T-

state hemes because R-state like hemes, where nitrite binds preferentially, have lower redox

potentials.8 Chemical modifications, such as addition of PEG chains, which destabilize the

deoxyHb conformation, enhance nitric oxide production.23 It should be noted that the physiological

significance of nitrite reductase activity within the red blood cell is still not clear because this

pathway does not necessarily provide for the distribution of localized nitric oxide from nitrite

stores in regions of low oxygen pressure.10 Since NO rapidly binds to a second equivalent of

deoxyHb to form iron-nitrosyl-Hb, the mechanism by which NO leaves the red blood cell remains

a subject of interest.10 Oxidation of iron-nitrosyl-Hb mediated by an oxyHb/nitrite reaction radical

7

intermediate would release NO from the heme-bound ferrous iron. The metabolism of nitric oxide

to nitrate (NO3-) by oxyHb is then a further concern. An alternative theory has been proposed

where N2O3, an unstable precursor of nitric oxide, is the diffusing species.10

Identifying systems with appropriate functional and structural properties is only a partial

solution. There is a significant impediment to commercialization of these non-vasoactive HBOCs

because neither coupled nor clustered Hbs can be made with the efficiency that industrial scale-up

will require. The Hb bis-tetramer is produced in a maximum 40% yield from a tetra-functional

reagent that is susceptible to hydrolysis and which produces off-target modifications.23 The Hb-

albumin cluster needs to be purified from a large excess of albumin and still contains some under-

modified heterotrimer (Hb-HSA2).24 Improving the manufacturing efficiency of these promising

materials is a significant chemical problem that forms the basis of our work.

1.3 Bioorthogonal protein modification

The general idea of “bioorthogonal” processes was proposed by Sletten and Bertozzi27

and has been used to identify reactions that occur in the presence of biological molecules without

reacting with native functional groups in those molecules. Coupling two large proteins requires

attaching a chemical handle to surface-exposed residues of those proteins without affecting the

function of the proteins. There are many examples of bioorthogonal reactions that have been

successfully applied to the conjugation of proteins with small molecules: Staudinger ligation28,

tetrazole photo-click cycloadditions29, tetrazine ligation30, to name a few. We chose to focus on

reactions that are robust and wide in scope: the copper-catalyzed azide-alkyne cycloaddition

(CuAAC) and the strain-promoted alkyne-azide cycloaddition (SPAAC). The avidin-biotin

interaction is included as it is orthogonal with respect of Hb, although it is not bioorthogonal in

general.

1.3.1 Copper-catalyzed azide-alkyne cycloaddition (CuAAC)

Sharpless31 and Meldal32 independently discovered CuAAC in 2002. Sharpless coined the

term ‘click’ chemistry33 to describe these types of reactions that are high yielding with simple

conditions that form C-N bonds. CuAAC is one of several bioorthogonal reactions because the

participating moieties are not found in biomolecules.

8

The Huisgen 1,3-dipolar cycloaddition34 has been known since the 1960’s but requires

elevated temperatures and produces a mixture of regioisomers (Scheme 1.1). Sharpless had

proposed this as a potential “click” process but its properties did not lend themselves to that

application. The addition of Cu(I) to the reaction solution transforms the process dramatically.

Scheme 1.1: Huisgen cycloaddition.

CuAAC proceeds rapidly and regiospecifically at room temperature to give exclusively the

1,4-disubstituted isomer. The mechanism probably proceeds by terminal alkyne activation by a

copper-catalyst (Scheme 1.2).35 Propiolamide derivatives were found to be the ideal dipolarophiles

because electron withdrawing character lowers the lowest unoccupied molecular orbital (LUMO),

although enhanced reactivity of the alkyne as a Michael acceptor is an inevitable consequence.36

Scheme 1.2: CuAAC mechanism. Image reproduced with permission from Meldal et al.35

9

CuSO4 (in the presence of ascorbic acid as a reducing agent) is a popular choice of the

Cu(I) source for bioconjugation.37 The presence of ligands such as bathophenanthroline or tris(3-

hydroxypropyltriazolylmethyl)amine (THPTA) that stabilize the Cu(I) state accelerates the

reaction and protects the proteins from oxidative damage. The formation of reactive oxygen

species is a significant drawback of this seemingly convenient reaction. Ascorbic acid behaves as

a pro-oxidant in the presence of copper (Scheme 1.3)38, which initiates methemoglobin (metHb)

formation and denaturation of non-cross-linked proteins.39, 40 We find from quantification of free

thiols with Ellman’s reagent that under the CuAAC conditions the β-cys-93 residues are oxidized

to disulfides. Native Hb cysteines are oxidized to a greater extent than the cysteines of β-subunit

cross-linked Hb, suggesting that there is a different response to oxidizing species.

Scheme 1.3: The formation of reactive oxygen species in the presence of copper: AH- + Cu2+ →

A∙- + Cu+ + H+.38

Nonetheless, constructing Hb bis-tetramers by CuAAC does elicit control over product

formation, with yields between 40-50% that are excellent for this challenging combination.40, 41

Literature reports of quantitative yields are inflated because the authors failed to account for

CuAAC-mediated denaturation. In general, azide-modified proteins are coupled via an excess of

bis-alkyne by a solubility controlled process (Scheme 1.4). Once the insoluble bis-alkyne is

solubilized by the first modification (“Click 1”), a second protein rapidly adds (“Click 2”). The

128 kDa species is not detected if 0.5 eq. of bis-alkyne is used.

10

Scheme 1.4: Phase-direction copper-catalyzed azide-alkyne cycloaddition (PDCuAAC). Image

reproduced with permission from Foot et al.40

The challenge is the site-specific incorporation of a single azide. Foot et al.40 synthesized

an azide-containing reagent structurally analogous to TTDS, which minimizes but does not

eliminate off-target modifications. Yang et al.41 ensured site-specific modification by adding an

amine-azide immediately after Hb is treated with TTDS. However, hydrolysis is significant, even

with a large excess of amine-azide.

Alkyne homocoupling from Glaser42 (and the related Hay43) and/or Eglinton44 type

reactions are possible unwanted side-products of CuAAC (Scheme 1.5).45 Lampkowski et al.46

recently improved the oxidative coupling of terminal alkynes by adapting the reaction for

bioconjugation in aqueous medium. CuI is insoluble but the complex with TMEDA

(tetramethylethylenediamine) is soluble. Self-coupling of alkyne-derivatized proteins is a potential

route to simplifying coupling.

Scheme 1.5: Glaser-Hay homocoupling with CuI/TMEDA (tetramethylethylenediamine).

1.3.2 Strain-promoted alkyne-azide cycloaddition (SPAAC)

SPAAC is a metal-free alternative to CuAAC bioconjugation developed by Bertozzi

(Scheme 1.6).47 The reaction proceeds at room temperature and does not require a catalyst, making

11

it especially useful for the chemical labeling of living systems. The cyclooctyne moieties do not

appear to present any in vivo toxicity in mice.

Scheme 1.6: SPAAC reaction.

OCT was the first cyclooctyne substrate developed by Bertozzi (Figure 1.8). DIBO and

DIBAC experience faster kinetics because of the additional sp2 character.27 Increasing reactivity

produces side reactions, exemplified by BARAC, which undergoes hydrolysis in PBS buffer with

a half-life of 24 hours.

Figure 1.8: Second order rate constants for reactions of strained cyclooctynes OCT (cyclooctyne),

DIBO (dibenzocyclooctyne), DIBAC (dibenzoazacyclooctyne) and BARAC

(biarylazacyclooctynone) with a common azide.27

1.3.3 The high affinity avidin-biotin interaction

Avidin is an approximately 67 kDa tetrameric protein in egg white with an affinity for

biotin that is one of the strongest non-covalent interactions known.48 Numerous biotechnologies

are based on this efficient association and a recent example is in the field of cancer therapeutics.49

12

Biotinylated radiolabels can be amplified at the site of a cancer targeted antibody linked to avidin.

The foreign object in circulation does not appear to invoke a clinically relevant immune response.49

Based on this example, it was reasonable to consider applying this in an approach to HBOC

formation.

Chemists have approached the HBOC challenge by assembling Hb microspheres.50 Avidin

(pI ≈ 10) and oppositely charged ovalbumin (pI ≈ 4.6) can self-assemble into microspheres with

charge compensation driving the combination (Figure 1.9).51 Similar assemblies form with

lysozyme (pI ≈ 11)/ovalbumin and lysozyme/bovine serum albumin (pI ≈ 5) mixtures. Self-

assembled structures derived from avidin/Hb (pI ≈ 6.9) could be the starting point for stabilized

materials.

Figure 1.9: pH dependent formation of avidin/ovalbumin microspheres as visualized by phase-

contrast micrograhs. Image reproduced with permission from Desfougeres et al.51

1.4 Purpose of thesis

The structural criteria for non-vasoactive Hb-based oxygen carriers (HBOCs) can be

derived from working models with tetramer stabilization and volume amplification established as

essential elements. We now explore the adaptation of high efficiency reactions to the chemical

modification of Hb for the purpose of developing a scalable protein coupling method. ‘Click’

chemistry envelopes a diverse scope of economical bioorthogonal reactions with CuAAC and

SPAAC as prime examples. In tailoring these reactions for Hb conjugation, we learned that

SPAAC is particularly versatile and gives access to Hb-albumin clusters as an HBOC alternative.

13

The result is a transfusion-ready material rapidly assembled from raw materials by an operationally

non-complex approach that requires minimal purification. The scope of protein coupling recipes

need not be limited to bioorthogonal ‘click’ reactions: an entirely different approach based on

avidin-Hb self-assembly also produce novel materials with even greater efficiency. The special

considerations taken to secure 1) site-specific addition of a unique chemical handle; 2) an efficient

Hb-specific coupling method; and 3) synthetic ease provide general lessons in bringing about

precise conjugation of similarly modified proteins with optimum control over products.

14

Chapter 2

Hemoglobin bis-Tetramers by the Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC)

Figures in this chapter were reproduced with permission from: Singh, S., Dubinsky-Davidchik,

I. S., Yang, Y., and Kluger, R. (2015) Subunit-directed click coupling via doubly cross-linked

hemoglobin efficiently produces readily purified functional bis-tetrameric oxygen carriers,

Organic & Biomolecular Chemistry 13, 11118-11128.

2.1 Results and Discussion

Addition of azido functionalized analogues of TTDS to native deoxyHb give undesired

modifications within both the β- and α-subunits.40 Azides within the α-subunits do not participate

in CuAAC coupling, probably because their orientation is internal to the protein. To re-direct the

undesirable modifications, we opted to install a fumaryl bridge as an α-subunit amino-blocking

group.

It was not known if doubly cross-linked Hb could be prepared efficiently by this route. A

modest yield is reported for the synthesis α99-fumaryl-α99, β82-trimesoyl-β82 Hb from α2β82-

trimesoyl-β82 Hb.52 The formation of a doubly cross-linked species is non-commutative in that

the doubly cross-linked Hb can be assembled quantitatively if the α-subunits are linked first

(Figures 2.1, Scheme 2.1).52, 53 The second cross-link is sensitive to the conformation of the pre-

cursor, which is native-like in the case of α99-fumaryl-α99, β2 deoxyHb.54

15

Figure 2.1: Reverse-phase HPLC trace under dissociating conditions of α99-fumaryl-α99, β82-

trimesoyl-β82. Peaks are as follows: heme (10 min.); β-subunit cross-linked subunits (56 min.); α-

subunit cross-linked subunits (76 min.).

Scheme 2.1: Doubly cross-linked Hb can be prepared quantitatively if the α-subunits are modified

first.52, 53

An α-subunit protecting group strategy should then produce a well-defined azide-activated

protein. The rigid linker 1 (Scheme 2.2) was selected from a library of compounds for its superior

site-selectivity.53 The seven step synthesis of this compound is shown, starting from the boc-

protected amine, and details are reported in Singh et al.53

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.0 20.0 40.0 60.0 80.0

Absorb

ance

Time (min.)

16

Scheme 2.2: Synthesis of azide cross-linker 1.

When linker 1 is combined with α99-fumaryl-α99, β2 Hb, additional modifications are

produced (Scheme 2.3, Figure 2.2). Fortunately, the undesired modifications can be suppressed

by reducing the amount of linker from 16 eq. to 8.5 eq. (Figure 2.3). The identity of the peak from

the cross-linked β-subunits at 56 min. in the reverse-phase HPLC trace, which should represent

half of the total β-subunits, was confirmed by mass analysis (Figure 2.15).

17

Scheme 2.3: Addition of azide cross-linker 1 to α-subunit-protected Hb gives α99-fumaryl-α99,

β82-azido-β82.

Figure 2.2: Reverse-phase HPLC trace under dissociating conditions of the product of the reaction

of α99-fumaryl-α99, β2 with linker 1. Peaks: heme (10 min.); β-subunits cross-linked (52 min.); α-

subunits cross-linked (62 min.).

Figure 2.3: Reverse-phase HPLC trace under dissociating conditions of the product of the reaction

of α99-fumaryl-α99, β2 with linker 1. Peaks are due to: heme (10 min.); β-subunits (30 min.); β-

subunits cross-linked (56 min.); α-subunits cross-linked (73 min.).

The doubly cross-linked tetramers were coupled by the CuAAC conditions with 20 eq. of

bis-alkyne 2, 4 eq. of bathophenanthroline ligand, 2 eq. of CuSO4 and 40 eq. of ascorbic acid

(Scheme 2.4). The achievement of a well-defined bis-tetramer is promising despite the moderate

0.00

0.05

0.10

0.15

0.20

0.0 20.0 40.0 60.0 80.0

Absorb

ance

Time (min.)

0.00

0.10

0.20

0.30

0.40

0.50

0.0 20.0 40.0 60.0 80.0

Absorb

ance

Time (min.)

18

yield (Figure 2.4). SDS-PAGE analysis reveals a band belonging to the tetra-β fragment (Figure

2.5). The small improvement in yield under deoxygenated conditions (Figure 2.4) probably results

from increased accessibility of the cationic funnel. A marginal increase was also observed in more

acidic buffers, consistent with a more reactive conformation.55 The addition of IHP had the reverse

outcome, probably due to blocking of the central channel. Protection of the β-subunit cysteines

with N-ethylmaleimide had no effect.

Scheme 2.4: CuAAC with bis-alkyne 2 to give the doubly cross-linked bis-tetramer.

Figure 2.4: Size-exclusion HPLC of the CuAAC mixture before purification. The peak at 32 min.

is due to the ~128 kDa bis-tetramer and the peak at 37 min. is due to the cross-linked starting

materials.

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

27.0 31.0 35.0 39.0 43.0

Ab

sorb

ance

Time (min.)

DeoxyHb

COHb

19

Figure 2.5: SDS-PAGE analysis of the bis-tetramer before purification. Lane 1: protein ladder;

Lane 2: native Hb; Lane 3: α99-fumaryl-α99, β82-trimesyl-β82; Lane 4: α99-fumaryl-α99, β82-

azido-β82; Lanes 5 & 6: bis-tetramer containing mixture before purification.

This bis-tetramer is unique for its low oxygen affinity of P50 = 20.6 torr, which resembles

Hb’s oxygen affinity within the red blood cell of P50 = 30 torr (Figure 2.6, Table 2.1).56 The value

of intrinsic low oxygen affinity is emphasized by the insensitivity of β-subunit cross-linked

materials to anionic effectors. The raw oxygen binding data (not shown here) is notably disperse,

deviating slightly from the fitted curve.57

Figure 2.6: Oxygen binding curves of α99-fumaryl-α99, β82-trimesyl-β82 Hb and the bis-tetramer

(compared to native Hb).

20

Table 2.1: Oxygen binding properties of doubly cross-linked Hb and the bis-tetramer (compared

to native Hb).

Hb species P50 (torr) Fold-increase in

P50 after IHP n50

Native 5.0 ± 0.1 3.0 ± 0.1

Native + 1.2 eq. IHP 28.4 ± 0.2 5.7 1.8 ± 0.1

Native + 2.3 eq. IHP 40.0 ± 0.4 8.0 1.8 ± 0.1

α99-fumaryl-α99, β2 13.9 ± 0.3 2.6 ± 0.1

α99-fumaryl-α99, β2 + 2.3 eq. IHP 47.3 ± 0.3 3.4 1.9 ± 0.1

α2, β82-trimesyl-β82 4.8 ± 0.2 2.4 ± 0.1

α2, β82-trimesyl-β82 + 1.2 eq. IHP 9.9 ± 0.2 2.1 2.2 ± 0.1

α2, β82-trimesyl-β82 + 2.3 eq. IHP 22.9 ± 0.2 4.8 2.4 ± 0.1

α99-fumaryl-α99, β82-trimesyl-β82 20.6 ± 0.3 1.7 ± 0.1

α99-fumaryl-α99, β82-trimesyl-β82 + 2.3 eq. IHP 23.6 ± 0.4 1.2 1.8 ± 0.1

bis-tetramer 17.4 ± 0.5 1.5 ± 0.1

The chemical modifications do not impact the secondary structure of the protein, as can be

determined by CD spectroscopy. T-state character is evident by the depressions at 220 nm (Figure

2.7) and 285 nm (Figure 2.8), which occur when oxyHb is deoxygenated.58

Figure 2.7: CD spectra of α99-fumaryl-α99, β82-trimesyl-β82 Hb and the bis-tetramer (compared

to native carbonmonoxyHb) in the far UV.

-80.0

-60.0

-40.0

-20.0

0.0

20.0

40.0

60.0

80.0

203 213 223 233 243

Elli

pticity

(mdeg)

Wavelength (nm)

Native

Doubly cross-linked

Bis-tetramer

21

Figure 2.8: CD spectra of α99-fumaryl-α99, β82-trimesyl-β82 Hb and the bis-tetramer (compared

to native carbonmonoxyHb) in the near UV.

Purification of the doubly cross-linked material from singly cross-linked material by heat

treatment is not feasible based on an evaluation of their relative thermal stabilities under anaerobic

conditions (Table 2.2). Under aerobic conditions, fumaryl cross-linked proteins are vulnerable

because of their tendency to undergo auto-oxidation (Figure 2.9).59 In general, thermal stability

does not increase with additional cross-links.

Table 2.2: Minimum temperatures required for the denaturation of native and modified

carbonmonoxyHb species under anaerobic conditions in sodium phosphate buffer (0.01 M, pH

7.4). CarbonmonoxyHb species Temperature (°C)

Native 79

α99-fumaryl-α99, β2 90

α99-fumaryl-α99, β82-trimesyl-β82 94

α2, β82-trimesyl-β82 95

-15.0

-10.0

-5.0

0.0

5.0

10.0

15.0

240 260 280 300 320

Elli

pticity (

mdeg)

Wavelength (nm)

Native

Doubly cross-linked

Bis-tetramer

22

Figure 2.9: Turbidity curves for native and modified carbonmonoxyHb species under aerobic

conditions in 0.1 M sodium phosphate buffer (0.1 M, pH 7.4).

Nitrite reductase activity (NiR) is described by the rate constant for the second order

process,

Rate (metHb formation) = k[NO2-][HbFe2+]

The rate constant can be determined from initial rates in the presence of excess nitrite. In

this case, the observed rate constant, kobs, for the second order process is first order because the

concentration of nitrite is kept constant,

kobs = k[NO2-]initial

rate(metHb formation) = kobs[HbFe2+]

The nitrite reductase activity (NiR) of doubly cross-linked Hb and the PEG derivative were

measured by the method of Lui et al.23 HbFe2+ (deoxyHb), HbFe3+ (metHb), HbFe2+-NO (NOHb)

and HbFe2+-O2 (oxyHb) have unique absorbance profiles between 500-700 nm. Therefore, the

changes in concentration of these species can be tracked by absorbance spectroscopy.26 A program

designed originally by Dr. Francine Lui deconvolutes the absorbance spectra (Figure 2.10). The

bimolecular rate constant for the NiR reaction of α99-fumaryl-α99, β82-trimesyl-β82 deoxyHb

with nitrite is k = 0.46 +/- 0.03 M-1s-1 (Figure 2.11). Low-oxygen-affinity materials have little NiR

activity if cross-links prevent a complete R-state transition.6, 23

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

50 55 60 65 70 75 80 85Op

tica

l den

sity

(ab

sorb

ance

at

70

0 n

m)

Temperature (°C)

Native

α99-fumaryl-α99, β2

α2, β82-trimesoyl-β82

Doubly cross-linked

23

Figure 2.10: Progression of the reaction of deoxyHb (α99-fumaryl-α99, β82-trimesyl-β82) with

nitrite (deconvolution program originally developed by Dr. Francine Lui23). [deoxyHb] = 0.04 mM

and [nitrite] = 0.65 mM.

Figure 2.11: Initial rate plot of the NiR reaction of α99-fumaryl-α99, β82-trimesoyl-β82 deoxyHb

with nitrite. [deoxyHb] = 0.04 mM.

The two β-cys-93 residues of α99-fumaryl-α99, β82-trimesoyl-β82 Hb were modified with

maleimide-PEG5K (Figure 2.12). β-subunit cross-linked COHb substrates require heat for the

PEGylation reaction to proceed.23 The oxygen binding curve for this new derivative is shown in

Figure 2.13. PEG modification increases the oxygen affinity significantly, from P50 = 20.6 torr to

P50 = 11.6 torr (Table 2.3), consistent with the idea that PEG modification destabilizes the T-

state.23

R² = 0.9755

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

0.0 0.5 1.0 1.5 2.0

v o (n

M/s

)

[NO2] (mM)

24

Figure 2.12: Size-exclusion HPLC traces of the products from PEGylation of α99-fumaryl-α99,

β82-trimesoyl-β82 Hb at room temperature and 37 °C.

Figure 2.13: Oxygen binding curve of PEG modified α99-fumaryl-α99, β82-trimesoyl-β82 Hb.

The bimolecular NiR rate constant almost doubled, from k = 0.46 M-1s-1 to k = 0.87 M-1s-1

following PEG modification (Figure 2.14, Table 2.3). Within the data set presented, native Hb

and its PEGylated derivative define the lower and upper limit of NiR activity and the activities of

cross-linked materials are within that range. Unfortunately, T-stabilization appears to attenuate the

degree to which PEG can enhance the rate constant. In general, materials with high oxygen affinity

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

20.0 25.0 30.0 35.0 40.0 45.0

Ab

sorb

ance

(2

80

nm

)

Time (min.)

Doubly x-linked PEG (37 °C rxn)

Doubly x-linked PEG (RT rxn)

Doubly x-linked

25

and low cooperativity give the best NiR activity, presumably because hemes are locked in globins

in the preferred R-state.23, 60

Figure 2.14: Initial rate plot PEG modified α99-fumaryl-α99, β82-trimesoyl-β82 Hb. [deoxyHb]

= 0.04 mM.

Table 2.3: Comparison of oxygen affinity, cooperativity and nitrite reductase activity (NiR) of

doubly cross-linked Hb and its PEG modified derivative with other chemically modified species.23

Hb species NiR kint (M-1s-1) % increase in

NiR after PEG P50 (torr) n50

Native (α2β2) 0.25 +/-0.02 5 3

Native (α2β2-PEG5K2) 2.5 +/- 0.03 1000 3.6 1.8

α99-fumaryl-α99, β2 0.52 +/- 0.03 13.9 2.6

α99-fumaryl-α99, β2 PEG5K2 1.4 +/- 0.03 269 7.9 2.4

α99-fumaryl-α99, β82-trimesyl-β82 0.46 +/- 0.03 20.6 1.7

α99-fumaryl-α99, β82-trimesyl-β82

PEG5K2 0.87 +/- 0.05

189 11.6 1.8

Hb bis-tetramer (Lui et al.23) 0.7 +/- 0.05 9.3 2.7

Hb bis-tetramer-PEG5K4 (Lui et al.23) 1.8 +/- 0.05 257 4.1 2.4

2.2 Concluding remarks

Previous efforts undertaken to install a single azido functional group within Hb resulted

in the cross-linker reacting non-specifically at multiple sites on the protein. We’ve solved this

challenging dilemma by invoking a protecting group strategy to block potential off-target

modifications. The double cross-linking approach permits efficient modification of the protein in

a sequence that accommodates our synthetic needs. We are able to secure homogeneity of the

resultant Hb-N3 construct by harnessing precise control over the reaction conditions. The yield of

the subsequent click reaction is somewhat improved by accessing a protein conformation that

R² = 0.9901

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0.0 0.5 1.0 1.5 2.0

v o (n

M/s

)

[NO2] (mM)

26

improves the accessibility of the central channel. The end result of a defined species that can be

prepared reproducibly with confidence is an essential achievement. Furthermore, characterization

of this multiply cross-linked bis-tetrameric species revealed unique properties. The influence of

the fumaryl bridge dramatically shifts the oxygen dissociation curve to the right that is further

augmented by the rigid double cross-link framework. The protein is fixated to favor the T-state

and this is recognizable from the CD spectrum. The impact of the fumaryl bridge is also reflected

by the apparent thermal stability of the protein under aerobic conditions. NiR remains

competitive with that of the native tetramer despite the apparent low oxygen affinity. We expect

that this new bis-tetramer type should support useful oxygen delivery at oxygen deficient tissues.

The bis-tetramer’s physical properties may be further fine-tuned by a facile PEG modification.

The necessary steps taken to secure homogeneity while simultaneously adjusting the physical

properties of the target protein may be generally applicable to other like systems.

2.3 Experimental

2.3.1 General

Azide cross-linker 1 was synthesized according to Singh et al.53 Additional reagent, Hb

deoxygenation, HPLC, mass spectrometry, PAGE and oxygen binding details are described in the

General Experimental Methods chapter of this thesis.

2.3.2 Sequentially cross-linked Hb (α99-fumaryl-α99, β82-trimesoyl-β82)

To deoxygenated α99-fumaryl-α99, β2 Hb (0.05 mM, 5 mL in sodium borate buffer (50

mM, pH 9.0)) was added TTDS (5 eq., 1.3 mg) and this mixture was stirred under a nitrogen

atmosphere overnight at 37 °C. The solution was then flushed with carbon monoxide and passed

through a Sephadex G-25 column equilibrated with MOPS buffer (0.1 M, pH 8.0). The protein

was concentrated by centrifugation through a membrane filter (30 kDa cut-off) then stored at 4 °C

under an atmosphere of carbon monoxide. The products were analyzed by reverse-phase HPLC

and mass spectrometry analysis.

α99-fumaryl-α99, β82-trimesoyl-β82 was modified with maleimide-PEG (MW 5K) by the

conditions described in Lui et al.23 The products were analyzed by size-exclusion HPLC.

27

2.3.3 Preparation of a doubly cross-linked Hb bis-tetramer

To deoxygenated α99-fumaryl-α99, β2 Hb (0.02 mM, 18.4 mL in sodium borate buffer (50

mM, pH 9.0)) was added azide 1 (8.5 eq, 50 μL, 0.0513 M in DMSO) and this mixture was stirred

under a nitrogen atmosphere for 5 hours at 37 °C. The solution was then flushed with carbon

monoxide and passed through a Sephadex G-25 column equilibrated with MOPS buffer (0.1 M,

pH 8.0). The protein was concentrated by centrifugation through a membrane filter (30 kDa cut-

off) then stored at 4 °C under an atmosphere of carbon monoxide. The products were analyzed by

reverse-phase HPLC and mass spectrometry analysis.

To the modified carbon monoxide-bound protein (0.13 mM, 1.65 mL in sodium phosphate

buffer (0.02 M, pH 7.4)) was added alkyne 2 (20 eq., 43 μL, 20 mM in DMSO),

bathophenanthroline ligand (4 eq., 43 μL, 20 mM in H2O), CuSO4 (2 eq., 22 μL, 20 mM in H2O)

and L-ascorbic acid (40 eq., 86 μL, 100 mM in H2O). The vial containing this mixture was crimp

sealed, flushed with carbon monoxide and stirred for 4 hours. The mixture was passed through a

Sephadex G-25 column equilibrated with MOPS buffer (0.1 M, pH 7.2). The protein was

concentrated by centrifugation through a membrane filter (30 kDa cut-off). The bis-tetramer was

purified by passing the mixture through a Sephadex G-100 column equilibrated with Tris-HCl

(37.5 mM, pH 7.4) containing magnesium chloride (0.5 M). The first fraction (containing the bis-

tetramer) was concentrated by centrifugation through a membrane filter (30 kDa cut-off) then

stored at 4 °C under an atmosphere of carbon monoxide. The bis-tetramer (before and after

purification) was analyzed by size-exclusion HPLC. The CuAAC coupling reaction was also

performed on the deoxygenated protein.

2.3.4 Circular dichroism (CD) spectroscopy

Scans were acquired in triplicate using a Jasco J-710 CD spectrophotometer. Hb bound

with carbon monoxide (5 μM heme in sodium phosphate buffer (0.01 M, pH 7.4)) was submitted

to far UV (200-260 nm) measurements. 50 μM heme samples were prepared for UV-vis (245-470

nm) measurements.

28

2.3.5 Thermal denaturation and turbidity

Thermal denaturation: A headspace vial containing the sample of carbon monoxide-bound

Hb (2.0 mL, 0.02 mM in sodium phosphate buffer (0.01 M, pH 7.4)) was crimp-sealed then flushed

with carbon monoxide. The sample was stirred in the vial, which was immersed in a heated

silicone oil bath for 30 min., then cooled in an ice bath for 10 min. The resulting heterogeneous

solution, containing denatured protein as a precipitate, was concentrated through a membrane (30

kDa cutoff) by centrifugation (14,000 × g for 15 min.). The supernatant was collected and the final

Hb concentration was determined from measuring absorbance at 540 nm.

Turbidity: The samples of native and modified carbon monoxide-bound Hb (2 mL, 0.02

mM in sodium phosphate buffer (0.01 M, pH 7.4)) were contained in quartz cuvettes. Absorbance

at 700 nm was determined at 1.0 min. intervals with a double beam spectrophotometer (GBC

Cintra 40). The temperature was ramped at a rate of 5 °C/min. from 25 °C to 40 °C, then ramped

at 1 °C/min. from 40 °C to 90 °C.

2.3.6 Nitrite reductase activity

The nitrite reductase activity of α99-fumaryl-α99, β82-trimesoyl-β82 and PEG modified

α99-fumaryl-α99, β82-trimesoyl-β82 were measured by the procedure of Lui et al.23 The

deconvolution program was developed originally by Dr. Francine Lui. The deoxyHb concentration

was held constant at 0.04 mM and nitrite concentrations were varied between 0.5 and 1.8 mM.

29

2.4 Supplemental Information

Figure 2.15: Mass spectrum of peak collected from reverse-phase chromatography corresponding

to β82-azido-β82. (15867.22 Da – 1.01) x 2 β-subunits + C16H9O3N4Br (385.19 Da) = 32117.61

Da.

30

Chapter 3

Physiological Responses of a Bioorthogonally Coupled Hemoglobin bis-Tetramer in Circulation


This work is the result of a collaboration with the research group headed by Professor Warren

Zapol (Massachusetts General Hospital, Harvard Medical School, Boston, USA).

The bis-tetramer derived from doubly cross-linked Hb (Chapter 2) demonstrated a low

oxygen affinity as observed for Hb within the red blood cell.53 However, the nitrite reductase

activity of that material (k = 0.46 M-1s-1 or 0.87 M-1s-1 if PEGylated) was incomparable to

previously studied bis-tetramers (k = 0.7 M-1s-1 or 1.8 M-1s-1 if PEGylated).23 Therefore, we chose

the singly cross-linked bis-tetramer formulated by Yang et al.41 (P50 = 6.0) for physiological testing

in mice (Scheme 3.1). The synthesis was optimized to decrease hydrolysis and improve the overall

yield (see supplemental information for details). We expected that this material would maximize

production of nitric oxide at deoxygenated sites compared to other bis-tetramers, which is a

desirable property.

31

Scheme 3.1: Hb bis-tetramer prepared by the PDCuAAC.

We sought to characterize the hemodynamic responses of this new bis-tetramer constructed

from triazole scaffolds in both wild-type and physiologically stressed diabetic (db/db) mice. These

diabetic mice are sensitive to hypertension related NO-scavenging from endothelial nitric oxide

synthase (eNOS).61 Oxidative stress from hyperglycemia in these animals depletes the eNOS

cofactor tetrahydrobiopterin, leading to uncoupling of eNOS and production of superoxide.62

Changes in blood pressure of male 8- to 10-week-old wild-type (Figure 3.1) and db/db mice

(Figure 3.2) were monitored over a period of an hour following a 15% top load infusion of Yang’s

bis-tetramer. Control experiments utilizing modified PBS buffer containing N-acetyl cysteine,

native Hb and singly β-subunit cross-linked Hb (α2β82-trimesoyl-β82) were also conducted.

Protein solutions (3.5 g/dL) were introduced by tail vein injection at a dosage of 0.4 g/kg (e.g. 300

µL in a 25 g mouse). A more concentrated solution (7 g/dL) was prepared for experiments with

32

db/db mice to maintain a similar percent top-load. It is well-documented that the introduction of

native Hb or cross-linked Hb is accompanied by a significant spike in systolic blood pressure

(SBP).11, 63, 64 The 64 kDa species are likely to be small enough to be able to pass through

endothelial junctions, scavenging nitric oxide from nitric oxide synthase (eNOS).12 The localized

depletion of nitric oxide causes the surrounding smooth muscle to remain contracted. In both wild-

type and db/db mice, SBP measurements generally exceeded 120 mmHg. The effect was slightly

more sustained in db/db mice. These nitric oxide depleted subjects are sensitive to HBOC-induced

toxicity.61

In contrast, SBP remains in a normal range below 120 mmHg following treatment with the

bis-tetramer. The outcome was comparable to treatment with buffer alone (PBS buffer containing

N-acetyl cysteine). These data confirm that the size of a Hb bis-tetramer is sufficient to evade

extravasation. We can also affirm that the new structural features built from bioorthogonal ‘click’

chemistry are tolerated in vivo. The comparison between the SBP responses to the bis-tetramer and

the cross-linked tetramer is critical because the oxygen-binding properties of the two materials are

otherwise nearly identical.

Winslow and coworkers had proposed that the vasoactivity of cross-linked tetramers is

from a homeostatic response to excess oxygen.65, 66 The ‘autoregulation theory’ is based on

observations that materials with high oxygen affinity and/or diffusion barriers cause less

hypertension in small animal models.66 Olson and co-workers found that the physiological

outcome cannot be predicted from P50 alone as similarly high oxygen affinity Hb mutants with

varying nitric oxide dioxygenase activity had varying capacities to induce hypertension.67 The

excessive oxygen delivery hypothesis also does not apply for our data since the bis-tetramer is

fully capable of oxygen delivery but is not vasoactive. Rather, the lack of vasoactivity of the bis-

tetramer is related to its increased dimensions compared to tetramers with the source of toxicity

elated to their size-dependent penetration of the endothelium.12, 68

33

Figure 3.1: Tail systolic blood pressure (SBP) in awake wild-type mice following tail-vein


Protein solutions (3.5 g/dL) were administered at a dosage of 0.4 g/kg.

Figure 3.2: Measurement of tail systolic blood pressure (SBP) in awake db/db mice following tail-


Protein solutions (7 g/dL) were administered at a dosage of 0.4 g/kg.

Whole arterial blood was extracted by open chest cardiac puncture two hours after

injection. The total plasma-free Hb was quantified to confirm that the bis-tetramer was still present

in circulation (Figure 3.3 and Figure 3.4). Considering that we administered 300 µL of a 3.5 g/dL

protein solution in a 25 g mouse with a total blood volume of approximately 2 mL, we would

expect a plasma concentration around 0.5 g/dL. Diabetic mice were administered a higher protein

80

90

100

110

120

130

140

150

0 10 20 30 40 50 60

Syst

olic

blo

od

pre

ssu

re (

mm

Hg)

Time (min.)

*

Native Hb (n=6)

X-linked Hb tetramer (n=4)

Modified PBS buffer (n=6)

Bis-tetramer (n=6)

80

90

100

110

120

130

140

150

0 10 20 30 40 50 60

Syst

olic

blo

od

pre

ssu

re (

mm

Hg)

Time (min.)

*

X-linked Hb tetramer (n=5)

Modified PBS buffer (n=5)

Bis-tetramer (n=5)

34

dosage, despite having a similar total blood volume. Therefore, we recovered twice the

concentration of bis-tetramer in the plasma. The bis-tetramer, as well as the cross-linked tetramer,

remained in circulation hours after addition. The cross-linked Hb derivatives are likely to be

metabolized via the liver by a pathway that is mediated by haptoglobin.69 In contrast, native Hb

upon dissociation into dimers may also be removed rapidly through the kidneys leading to

significantly less protein being detected in the plasma. The results are consistent with the longer

half-lives in circulation of cross-linked derivatives compared to that of native Hb.69, 70

Figure 3.3: Total plasma free Hb from arterial whole blood of wild-type mice two hours after tail


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Modified PBS buffer(n=5)

Native Hb (n=5) Bis-tetramer (n=5)

Pla

sma

Hb

(g/

dL)

Hb species

35

Figure 3.4: Total plasma free Hb from arterial whole blood of db/db mice two hours after tail vein


The fraction of Hb bis-tetramer oxidized to metHb after the two-hour period in arterial

plasma was measured by deconvolution of the absorption spectra of the samples (Figure 3.5 and

Figure 3.6). The bis-tetramer heme iron remains in the functionally active ferrous form. Within

the erythrocyte, metHb reductase minimizes the amount of oxidized Hb. Ascorbic acid and

glutathione are normal components of plasma that should reduce ferric forms of HBOCs to the

ferrous state.71 In addition, the protein solutions in this study were suspended in buffer containing

a powerful anti-oxidant, N-acetyl cysteine.72 Experimental evidence suggests that erythrocytes

may also participate in the overall reduction system.73 Furthermore, ferrous heme may be

regenerated by Hb’s reductase/anhydrase activity.74 We conclude that our Hb bis-tetramer is

subject to the same environmental factors as its native and cross-linked counterparts. Since all of

the recovered plasma Hb is then potentially active to NO-scavenging, the blood pressure

measurements will properly reflect their activity in circulation.

0

0.2

0.4

0.6

0.8

1

1.2

1.4


X-linked Hb tetramer(n=4)

Bis-tetramer (n=4)

Pla

sma

Hb

(g/

dL)

Hb species

36

Figure 3.5: Total plasma metHb from arterial whole blood of wild-type mice two hours after tail

vein injection of the modified PBS buffer containing N-acetyl cysteine, native Hb or Hb bis-

tetramer.

Figure 3.6: Total plasma metHb from arterial whole blood of db/db two hours after tail vein


The oxidation of oxyHb to metHb by NO is very fast with a reported rate constant of 3.7

×107 M-1s-1.75 To test our hypothesis that the bis-tetramer avoids nitric oxide scavenging, we tested

its facility to bind NO using an NO chemiluminescence analyzer (Figure 3.7). The NO donor

DETA NONOate was selected to saturate the oxygenated protein solutions. The depletion of NO

is indicative of a binding event. We prepared a calibration curve using known concentrations of

Hb (Figure 3.8). We find that the bis-tetramer binds nitric oxide to the same extent as does native

0

0.5

1

1.5

2

2.5


Native Hb (n=4) Bis-tetramer (n=4)

Pla

sma

met

Hb

(%

)

Hb species

0

0.5

1

1.5

2


X-linked Hb tetramer(n=4)

Bis-tetramer (n=4)

Pla

sma

met

Hb

(%

)

Hb species

37

Hb (Figure 3.9). This shows that the bis-tetramer is fully capable of binding NO and that its lack

of vasoactivity is not due to any lack of affinity for NO.

Figure 3.7: NO consumption by native Hb standards and the bis-tetramer from the

chemiluminescent-detection nitric oxide analyzer.

Figure 3.8: NO consumption by native Hb standards (3.25, 6.5, 13, 26 and 52 µM heme).

y = 140.67x + 116.74R² = 0.9496

0

1000

2000

3000

4000

5000

6000

7000

8000

0 10 20 30 40 50 60

Are

a U

nd

er C

urv

e (m

V/m

in.)

Heme concentration (μM)

38

Figure 3.9: NO consumption by the bis-tetramer (3.25, 6.5, 13, 26 and 52 µM heme) in solution.


These results provide a unique opportunity to directly compare the influence that an

increase in molecular size has on extravasation properties. The Hb bis-tetramer prepared by

bioorthogonal ‘click’ chemistry did not cause increases in blood pressure associated with

vasoconstriction in both wild-type and db/db mice. Accordingly, the potential HBOC remained

present and functional in the plasma after a two hours in circulation. The 128 kDa dumbbell shaped

assembly is likely too bulky to substantially invade the endothelial pores lining the blood vessel

wall. It also appears that the unique structures that compose this bis-tetramer are compatible with

the circulatory system. These include the cross-link that stabilizes the tetramer from dissociation

into dimers and the triazole moieties that bridge the two tetramers together. The differential effects

of cross-linked tetramer compared to bis-tetramer provide a basis for altering protein size to evade

extravasation. We selected this triazole-linked bis-tetramer as a promising candidate in the first

place for its ideal properties and efficient synthesis. It is a product that is now readily transferable

for future work involving more in depth clinical investigations.

y = 1.1549x + 1.4124R² = 0.9623

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60

NO

co

nsu

mp

tio

n (

µM

)

Heme Concentration (μM)

39

3.3 Experimental

3.3.1 General

The bis-tetramer was prepared as described in Yang et al.41 with slight optimization: a

solution of Hb (0.5 mM in 1.5 mL of 50 mM sodium borate buffer, pH 9.0) was oxygenated by

stirring under a stream of oxygen with photoirradiation for 2 hours at 4 °C. The sample was then

deoxygenated by stirring under a stream of nitrogen for 2 hours at 37 °C. Trimesoyl tris(3,5-

dibromosalicylate) (TTDS) was added (2.0 eq. of 0.2 M solution in DMSO) and the mixture was

stirred for 12 min. 4-azidomethyl-benzylamine was added (40 eq. of 1.0 M solution in DMSO)

and the sample flushed with carbon monoxide. After 1 hour stirring at room temperature, the

mixture was passed through a Sephadex G-25 column equilibrated with MOPS buffer (0.1 M, pH

8.0). The collected fractions were concentrated by centrifugation through a filter (30 kDa cut-off)

and stored under carbon monoxide at 4 °C. See General Experimental Methods chapter of this

thesis for further details.

3.3.2 Preparation of protein solutions for transfusion

The protein solutions (native Hb and Hb bis-tetramer) were exchanged into a modified

phosphate buffered saline (PBS) buffer (pH 7.4) with 0.01 g/dL N-acetyl cysteine using a

Sephadex G-25 column. The final concentration of each solution was adjusted to 3.5 g/dL for

experiments with wild-type mice. The final concentration of each solution was adjusted to 7 g/dL

for experiments with db/db mice. The percent metHb in each sample was quantified at less than

3%. Protein solutions were oxygenated by photoirradiation for 1 hour at 4 °C.

3.3.3 Preparation of mice

This study was approved by the Subcommittee on Research Animal Care of Massachuetts

General Hospital. Mice were obtained from the Jackson Laboratory. The mice in this study were

8- to 10-week-old (25-30 g) male C57BL/6J mice and B6.Cg-m+/+Leprdb/J (C57BL/6J

background) db/db mice (50-55 g).

40

3.3.4 Blood pressure measurements

Systolic blood pressures (SBP) were collected using a tail cuff apparatus (CODA non-

invasive blood pressure system, Kent Scientific). The mice (wild-type or db/db) were restrained in

a tube holder (Kent Scientific) then the desired solution (either buffer, native Hb or Hb bis-

tetramer) was introduced by tail vein injection. The protein solutions (3.5 g/dL) were transfused at

a dosage of 0.4 g/kg (e.g. 300 µL in a 25 g mouse). This is a 15% top-load infusion in wild-type

mice if we assume a mouse blood volume of 80 µL/g (e.g. 2 mL in a 25 g mouse). Db/db mice

were also administered 0.4 g of protein/kg. However, the protein solutions were more concentrated

(7 g/dL) to maintain the same percent top-load (e.g. 300 µL in a 50 g mouse).

3.3.5 Blood gas and methemoglobin analysis

Mice were anaesthetized by intraperitoneal (IP) injection using a pre-prepared drug

cocktail containing 12 mg/mL ketamine and 9 µg/mL fentanyl, at a dosage of 10 µL/g of mouse

body weight - e.g. 250 µL in a 25 g mouse). A twice concentrated drug mixture was prepared for

anesthesia of db/db mice. Whole arterial blood was obtained by open chest cardiac puncture with

a heparinized syringe. Blood plasma was collected by centrifugation at 4000 × g at 4 °C for 8 min.

Total plasma Hb was measured using a blood gas analyzer (ABL800 Radiometer, Copenhagen,

Denmark). Percent metHb was calculated by deconvolution of the UV-vis spectrum (Biochrom

Libra S70 double beam spectrophotometer).

3.3.6 Nitric oxide consumption assay

Nitric oxide (NO) scavenging was measured using an NO chemiluminescence analyzer

(Sievers 280i Nitic Oxide Analyzer). Solutions of (oxygenated) native Hb and bis-tetramer were

prepared at varying concentrations (0, 3.25, 6.5, 13, 26 and 52 µM heme). An aliquot (10 µL) of

each protein solution was introduced per injection. DETA NONOate (Cayman Chemicals)

provided the source of nitric oxide.

3.3.7 Statistical analysis

Data is expressed as mean ± standard deviation. Data analysis was by repeated measures

two-way analysis of variance (ANOVA). A p-value less than 0.05 was considered significant.

41

Chapter 4

Assembly of Hemoglobin bis-Tetramers and Hemoglobin-Albumin Clusters by Metal-free Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC)


Hb bis-tetramers. Bioorthogonal coupling of proteins can lead to the controlled assembly

of a single entity from several proteins.22, 41 However, CuAAC is a bioorthogonal approach that is

not entirely suitable for Hb-based applications. The occurrence of metHb formation and

subsequent denaturation is the result of the pro-oxidant effects of ascorbic acid in the presence of

copper.38 Furthermore, copper ions are difficult to separate from the end product because of their

association with the protein.76 As an alternative, we adapted SPAAC to couple proteins without

added metal ions while retaining high efficiency and specificity. The target architecture of a

functional bis-tetramer is two tetramers cross-linked between the β-subunits and linked at this

interface.20 We modified Hb site-specifically with a cyclooctyne/azido moiety by addition of

amine-cyclooctyne/amine-azide to cross-linked Hb (Scheme 4.1). The reverse-phase HPLC trace

of Hb-cyclooctyne (Figure 4.1) and Hb-azide (Figure 4.2) show the products are pure except for

small amounts of impurities from competitive hydrolysis of the ester of the reactant.

Scheme 4.1: Preparation of Hb-cyclooctyne (top) and Hb-azide (bottom).

42

Figure 4.1: Reverse-phase HPLC trace of cross-linked Hb-cyclooctyne under dissociating

conditions. Peaks are as follows: heme (10 min.); α-subunits (38 min.); cross-linked β-subunits

(hydrolysis product, 52 min.); cross-linked β-subunits (cyclooctyne modified, 55 min.).

Figure 4.2: Reverse-phase HPLC trace of Hb-azide under dissociating conditions. Peaks are as

follows: heme (10 min.); α-subunits (45 min.); cross-linked β-subunits (hydrolysis product, 62

min.); cross-linked β-subunits (azide modified, 64 min.).

The cycloaddition reaction was initiated by combination of CO-bound Hb-cyclooctyne and

Hb-azide (Scheme 4.2). After incubation of the modified Hb solution at 4 °C for 12 days, we

obtained ~72% bis-tetramer (Figure 4.3). This yield takes into account a 10% impurity in each

starting material. Native gel analysis confirms that the peak eluting earlier in the size-exclusion

HPLC trace is due to the 128 kDa species (see SI).

It is apparent from the less than quantitative yield that the coupling reaction probably does

not proceed to completion. This appears to be unrelated to the stability of the starting materials:

the coupling yield can be reproduced using Hb-cyclooctyne and Hb-azide that had been stored in

sodium phosphate buffer at 4 °C for 30 days. We also confirmed that amine-cyclooctyne is

unaffected by the higher pH protein preparation conditions. It is possible that not every reaction

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0A

bso

rban

ce (

22

0 n

m)

Time (min.)

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

Ab

sorb

ance

(2

20

nm

)

Time (min.)

43

site is conformationally accessible for coupling. We tried preparing the protein starting materials

in a different way, specifically, by adding amine-cyclooctyne/amine-azide to the β-subunit cross-

linked protein after 10 min. of flushing with carbon monoxide. This attempt to secure

conformational uniformity did not improve the coupling yield. Perhaps installing an α-subunit

cross-linker first would block the bioorthogonal group from the interior of the protein by limiting

directional variability. The 72% yield was improved to 76% by coupling the proteins in the

deoxygenated state for 4 days (after 12 days under carbon monoxide). Access to the central channel

is improved in the deoxy conformation.22 However, this tactic is hampered by competitive metHb

formation. We are nonetheless pleased with the current state of the reaction and the simplicity of

the overall preparation.

If we assume that only a fraction of the starting material is available for coupling we can

obtain a second order rate constant of about 2.9×10-7 M-1s-1. This is significantly less than a typical

reported value27, which tells us that the protein environment has a profound effect on the reaction

kinetics. As expected, the rate of the reaction increases at higher concentrations of Hb. However,

there is no benefit obtained from heating the solution at 70 °C for 30 min. Replacing Hb-azide

with the more flexible Hb-PEG10-N3 also failed to enhance the rate.

Scheme 4.2: Copper-free click of Hb-cyclooctyne with Hb-azide.

44

A) B)

C) D)


Hb-azide. The peak at 33 min. is due to the ~128 kDa Hb bis-tetramer and the peak at 38 min. is

due to the ~64 kDa cross-linked starting materials. B) Percent yield based on the theoretical

maximum. C) Consumption of starting material fit to second order kinetics. D) Linear inverse plot.

The SPAAC process was compared with the analogous CuAAC reaction by coupling Hb-

alkyne and Hb-azide (Scheme 4.3). Bathophenanthroline ligand (4 eq.), CuSO4 (2 eq.) and

ascorbic acid (40 eq.) were added to the protein mixture. This ratio of reagents is effective for the

coupling of modified Hbs to bis-alkynes.40 In this case, the conditions were destructive. After one

hour, only a small percentage of protein had coupled (Figure 4.4). Leaving the reaction for longer

resulted in significant metHb formation and denaturation. The same outcome was observed when

we attempted to couple the Hb-alkyne to the bis-azide, indicating that CuAAC of alkyne-

containing Hbs activates a protein degradation pathway.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

25.0 30.0 35.0 40.0 45.0 50.0

No

rmal

ized

ab

sorb

ance

(2

80

nm

)

Time (min.)

1 day

6 days

12 days

12 days COHb,4 days deoxyHb

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15

% y

ield

bis

-tet

ram

er

Time (days)

[A]o = 0.32 mM

0

0.05

0.1

0.15

0.2

0.25

0 5 10 15 20 25

[A]

(mM

)

Time (days)

y = 23.7x + 4.74R² = 0.96

0

50

100

150

200

250

300

350

0 5 10 15

1 /

[A

] (m

M-1

)

Time (days)

45

Scheme 4.3: CuAAC of Hb-alkyne with Hb-azide.

Figure 4.4: Size-exclusion HPLC trace under high salt conditions of the products of CuAAC of

Hb-alkyne with Hb-azide. The peak at 34 min. is due to the ~128 kDa Hb bis-tetramer and the

peak at 39 min. is due to the ~64 kDa cross-linked starting materials.

Only Hb-cyclooctyne is required if coupling is mediated by a bis-azide that is not on a

protein. (Scheme 4.4). 4,4’-Diazidediphenylsulfone (0.45 eq.) was added to a solution of Hb-

cyclooctyne. Approximately 63% bis-tetramer was obtained after incubation at 4 °C for 12 days

(Figure 4.5). Native gel electrophoresis analysis confirmed that the species eluting first in the size-

exclusion HPLC is due to the approximately 128 kDa construct (see SI). The bis-azide must be

able to react with the entire pool of Hb-cyclooctyne, including the approximately one-third that is

unavailable for protein-protein coupling. Therefore, half of the total protein is modified with the

bis-azide. The outcome of the bis-tetramer forming reaction is then analogous to the combination

of Hb-azide with Hb-cyclooctyne.

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.10

30.0 35.0 40.0 45.0 50.0

Ab

sorb

ance

(2

80

nm

)

Time (min.)

46

Replacing the rigid bis-azide with a longer and more flexible alternative, (3,6,9,12,15-

pentaoxaheptadecane-1,17-diyl bis-azide) failed to enhance the rate. The addition of 2.0 or 10.0

eq. of bis-azide resulted in attachment of only the small molecule to Hb-cyclooctyne, indicating

that phase-directed coupling40 cannot be exploited here.

Scheme 4.4: Preparation of Hb bis-tetramer by copper-free coupling of Hb-cyclooctyne with bis-

azide.

47

A) B)

C) D)


bis-azide. The peak at 33 min. is due to the ~128 kDa Hb bis-tetramer and the peak at 38 min. is

due to the ~64 kDa Hb-cyclooctyne starting material. B) Percent yield based on the theoretical

maximum. C) Consumption of starting material fit to second order kinetics. D) Linear inverse plot.

Hb clusters. We are able to prepare entirely uniform Hb bis-tetramers in the highest yields

ever observed using strain-promoted bioorthogonal protein coupling. Combining heme proteins

under a carbon monoxide atmosphere in a stable non-denaturing environment ensures a high

quality end product that is likely to be suitable for clinical evaluation However, the incomplete

conversion of cross-linked tetramer to bis-tetramer results in an unacceptable percentage of

unwanted material. We wished to develop SPAAC technology further to prepare an HBOC in a

quantitative yield by the least demanding synthetic sequence possible. A significant advantage of

SPAAC is that linkages between two large proteins in solution can be formed because the reactive

moieties remain stable in solution, permitting long reaction times. We built on this feature by

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

26.0 31.0 36.0 41.0 46.0

No

rmal

ized

ab

sorb

ance

(2

80

nm

)

Time (min.)

6 days

1 day

12 days

15 days

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15

% y

ield

bis

-tet

ram

er

Time (days)

[A]o = 0.16 mM

0

0.02

0.04

0.06

0.08

0.1

0.12

0 5 10 15 20 25

[A]

(mM

)

Time (days)

y = 13.8x + 10.4R² = 0.93

0

50

100

150

200

250

0 5 10 15 20

1 /

[A

] (m

M-1

)

Time (days)

48

assembling higher order structures of Hb and albumin using as small an excess of the proteins as

possible. The previously reported Hb-albumin cluster (HemoAct) requires purification from

excess albumin and must also be separated from partially modified species.24 Using SPAAC as a

protein-clustering tool, we combine a specifically derivatized Hb with complementary shielding

proteins in a quantitative manner to minimize the need for post-production protein purification.

We surveyed a variety of possible cluster architectures before the final optimized assembly

was realized. Initially, we wished to shield the central oxygen carrying protein with cross-linked

Hb derivatives to maximize the oxygen-carrying capacity of the overall structure. We were

successful in appending multiple cross-linked Hb-azide derivatives to a core protein with

conjugates of multiple dibenzocyclooctyne moieties. Hb (non-cross-linked and β-subunit cross-

linked) were non-specifically acylated with NHS-DIBO (dibenzocyclooctyne) to give Hb-

DIBO/xlHb-DIBO, respectively (Scheme 4.5). The reverse-phase HPLCs of these products

(Figure 4.6 and Figure 4.7) reveal the impact that this modification has on the surface character

of the protein. Addition of non-polar appendage to Hb rendered the surface hydrophobic such that

most of the protein elutes very late and non-separated. Mass spectral analysis of the fractions

collected from reverse-phase HPLC proved to be challenging; we assume that 1-3 cyclooctynes

are appended per subunit based on the pattern of modification of Hb with the NHS-alkyne (see SI

for HPLC and mass spectrometry data).

Scheme 4.5: Decoration of native Hb with NHS-DIBO to give Hb-DIBO. The number of

cyclooctynes appended per Hb subunit (n) could not be determined by mass spectrometry

analysis.

49

Figure 4.6: Reverse-phase HPLC trace of Hb-DIBO under dissociating conditions.

Figure 4.7: Reverse-phase HPLC trace of xlHb-DIBO under dissociating conditions.

Hb-DIBO/xlHb-DIBO was combined with Hb-azide to give the product proposed in

Scheme 4.6. Size-exclusion HPLC analysis of the Hb-DIBO/Hb-azide product under high salt

conditions (Figure 4.8) reveals that the reaction proceeds to near completion in one day at 4 °C.

The very small amount of the 32 kDa αβ dimer peak suggests that Hb-cyclooctyne is appended to

at least two Hb-azide tetramers. This geometry maximizes the shielding of the central tetramer and

ensures that each dimer is affixed to a larger structure. Products with either one or two tetramers

linked to the central scaffold were obtained when xlHb-DIBO was the substrate (Figure 4.9).

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.0 20.0 40.0 60.0 80.0 100.0

Ab

sorb

ance

(2

20

nm

)

Time (min.)

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

0.0 20.0 40.0 60.0 80.0 100.0

Ab

sorb

ance

(2

20

nm

)

Time (min.)

50

Scheme 4.6: Copper-free click of Hb-DIBO with Hb-azide. The number of cyclooctynes appended

per Hb subunit (n) could not be determined by mass spectrometry analysis. The number of cross-

linked tetramers per Hb dimer (m) = 1 to 2.


free click of Hb-DIBO with Hb-azide. The (impure) Hb bis-tetramer reference (~128 kDa) elutes

at 34 min. while cross-linked starting material (~64 kDa) elutes at 38 min. Hb αβ dimer (~32 kDa)

elutes at 41 min.

0.00

0.20

0.40

0.60

0.80

1.00

1.20

25.0 30.0 35.0 40.0 45.0 50.0

Ab

sorb

ance

(2

80

nm

)

Time (min.)

Hb-DIBO + Hb-azide

Hb-DIBO

Hb-bistetramerreference

51


free click of xlHb-DIBO with Hb-azide. The (impure) Hb bis-tetramer reference (~128 kDa) elutes

at 34 min. while cross-linked starting material (~64 kDa) elutes at 38 min.

Native gel analysis confirmed the identities of the peaks in size-exclusion HPLC (see SI).

In both ensembles (incorporating Hb-DIBO or xlHb-DIBO), we see bands due to the ~128 kDa

bis-tetramer and a higher molecular weight species, which we can assume is an ensemble formed

from three Hbs.

The analagous CuAAC coupling of the product of the reaction of Hb with NHS-alkyne

(Hb-alkyne(s)) and Hb-azide failed to yield high molecular weight species (Scheme 4.7). The

reaction proceeds with significant denaturation, once again confirming that it is detrimental to

incorporate alkynes into heme proteins undergoing CuAAC.

Scheme 4.7: CuAAC of Hb-alkyne(s) with Hb-azide.

SPAAC assembly of Hb clusters is advantageous because the many surface-accessible

strained moieties in conjunction with the inherent enhanced reactivity of dibenzocyclooctynes

0.00

0.20

0.40

0.60

0.80

1.00

1.20

25.0 30.0 35.0 40.0 45.0 50.0

Ab

sorb

ance

(2

80

nm

)

Time (min.)

xlHb-DIBO + Hb-azide

xlHb-DIBO

Hb-bistetramer reference

52

compared to cyclooctynes77 ensures the rapid and complete functionalization of Hb. We should

also be able to append additional shielding groups and useful small molecules to the remaining

reactive groups. However, we sought to simplify the procedure further by avoiding production of

cross-links.

Substitution of Hb-azide with the non-vasoactive 67 kDa albumin is the logical solution

(Scheme 4.8). Albumin is a major constituent of blood plasma at a normal concentration of 4

g/dL.78 Cys-34 residues that were not blocked by post-translational cysteinylation79 were

modified with maleimide-azide, which is unstable and must be prepared from azido-PEG3-amine

and maleimide-NHS-ester. The partial purity of the product albumin-azide was assessed based

on analysis by mass spectrometry (see SI). Nonetheless, Hb-albumin clusters can be prepared by

this method (Figure 4.10, Figure 4.11). We note that a single αβ dimer within the tetramer can

accommodate up to at least two albumin proteins. The major peaks at 28 min. and 33 min. and

are due to (αβ dimer + one albumin) and (αβ dimer + two albumins), respectively. Constructs of

albumin-azide and Hb-cyclooctyne were also prepared (see SI).

53

Scheme 4.8: SPAAC of Hb-DIBO with albumin-azide. The number of cyclooctynes per Hb

subunit (n) could not be determined by mass spectrometry analysis. The number of albumin

proteins per Hb dimer (m) = 1 to 3 based on the size-exclusion HPLC.

Figure 4.10: Size-exclusion HPLC trace under high salt conditions of the products of the reaction

between albumin-azide and Hb-DIBO (3 equiv albumin to 1 equiv Hb). Peaks are as follows: 28

min. (αβ dimer + 2×albumin), 33 min. (αβ dimer + 1×albumin), 35 min. (albumin), 41 min. (αβ

dimer).

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

10.0 20.0 30.0 40.0 50.0 60.0

No

rmal

ized

Ab

sorb

ance

(2

80

nm

)

Time (min.)

1 hour

3 hours

1 day

3 days

8 days

Albumin-azide

Hb-DIBO

54


reaction between albumin-azide and Hb-DIBO (6 equiv albumin to 1 equiv Hb). Peaks are as

follows: 28 min. (αβ dimer + 2×albumin), 33 min. (αβ dimer + 1×albumin), 35 min. (albumin),

41 min. (αβ dimer).

Although Hb-DIBO and albumin-azide did readily combine, we would not be able to exert

complete control over the outcome of the reaction using a heterogeneous and undefined albumin-

azide starting material. It was by reversing the modification scheme and functionalizing the surface

of Hb with azides that we were able to produce our highest value product. First, we submitted

native Hb in the carbon monoxide-bound state to reaction with the small molecule NHS-azide

(Scheme 4.9). The protein surface of the resultant Hb-PEG-azide is significantly less hydrophobic

than that of Hb-DIBO based on the elution pattern of the reverse-phase HPLC (Figure 4.12).

Scheme 4.9: Preparation of Hb-PEG-azide by treatment of native Hb with NHS-azide. The number

of azides appended per Hb subunit (n) = 1 to 6.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

10.0 20.0 30.0 40.0 50.0 60.0

No

rmal

ized

Ab

sorb

ance

(2

80

nm

)

Time (min.)

4 days

Albumin-azide

Hb-DIBO

55

Figure 4.12: Reverse-phase HPLC trace of Hb-PEG-azide under dissociating conditions.

Albumin-DIBO was prepared as the complementary reactive partner and combined with

Hb-PEG-azide (Scheme 4.10). Unlike albumin-azide, albumin-DIBO is a well-defined starting

material and is well characterized by mass spectrometry. It is apparent from the mass spectrum of

albumin-DIBO that the cysteinylated albumin portion is not modified by the reagent at the surface-

accessible residue (see SI). Therefore, only approximately half of the total albumin participates in

SPAAC. Cysteinylated albumin can be stabilized in the reduced form by careful chemical

treatment80, but we decided to work with the protein as is because the minor non-vasoactive

contamination should not have an adverse effect in vivo. We were able to fully modify Hb-PEG-

azide using a small excess of albumin-DIBO (Figure 4.13). Complete modification is defined by

addition of at least one albumin to each αβ dimer within the tetramer so that the dissociated

construct exceeds the size threshold for renal filtration and extravasation. Based on the size-

exclusion HPLC, a maximum of three albumin proteins adhere to one αβ-dimer within the overall

tetramer. The utility of SPAAC to bring together large proteins without competition by hydrolysis

or denaturation is a significant advantage of this approach. The Hb-albumin cluster of Hosaka et

al.24 that is assembled using SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-

carboxylate) requires purification from a large excess of albumin. In our case, only a small excess

of albumin is present. Therefore, this material could be tested as an oxygen carrier immediately

without further purification.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.0 20.0 40.0 60.0 80.0 100.0 120.0

Ab

sorb

ance

(2

20

nm

)

Time (min.)

56

Scheme 4.10: SPAAC of Hb-PEG-azide with albumin-DIBO to assemble the Hb-albumin cluster.

Figure 4.13: Size-exclusion HPLC trace under high salt conditions of the products of the reaction

between albumin-DIBO and Hb-PEG-azide after 4 days. Peaks are as follows: 28 min. (αβ dimer

+ 3×albumin), 33 min. (αβ dimer + 2×albumin), 36 min. (αβ dimer + 1×albumin), 38 min.

(albumin), 44 min. (αβ dimer).

0.0

0.2

0.4

0.6

0.8

1.0

15 25 35 45 55

No

rmal

ized

Ab

sorb

ance

(2

80

nm

)

Time (min.)

1:2 Hb-PEG-azide:albumin-DIBO



Hb-PEG-azide

albumin-DIBO

57

In assembling a product with a predictable composition by a method that is both simple

and direct, we prepared a product with high therapeutic potential. Assembly of Hb-albumin

clusters using SPAAC as the primary synthetic tool to bring together large proteins has several

advantages over competitive preparations: 1) The protein is manipulated exclusively in the stable

CO-bound state; 2) There is no risk of potentially vasoactive contaminants; 3) Wasted by-

products are minimized 4) The material can be used as an oxygen carrier immediately without

further purification; and 5) The composition is defined so that the physiological outcomes will be

related to consistent materials.

The material modified to completion by albumin-azide (Figure 4.13, 1:4 Hb-PEG-

azide:albumin-DIBO) was analyzed for oxygen binding without purification. The product mixture

yields a heterogeneous binding curve with an oxygen affinity similar to that of native Hb (P50 =

6.2 +/- 0.4) and moderate cooperativity (n50 = 1.5 +/- 0.1) (Figure 4.14).

Figure 4.14: Oxygen binding curve of Hb-albumin cluster prepared from combination of Hb-PEG-

azide with albumin-DIBO.


SPAAC provides a practical and convenient route for the precise coupling and clustering

of heme proteins. Metal free ‘click’ chemistry ensures that the heme is not harmed by the reaction

conditions. Bis-tetramers can be constructed efficiently either by coupling bioorthogonally

modified proteins directly or by linking tetramers via a small molecule bis-azide bridge. The

architecture of two coupled Hb tetramers cross-linked between the β-subunits should support safe

and effective oxygen delivery. There may also exist the potential to remove unreacted materials

58

by an avidin affinity column if the free bioorthogonal moieties are modified with biotin. Hb-

albumin clusters are a readily accessible alternative that can be prepared by shielding the central

protein with neighboring accessory proteins. The improved approached is enticing for its large

scale manufacturing potential. The protein therapeutic would be assembled by a synthetic sequence

that converts raw materials directly to the final product with minimal complexity. Considering the

simplicity of the preparation and the high quality of the product, we expect that these innovations

will translate readily to industrial scale-up.

4.3 Experimental

4.3.1 General

Human serum albumin (HSA), N-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-

ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane (amine-cyclooctyne), 3,6,9,12,15-

pentaoxaheptadecane-1,17-diyl bis-azide, amino-PEG4-alkyne (amine-alkyne),

dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (NHS-DIBO), alkyne-PEG5-N-

hydroxysuccinimidyl ester (NHS-alkyne) and dibenzocyclooctyne-PEG4-maleimide (maleimide-

DIBO) were purchased from Sigma-Aldrich. Azido-PEG3-maleimide (maleimide-azide, prepared

from azido-PEG3-amine and maleimide-NHS-ester) was purchased from Alfa Aeser. NHS-PEG4-

azide (NHS-azide) was purchased from Thermo Fisher Scientific. 4,4’-Diazidediphenylsulfone

(bis-azide) was synthesized according to Li et al.81 4-azidomethyl-benzylamine (amine-azide) and

the bis-tetramer reference were prepared as described by Yang et al.41 PBS buffer refers to

phosphate buffered saline. Additional reagent, Hb deoxygenation, HPLC, mass spectrometry and

PAGE details are described in the General Experimental Methods chapter of this thesis.

4.3.2 Hb bis-tetramers (Hb-azide)

To deoxyHb (0.5 mM in 1.5 mL of 50 mM sodium borate buffer, pH 9.0) was added TTDS

(2 eq. of a 0.2 M solution in DMSO) and this mixture was stirred for 12 min. Then, amine-

cyclooctyne was added (40 eq. of a 1 M solution in DMSO) and the sample was flushed with

carbon monoxide. After 1 hour of stirring at room temperature, the mixture was passed through a

Sephadex G-25 column equilibrated with sodium phosphate buffer (0.02 M, pH 7.4). The collected

fraction was concentrated by centrifugation through a filter (30 kDa cut-off) and stored under an

59

atmosphere of carbon monoxide at 4 °C. The products were analyzed by reverse-phase HPLC and

mass spectrometry.

The procedure for the preparation of Hb-azide is an optimization of the method originally

reported by Ying et al.41 and is identical to the preparation of Hb-cyclooctyne with the following

exceptions: amine-azide was added instead of amine-cyclooctyne; the mixture was passed through

a G-25 column equilibrated with MOPS buffer (0.1 M, pH 8.0) instead of sodium phosphate buffer.

The products were analyzed by reverse-phase HPLC and mass spectrometry.

Hb-cyclooctyne (1 eq., 100 µL of a 0.32 mM stock solution in 0.02 M sodium phosphate

buffer, pH 7.4) was combined with Hb-azide (1 eq., 133 µL of a 0.24 mM stock solution in 0.02

M sodium phosphate buffer, pH 7.4) and the total volume was adjusted to 100 µL to give a total

Hb concentration of 0.64 mM. This mixture was incubated for ~2 weeks at 4 °C under an

atmosphere of carbon monoxide. The products were analyzed by size-exclusion HPLC.

4.3.3 Hb bis-tetramers (bis-azide linker)

To Hb-cyclooctyne (1 eq., 100 µL, 0.32 mM in 0.02 M sodium phosphate buffer, pH 7.4)

was added 0.45 eq. of bis-azide (4.8 µL of a 3 mM stock solution in DMSO) and this mixture was

incubated for ~3 weeks at 4 °C under an atmosphere of carbon monoxide. The products were

analyzed by size-exclusion HPLC.

4.3.4 Preparation of Hb-DIBO and Hb-exclusive clusters

To native Hb (0.1 mM in 1.6 mL of PBS buffer, pH 7.4) was added NHS-DIBO (30 µL of

a 100 mM solution in DMSO). This mixture was stirred for 2 hours at room temperature then

passed through a Sephadex G-25 column equilibrated with 0.02 M sodium phosphate buffer, pH

7.4. The collected fraction was concentrated by centrifugation through a filter (30 kDa cut-off) and

stored under an atmosphere of carbon monoxide at 4 °C. The same procedure was followed to

modify β-subunit cross-linked (α2β82-trimesoyl-β82) Hb to give xlHb-DIBO. The products were

analyzed by reverse-phase HPLC and mass spectrometry.

Hb-DIBO/xlHb-DIBO (1 eq., 62 µL of a 0.1 mM stock solution in 0.02 M sodium

phosphate buffer, pH 7.4) and Hb-azide (approx. 4 eq. of a 0.62 mM stock solution in 0.02 M

60

sodium phosphate buffer, pH 7.4) were incubated together for 1 day at 0 °C under an atmosphere

of carbon monoxide. The products were analyzed by size-exclusion HPLC.

4.3.5 Preparation of albumin-azide

The azido-PEG3-amine (1.1 eq., oil) was dissolved in 1 mL DMSO and this solution was

added to the maleimide NHS-ester (1 eq., solid). We stirred the mixture for 30 min. at room

temperature then added it directly to albumin (the reagent must be made fresh). The maleimide-

azide (30 eq., 24 µmol, 0.32 mL of a 75 mM maleimide-azide solution in 1 mL DMSO) was added

to albumin (1 eq., 0.79 µmol, 0.15 mM solution in 5.24 mL 50 mM sodium phosphate buffer, pH

6.5). This mixture was stirred for 2 hours at room temperature. The solution was diluted to 15 mL

with 0.02 M sodium phosphate buffer, pH 7.4 then concentrated by centrifugation through a filter

(30 kDa cut-off). This process was repeated 3 times. The albumin-azide was stored in the fridge at

4 °C. The concentration of the stock solution was determined using the extinction coefficient of

HSA at 280 nm of 36500 M-1 cm-1. The whole protein was submitted to mass spectrometry

analysis.

4.3.6 Preparation of Hb-albumin cluster from albumin-azide

Hb-DIBO (1 eq., 0.008 µmol, 100 µL of a 0.08 mM stock solution in 0.02 M sodium

phosphate buffer, pH 7.4) was added to albumin-azide (approx. 4 eq. of a 0.23 mM stock solution

in 0.02 M sodium phosphate buffer, pH 7.4). The final volume of the solution was adjusted to 100

µL by concentration through a filter (30 kDa cut-off). The mixture was stirred at room temperature

under an atmosphere of carbon monoxide for 2 hours then incubated at 4 °C for ~ 1 week. The

products were analyzed by size-exclusion HPLC. Clusters were also prepared using ~ 8 eq. of

albumin.

4.3.7 Preparation and SPAAC of Hb-PEG-azide and albumin-DIBO

The procedure for the preparation of Hb-PEG-azide is identical to the preparation of Hb-

DIBO except NHS-DIBO was replaced with NHS-azide. The products were analyzed by reverse-

phase HPLC and mass spectrometry as previously described.

61

The procedure for the preparation of albumin-DIBO is identical to the preparation of

albumin-azide except maleimide-azide was replaced with maleimide-DIBO. The whole protein

was submitted to mass spectrometry analysis.

The procedure for SPAAC of Hb-PEG-azide with albumin-DIBO is identical to SPAAC

of Hb-DIBO with albumin-azide except Hb-DIBO was replaced with Hb-PEG-azide and albumin-

azide was replaced with albumin-DIBO. The products were analyzed by size-exclusion HPLC as

previously described.


Figure 4.15: Mass spectrum of Hb-cyclooctyne (β-subunits cross-linked). ((β-subunit (15867.22-

1.01) × 2) + C26O7N2H30 (482.58)) = 32215.00 g/mol.

Figure 4.16: Mass spectrum of Hb-azide (β-subunits cross-linked). (β-subunits ((15867.22-1.01)

× 2) + C17O3N4H12 (320.33)) = 32052.75 g/mol.

62

Figure 4.17: Mass spectrum of Hb-cyclooctyne (β-subunits cross-linked) modified with bis-azide

(4,4’-diazidediphenylsulfone) only. ((β-subunit (15867.22-1.01) × 2) + C26O7N2H30 (482.58) +

C12SO2N6H8 (300.30)) = 32515.00 g/mol.

Figure 4.18: Native PAGE analysis of the products of the copper-free click reactions after

approximately half the starting material is consumed. Lane 1: Native Hb; Lane 2: Pure Hb bis-

tetramer reference; Lanes 3 and 5: Products of the reaction of Hb-cyclooctyne with bis-azide;

Lanes 4 and 6: Products of the reaction of Hb-cyclooctyne with Hb-azide.

63

Figure 4.19: Mass spectra of Hb-DIBO (the pre-deconvolution spectra were too complex to yield

accurate masses). (α-subunit (15126.36-1.01) + C30N2O7H35 (535.67)) = 15661.02 g/mol; 2

additions is 16195.68 g/mol; 3 additions is 16730.34 g/mol; (β-subunit (15867.22-1.01) +

C30N2O7H35 (535.67)) = 16401.88 g/mol; 2 additions is 16401.88 g/mol; 3 additions is 17471.20

g/mol. We can guess: 15804 corresponds to α-subunit (1 addition) + ~143 mass adduct; 16548

corresponds to β-subunit (1 addition) + ~147 mass adduct; 16874 corresponds to α-subunit (3

additions) + ~144 mass adduct.

Figure 4.20: Mass spectrum of TTDS cross-linked Hb-DIBO (the pre-deconvolution spectra were

too complex to yield accurate masses). (α-subunit (15126.36-1.01) + C30N2O7H35 (535.67)) =

15661.02 g/mol; 2 additions is 16195.68 g/mol; 3 additions is 16730.34 g/mol; (β-subunits

((15867.22-1.01) × 2) + trimesoyl bridge C9O4H4 (176.13) + C30N2O7H35 (535.67)) = 32443.21

g/mol; 2 additions is 32977.87 g/mol; 3 additions is 33512.53 g/mol.

64

Figure 4.21: Native PAGE analysis of the Hb-exclusive clusters after incubation for 1 day at 4

°C. Lane 7: Native Hb; Lanes 1, 4: Pure Hb bis-tetramer reference (~128 kDa); Lanes 3, 6: Hb-

DIBO ensemble; Lanes 2, 5: xlHb-DIBO ensemble.

Figure 4.22: Mass spectrum of native albumin as purchased. The mass addition of 119.03 Da is

due to cysteinylation.

65

Figure 4.23: Mass spectrum of maleimide-azide reagent. [M+H+] = 370 g/mol.

Figure 4.24: Mass spectrum of albumin-azide (the pre-deconvolution spectrum was too complex

to yield accurate masses).

66

Figure 4.25: Native PAGE analysis of the Hb-albumin clusters prepared from SPAAC of Hb-

DIBO with Hb-PEG-azide. Lane 1: Native albumin (HSA); Lane 2: Products of the reaction

between albumin-azide and Hb-DIBO (6 equiv albumin to 1 equiv Hb); Lane 3: Products of the

reaction between albumin-azide and Hb-DIBO (3 equiv albumin to 1 equiv Hb); Lane 4: Pure Hb

bis-tetramer reference; Lane 5: Native Hb.

Figure 4.26: Native PAGE analysis of the products of the SPAAC coupling of albumin-azide with

Hb-cyclooctyne. Lane 1: Native albumin (HSA); Lane 2: albumin-azide; Lane 3: Hb-cyclooctyne;

Lane 4: Products of the reaction between albumin-azide and Hb-cyclooctyne (1 equiv albumin to

2 equiv Hb); Lane 5: Products of the reaction between albumin-azide and Hb-cyclooctyne (1 equiv

albumin to 1 equiv Hb); Lane 6: Products of the reaction between albumin-azide and Hb-DIBO (3

equiv albumin to 1 equiv Hb); Lane 7: Products of the reaction between albumin-azide and Hb-

DIBO (6 equiv albumin to 1 equiv Hb); Lane 8: Hb-DIBO; Lane 9: Pure Hb bis-tetramer reference;

Lane 10: Native Hb.

67

Figure 4.27: Mass spectra of Hb-PEG-azide. Elution times from HPLC: top) 36-42 min.; middle)

42-56 min.; bottom) 56-75 min. α-subunit (15126.36-1.01) + C11H20N3O5 (274.31) = 15399.67

g/mol; 2 additions is 15672.98 g/mol; 3 additions is 15946.29 g/mol; 4 additions is 16219.6 g/mol;

5 additions is 16492.91 g/mol; β-subunit (15867.22-1.01) + C11H20N3O5 (274.31) = 16140.53

g/mol; 2 additions is 16413.84 g/mol; 3 additions is 16687.15 g/mol; 4 additions is 16960.46

g/mol; 5 additions is 17233.77 g/mol.

68

Figure 4.28: Mass spectrum of albumin-DIBO. Albumin blocked by cysteinylation (66558.86 Da

accounts for mass addition of 119 Da). Albumin (66439.83 Da) + C36H42N4O9 (674.74 g/mol) =

67114.57 Da. Albumin-cysteinylated (66558.86 Da) + C36H42N4O9 (674.74 g/mol) = 67233.60 Da.

Albumin (66439.83 Da) + 2 × C36H42N4O9 (674.74 g/mol) = 67789.31 Da.

Figure 4.29: Native PAGE analysis of the products of SPAAC of Hb-PEG-azide and albumin-

DIBO. Lane 1: Native albumin (HSA); Lane 2: Products of the reaction between albumin-DIBO

and Hb-PEG-azide (4 equiv albumin to 1 equiv Hb); Lane 3: Products of the reaction between

albumin-DIBO and Hb-PEG-azide (3 equiv albumin to 1 equiv Hb); Lane 4: Products of the

reaction between albumin-DIBO and Hb-PEG-azide (2 equiv albumin to 1 equiv Hb); Lane 5:

albumin-DIBO; Lane 6: Pure Hb bis-tetramer reference; Lane 7: Native Hb.

69

Figure 4.30: Reverse-phase HPLC of Hb-alkyne under dissociating conditions. Peaks are as

follows: heme (10 min.); α-subunits (40 min.); β cross-linked subunits (alkyne modified, 54 min.).

Figure 4.31: Mass spectrum of Hb-alkyne (β-subunits cross-linked). ((β-subunits (15867.22-1.01)

× 2) + C20O7NH23 (389.44)) = 32121.86 g/mol.

Figure 4.32: Reverse-phase HPLC of Hb-alkyne(s) under dissociating conditions.

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

0.0 20.0 40.0 60.0 80.0 100.0

Ab

sorb

ance

(2

20

nm

)

Time (min.)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.0 20.0 40.0 60.0 80.0 100.0

Ab

sorb

ance

(2

20

nm

)

Time (min.)

70

Figure 4.33: Mass spectra of Hb-alkyne(s). (α-subunit (15126.36-1.01) + C14O6H23 (287.37)) =

15412.72 g/mol; 2 additions is 15699.08 g/mol; 3 additions is 15985.44 g/mol; 4 additions is

16271.80 g/mol; (β-subunit (15867.22-1.01) + C14O6H23 (287.37)) = 16153.58 g/mol; 2 additions

is 16439.94 g/mol; 3 additions is 16726.30 g/mol; 4 additions is 17012.66 g/mol.

71

Figure 4.34: Reverse-phase HPLC of TTDS cross-linked Hb-alkyne(s) under dissociating

conditions.

Figure 4.35: Mass spectra of TTDS cross-linked Hb-alkyne(s). (α-subunit (15126.36-1.01) +

C14O6H23 (287.37)) = 15412.72 g/mol; 2 additions is 15699.08 g/mol; 3 additions is 15985.44

g/mol; 4 additions is 16271.80 g/mol; (β-subunits ((15867.22-1.01) × 2) + trimesoyl bridge C9O4H4

(176.13) + C14O6H23 (287.37)) = 32194.91 g/mol; 2 additions is 32481.27 g/mol; 3 additions is

32767.63 g/mol; 4 additions is 33053.99 g/mol.

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.0 20.0 40.0 60.0 80.0 100.0A

bso

rban

ce (

22

0 n

m)

Time (min.)

72

Chapter 5

Self-assembly of Hemoglobin and Avidin

Figures in this chapter were reproduced with permission from: Singh, S., and Kluger, R. (2016)

Self-Assembly of a Functional Triple Protein: Hemoglobin-Avidin-Hemoglobin via Biotin–

Avidin Interactions, Biochemistry 55, 2875-2882.


We discovered a curious propensity for the oppositely charged proteins Hb and avidin to

self-assemble in solution.82 An increase in optical density (absorption at 700 nm) indicates

turbidity of the solution following a self-assembly process.51 There is no apparent association

between native Hb (pI = 6.9) and avidin (pI = 10.5). The seemingly inconsequential substitution

of native Hb for either fumaryl (α99-fumaryl-α99, β2) or trimesoyl (α2, β82-trimesoyl-β82) cross-

linked Hb dramatically impacts solution character (Figure 5.1). Turbidity varies with buffer ionic

strength and pH (Figure 5.2). In general, buffers of low ionic strength promote aggregation. We

can enhance the effect by increasing the Hb to avidin ratio (Figure 5.3) or by the addition of

inositol hexaphosphate (IHP) (Figure 5.4).

Figure 5.1: Absorbance at 700 nm of one to one mixtures of native or cross-linked Hb and avidin

in buffers of varying pH and ionic strength. Buffers: sodium phosphate, pH 7.4 (I=24 mM); Tris-

HCl, pH 8.3 (I=3 mM); Tris-HCl, pH 9.0 (I=1 mM); borate, pH 9.2 (I=54 mM).

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

7 8 9 10

Op

tica

l den

sity

(a

bso

rban

ce a

t 7

00

nm

)

pH

Fumaryl cross-linked Hb + Avidin

Native Hb + Avidin

Trimesoyl cross-linked Hb + Avidin

73

Figure 5.2: Absorbance at 700 nm of one to one mixtures of fumaryl cross-linked Hb and avidin

in buffers of varying pH and ionic strength.

Figure 5.3: Turbidity increase associated with increasing the fumaryl cross-linked Hb to avidin

ratio. Buffer is Tris-HCl, pH 9.0 (I=1 mM).

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

6 7 8 9 10

Op

tica

l den

sity

(a

bso

rban

ce a

t 7

00

nm

)

pH

Phosphate (I=13-27 mM)

PBS (I=163 mM)

Tris (I=1-3 mM)

Tris with NaCl (I=27 mM)

HEPES (I=2-7 mM)

Borate (I=54 mM)

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0 1 2 3 4 5 6

Op

tica

l den

sity

(ab

sorb

ance

70

0 n

m)

# fumaryl cross-linked Hb : 1 avidin

74

Figure 5.4: The turbidity changes associated with adding inositol hexaphosphate (IHP) to fumaryl

cross-linked Hb/avidin mixtures in HEPES, pH 7.2 (I=2 mM).

Substituting avidin for another high isoelectric point protein, lysozyme (pI = 11) for

example, does not guarantee self-assembly. As with native Hb-avidin mixtures, native Hb-

lysozyme mixtures failed to generate an effect. The absorbance changes recorded with cross-linked

Hb/lysozyme mixtures were minimal, with the absorbance at 700 nm reaching a maximum of only

0.044 OD when the lysozyme to Hb ratio was 7:1 (Figure 5.5).

Figure 5.5: Absorbance at 700 nm, associated with solution turbidity, of protein mixtures of

increasing lysozyme to native/cross-linked Hb ratio in Tris-HCl, pH 9.0 (I=1 mM).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Avidin + IHP Fumaryl cross-linkedHb + IHP

Fumaryl cross-linkedHb + avidin

Fumaryl cross-linkedHb + avidin + IHP

Op

tica

l den

sity

(a

bso

rban

ce a

t 7

00

nm

)

Protein mixture

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0 2 4 6 8

Op

tica

l den

sity

(a

bso

rban

ce a

t 7

00

nm

)

# lysozyme : 1 Hb species

Native Hb + Lysozyme

Fumaryl Cross-linked Hb + Lysozyme

75

The unique synergy between cross-linked Hb/avidin is probably in part due to charge

compensation.51 The properties in circulation and potential applications of these possibly

microsphere-like assemblies should be explored.50, 83

We fixed native Hb and avidin non-specifically with glutaraldehyde, under the assumption

that acylation drives aggregation. Native Hb treated with glutaraldehyde produces soluble

polymeric species.84 In contrast, polymerization of these oppositely charged proteins gives a turbid

sample. The mass collected upon concentration shatters into insoluble crystalline-like shards with

agitation (Figure 5.6).

Figure 5.6: Insoluble shards obtained following glutaraldehyde treatment of a solution of native

Hb and avidin.

We opted for an alternative stabilization strategy based on site-specific modification and

high affinity avidin-biotin interactions. Native Hb was treated with 20 eq. of the biotin-maleimide

reagent (Scheme 5.1). Subsequently, fumaryl and trimesoyl cross-linked Hbs were utilized in the

same protocol. Two biotin molecules were incorporated per Hb tetramer, as determined by mass

analysis (see SI), with the β-subunit cys-93 residues as the expected sites of modification.

Scheme 5.1: Biotinylation of Hb with the biotin-maleimide bifunctional reagent.

Modification of the solvent-accessible thiols, although convenient, does compromise the

protein’s intrinsic heat-stability. Native Hb maintains its structural stability upon heating at 60 °C

76

for ten min. (Figure 5.7, top panel). In contrast, biotinylated Hb denatures under the same

conditions and a turbid solution results from protein unfolding (Figure 5.7, bottom panel). Caccia

et al.85 described the same fate for PEG-modified Hb, noting increased tetramer dissociation as a

result of cysteine modification.

Figure 5.7: Spectral changes associated with heating native Hb (top panel) and native biotinylated

Hb (bottom panel) at 60 °C for 10 min.

Initially, we sought to modify avidin with the biotin-maleimide reagent and then introduce

Hb. However, this approach did not yield higher molecular weight species. The alternative

approach, incubating biotinylated Hb with avidin, gave the desired self-assembled triple protein

without any delay (Scheme 5.2).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

500 550 600 650 700

Ab

sorb

ance

Wavelength (nm)

0 min.

5 min.

10 min.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

500 550 600 650 700

Ab

sorb

ance

Wavelength (nm)

0 min.

5 min.

10 min.

77

Scheme 5.2: Self-assembly of biotinylated Hb with avidin.

Avidin’s biotin-binding sites exist as pairs located on opposing faces of the 67 kDa

protein.86 Thus, we expect a maximum of two neighboring Hb tetramers per avidin. Excess

biotinylated Hb, in combination with avidin, achieves saturation of the binding pockets. A single

high molecular weight product was obtained, as deduced from size-exclusion HPLC (Figure 5.8,

panel A). The product peak elutes earlier than the ~128 kDa Hb bis-tetramer reference at 32 min.

(Figure 5.8, panel B). This is as we would expect for a conjugate, with Hb:avidin 2:1 (~195 kDa)

as shown in Scheme 5.2. The peak at 40 min. is consistent with the 32 kDa αβ-dimer derived from

the biotinylated species not interacting with avidin.

When biotinylated Hb is combined with a large excess of avidin, an assembly with

Hb:avidin in a 1:1 ratio (~131 kDa) results (Figure 5.8, panel E). Scavenging of dissociating

biotinylated dimers by avidin during their passage through the column can account for the absence

of the 32 kDa peaks. These fragments were observed in native gel electrophoresis (Figure 5.12,

Lane 2), confirming that the tetramer is intact in the assembly. An alternative explanation is that

the direct interaction of the oppositely charged proteins stabilizes the tetramer against salt-induced

dissociation. This is unlikely, considering the importance of the Cys-93 modification.

Conjugates of fumaryl and trimesoyl cross-linked derivatives (Figure 5.8, panel C and

panel D) were also prepared. The cross-linker stabilizes the tetramer against dissociation into

dimers. Aggregation, related to the propensity for these species to interact and/or weak avidin-

biotin associations, produced an irregular tail to the major peak at 29 min. Unfolding of the protein

is less likely because the defect is a non-issue for the non-cross-linked derivative. Furthermore,

the shoulder is of a significant mass as it begins to elute with the void volume and remains present

where PBS buffer is the eluent. The effect is heavily exaggerated for the unsaturated fumaryl cross-

linked conjugate (Figure 5.8, panel F), which has its biotin pockets completely accessible to

approach. Ignoring the irregular segment, HPLC analysis reveals that the elution time of the cross-

linked conjugate is the same as that of the non-cross-linked one, providing further support for the

proposed route in Scheme 5.2.

78

Figure 5.8: Size-exclusion HPLC traces of Hb-avidin conjugates with Hb bis-tetramer as a

reference. Hb αβ-dimer (32 kDa, 40 min.); Hb cross-linked tetramer (64 kDa, 36 min.); Avidin (67

kDa, 35 min.); Hb bis-tetramer (128 kDa, 32 min.), Avidin + 1×Hb dimer (99 kDa, 30 min.);

Avidin + 2×Hb dimers (131 kDa, 29 min.); Aggregation > 131 kDa (< 28 min.).

It is unlikely that the Hb tetramer within the conjugate is cross-linked through avidin via

both biotin moieties on each tetramer interacting with a biotin-binding pocket because of

conflicting geometrical requirements. An assay with 4-hydroxyazobenzene-2-carboxylic acid

(HABA), which binds weakly to avidin’s biotin binding sites, reveals the number of unoccupied

sites. A binding event is accompanied by an increase in absorption at 500 nm. The spectral changes

associated with binding of HABA to avidin are presented in Figure 5.9.

79

Figure 5.9: Absorbance changes accompanying addition of HABA to avidin (5 µM avidin).

Avidin was in the reference beam such that the resulting spectra above are absorptions due to the

dye alone. Total [HABA]: 6, 13, 22, 37 or 55 μM.

Figure 5.10: Absorbance changes accompanying addition of HABA to the Hb-avidin conjugate

(1.3 µM avidin). The reference spectrum of the conjugate is provided as SI. The segment at 420

nm overlaps with the Hb Soret peak and can be disregarded because the spectrometer is at the limit

of detection. Total [HABA]: 5, 11, 19, 33 or 50 μM.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

220 270 320 370 420 470 520 570 620 670

Ab

sorb

ance

Wavelength

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

220 270 320 370 420 470 520 570 620 670

Ab

sorb

ance

Wavelength (nm)

80

A binding curve for the association of HABA with the conjugate was prepared and

compared against the HABA/avidin control (Figure 5.11). Saturation of avidin with HABA occurs

with approximately four equivalents of the dye bound (KD ~6µM).87 Saturation of the conjugate

occurs with approximately 1.1 equivalents of the dye bound, with an equal dissociation constant.

From our proposed assembly, we expect two equivalents to bind. We do not know the reason for

the deviation. Perhaps, access to the binding site depends on Hb’s docking mode, where spatial

crowding varies as a function of the number of significant orientations. The important conclusion

is that avidin’s binding sites are only partially saturated. Two Hb tetramers are likely linked to

avidin by two rather than four biotin-avidin associations to give HbAvHb, consistent with our

initial description of the conjugate.

Figure 5.11: Binding curves for the Hb-avidin conjugate and avidin titrated with HABA. Curves

are generated from an equation for hyperbolic saturation binding.

Native PAGE analysis provides our concluding piece of evidence. Gels (12%, see SI)

provide sufficient separation of native Hb (64 kDa) from the reference bis-tetramer (~128 kDa)

and demonstrate the reverse mobility of avidin (pI = 10.5). The progress of the conjugates in the

12% gel is severely hindered, probably because the small pores do not allow passage of the large

180 Å diameter constructs. A lower concentration (6%) separating gel was employed to

exaggerate the separation between the saturated and unsaturated conjugates (Figure 5.12). The

fully saturated conjugates (lanes 1, 3 and 5), with excess biotinylated Hb present in the mixture,

bear overall more negative surface charge. Therefore, they can progress through the gel despite

their larger size. Lanes 2 and 4 contain the unsaturated conjugates with Hb:avidin likely to be

present in a 1:1 ratio. With near neutral isoelectric points, their progression is severely retarded,

despite their smaller size. Thus, they remain at the origin.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20 25 30 35 40 45 50 55

[LR

]/[t

ota

l R]

[L] free (µM)

Avidin

Hb-avidinconjugateK

D ≈ 5 μM

81

Figure 5.12: Native PAGE analysis of the Hb-avidin conjugates (4% stacking gel, 6% separating

gel, 1 hour, 200 V). Lane 1: Native Hb-avidin conjugate (~195 kDa) with excess Hb; Lane 2:

Native Hb-avidin conjugate (~131 kDa) with excess avidin; Lane 3: Fumaryl cross-linked Hb-

avidin conjugate (~195 kDa) with excess Hb; Lane 4: Fumaryl cross-linked Hb-avidin conjugate

(~131 kDa) with excess avidin; Lane 5: Trimesoyl cross-linked Hb-avidin conjugate (~195 kDa)

with excess Hb.

A reverse polarity gel (run with cathode and anode switched) served as a control (see SI).

Bands from avidin are visible in that gel, while those from the saturated conjugates, which run in

the opposite direction, are not.

The unsaturated conjugate with a 1:1 ratio of (non-cross-linked) Hb:avidin (prepared using

an excess of avidin) was analyzed for its oxygen-binding properties. The heterogeneous oxygen-

binding curve yields an affinity (P50) of 3.4 torr and a Hill coefficient (n50) = 1.7 (Figure 5.13).

Cysteine modification generally results in high affinity with reduced cooperativity, as exemplified

by the HBOC Hemospan.16 Nonetheless, a degree of cooperativity and a usefully low oxygen

affinity are retained.

82

Figure 5.13: Raw oxygen binding data of the Hb-avidin conjugate (1:1 non-cross-linked

Hb:avidin) compared to the fitted curve for native Hb.


Protein coupling mediated by the biotin-avidin interaction provides a unique opportunity

to harness self-assembly for the preparation of a massive soluble 195 kDa triple protein construct.

We demonstrate that the specific attraction of the oppositely charged proteins does not include all

possible combinations (e.g. Hb and lysozyme) and is unique between Hb and avidin. Biotinylation

of Hb using a maleimide-functionalized reagent provides a methodology to install the orthogonal

group with unsurpassable efficiency and preparation ease. Future work may be conducted to install

the biotin moiety in a position that does not perturb the overall stability and cooperativity of the

tetramer. For example, biotin may be stabilized as an appendage on a cross-link instead. The

current methodology immediately affords the HbAvHb assembly and its impressive size can be

appreciated by its elution time in size-exclusion HPLC. The skew in isoelectric point is

immediately recognizable by its mobility during native gel electrophoresis. Binding experiments

provided the final piece of structural evidence to prove the availability of free binding pockets on

the central protein. The overall scheme represents the only true methodology to combine and

stabilize like proteins with the perfection that industrial scale-up would demand.

5.3 Experimental

5.3.1 General

Avidin from egg white was obtained from BioShop. Biotin-maleimide (N-Biotinoyl-N′-(6-

maleimidohexanoyl)hydrazide) was purchased from Sigma-Aldrich. Additional reagent, Hb

deoxygenation, HPLC, mass spectrometry, PAGE and oxygen binding details are described in the

General Experimental Methods chapter of this thesis.

83

5.3.2 Self-assembly of Hb and avidin

Optical density (absorbance at 700 nm) was acquired for Hb species/avidin mixtures and

Hb/lysozyme mixtures (5 μM of each protein in 1 mL of the specified buffer) using a Cintra 40

UV-vis spectrometer. The Hb species were in the CO-bound state. Independent studies with added

inositol hexaphosphate (IHP) (2 eq., 2.58 μL of a 4 mM solution in H2O) were conducted in the

same manner. In a separate experiment, a 1:1 mixture of native Hb and avidin was fixated using

13 eq. of glutaraldehyde (11 µL of a freshly prepared 0.05 M solution in water) for every 1.0 eq.

of Hb (0.2 mM in 210 µL of 10 mM HEPES buffer, pH 8.0). The reaction was terminated by the

addition of 100 µL of 1 M Tris-HCl, pH 8.0 then concentrated through a 30 kDa cut-off filter.

5.3.3 Biotinylation of Hb

The biotin-maleimide cross-linker (20 eq., 24 μL of a 62.5 mM stock solution in DMSO)

was added to COHb (0.075 μmol, 0.075 mM in 1 mL 50 mM sodium phosphate, pH 6.5) was

added. The Hb-containing solution was stirred at room temperature for one hour in a crimp-sealed

vial flushed with carbon monoxide. Excess reagent was removed from the protein solution by four

cycles of centrifugation (14000 × g, 5 min.) through a filter (30 kDa cut-off) followed by dilution

to the original volume with 50 mM sodium phosphate, pH 6.5. The biotinylated Hb species were

analyzed by reverse-phase HPLC. Biotinylated fumaryl/trimesoyl cross-linked Hb were prepared

by the same method.

The thermal stability of biotinylated Hb was determined using a UV-vis spectrometer with

a cell holder maintained at 60.0 °C. Protein solutions (10 μM in 1 mL of 0.01 M sodium phosphate

buffer, pH 6.5) were heated for 10 min. The absorbance spectrum (500 to 700 nm) was acquired

at 1 min intervals.

5.3.4 Hb-avidin conjugation

The solution of modified Hb (3 eq., 0.057 μmol, 710 µL of 0.08 mM solution in 50 mM

sodium phosphate, pH 6.5) was added to a solution of avidin (0.019 μmol, 86 μL of 0.22 mM in

50 mM sodium phosphate buffer, pH 6.5). The resulting solution was stirred at room temperature

for 1 hour in a crimp-sealed vial flushed with carbon monoxide. The resulting conjugates were

analyzed by size-exclusion HPLC using a Superdex G-200 HR size-exclusion column (10 mm ×

300 mm) and a Tris-HCl (37.5 mM, pH 7.4) elution buffer containing magnesium chloride (0.5

84

M). The eluent was monitored at 280 nm. Conjugates of fumaryl/trimesoyl cross-linked Hb and

avidin were prepared by the same method. Conjugates with 1:1 Hb:avidin were made by

combining 1 eq. of biotinylated Hb to approximately 5 eq. of avidin.

5.3.5 HABA occupancy assay

4’-hydroxyazobenzene-2-carboxylic acid (HABA) (1-100 µL of a 5 mM solution) was

added to independent solutions of native avidin and the Hb-avidin conjugate (5 µM or 1.3 µM of

avidin, respectively in 1.0 mL 50 mM sodium phosphate buffer, pH 6.5). The absorption spectrum

from 200-700 nm was acquired using a UV-vis spectrometer. An increase in absorbance at 500

nm (ε = 34500 M-1cm-1) is associated with bound HABA.88 Binding curves were derived from the

changes in absorbance using the Beer-Lambert law. The same assay was performed on non-

conjugated biotinylated Hb. The hyperbolic fitted curves were derived from the Michaelis-Menten

equation.


Figure 5.14: Reverse-phase HPLC traces of Hb species after treatment with biotin-maleimide

cross-linker. Peaks are as follows – Native: 20 min. (β-subunit modified), 30 min. (α-subunit).

Trimesoyl: 30 min. (α-subunit), 50 min. (β-subunits cross-linked and modified).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.0 20.0 40.0 60.0 80.0

Ab

sorb

ance

(2

20

nm

)

Time (min.)

Biotinylated native Hb

Biotinylated trimesoyl cross-linked Hb

85

Figure 5.15: Reverse-phase HPLC trace of fumaryl cross-linked Hb after treatment with biotin-

maleimide cross-linker. Peaks are as follows – 50 min. (β-subunit modified), 80 min. (α-subunits

cross-linked). Retention times vary due to solvent evaporation over time.

Figure 5.16: Biotinylated β-subunit (from modification of native Hb). 15867.22 g/mol +

C20H29N5O5S (451.54 g/mol) = 16318.76 g/mol.

Figure 5.17. Biotinylated trimesoyl cross-linked β-subunits. (2×(15867.22 g/mol-1.01 g/mol)) +

C9O4H4 (176.13 g/mol) + (2×C20H29N5O5S (451.54 g/mol)) = 32811.63 g/mol. The peak at

30251.72 g/mol is due to two native α-subunits.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.0 20.0 40.0 60.0 80.0

Ab

sorb

ance

(2

20

nm

)

Time (min.)

86

Figure 5.18: Biotinylated β-subunit (from modification of fumaryl Hb). 15867.22 g/mol +

C20H29N5O5S (451.54 g/mol) = 16318.76 g/mol.

Figure 5.19: Absorption spectrum of (non-cross-linked) Hb-avidin conjugate with excess

biotinylated Hb.

0

0.5

1

1.5

2

2.5

3

3.5

220 320 420 520 620

Ab

sorb

ance

Wavelength (nm)

Hb-avidin conjugate

(Non-conjugated) biotinylated Hb

87

Figure 5.20: Absorbance changes accompanying addition of HABA to non-conjugated

biotinylated Hb. Non-conjugated biotinylated Hb is the reference.

Figure 5.21: Native PAGE analysis of the Hb-avidin conjugates with a high percentage separating

gel. Lane 2 is empty because avidin runs in the opposite direction (pI = 10.5).

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

220 270 320 370 420 470 520 570 620 670

Ab

sorb

ance

Wavelength (nm)

88

Figure 5.22: Native PAGE analysis of the Hb-avidin conjugates. The anode and cathode are

reversed here.

Figure 5.23: Absorbance profile of the Hb-avidin conjugate (5 µM avidin) in the presence of

HABA (19 µM) compared to that of native avidin (5 µM) in the presence of the same

concentration of HABA. The dye does not bind to biotinylated Hb alone. The segment at 420 nm

overlaps with the Hb Soret peak and can be disregarded because the spectrometer is at the limit

of detection.

0

0.1

0.2

0.3

0.4

0.5

0.6

300 350 400 450 500 550 600 650 700

Ab

sorb

ance

Wavelength (nm)

Avidin + HABA

Hb-avidin conjugate + HABA

Hb-biotin + HABA

HABA only

89

Chapter 6

General Experimental Methods

6.1 Reagents

Purified Hb (carbon monoxide-bound, stored at 4 °C) was a gift from Oxygenix Co. Ltd.

Trimesoyl tris(3,5-dibromosalicylate) (TTDS) was synthesized and used to prepare α2β82-

trimesoyl-β82 Hb as reported in Kluger et al.89 Bis(3,5-dibromosalicyl) fumarate (DBSF) was

purchased from LKT Laboratories, Inc. and used to prepare α99-fumaryl-α99, β2 Hb as described

by Snyder et al.70

6.2 Hb oxygenation/deoxygenation

Carbon monoxide-bound Hb was converted to deoxyHb by stirring under a stream of

oxygen for 2 hours at 0 °C then stirring under a stream of nitrogen for 2 hours at 37 °C.

6.3 Reverse-phase HPLC

Protein analysis by “reverse-phase HPLC” was conducted using a 330 Å C-4 Vydac

column (4.6 × 250 mm) and a solvent gradient from 20 to 60% acetonitrile:water with 0.1%

trifluoroacetic acid (TFA). The eluent was monitored at 220 nm. Retention times drift because

solvents are mixed off-line.

Table 6.1: Reverse-phase HPLC elution gradient. Solvent A: 0.1% TFA, 20% acetonitrile in

water. Solvent B: 0.1% TFA, 60% acetonitrile in water.

Time (min.) v/v% Solvent A v/v% Solvent B

0.00 49.0 51.0

0.50 49.0 51.0

60.50 35.0 65.0

80.50 14.0 86.0

95.00 0.0 100.0

105.00 0.0 100.0

106.00 49.0 51.0

120.00 49.0 51.0

6.4 Size-exclusion HPLC

Protein analysis by “size-exclusion HPLC” was conducted using a Superdex G-200 HR

size-exclusion column (10 × 300 mm, VT = 24 mL, V0 = 8.5 mL) and a Tris-HCl (37.5 mM, pH

90

7.4) elution buffer containing magnesium chloride (0.5 M). The eluent (flow rate of 0.4 mL/min.)

was monitored at 280 nm.

6.5 Mass spectrometry analysis

The masses of protein fractions collected from reverse-phase HPLC were determined by

electrospray ionization (ESI) high-resolution mass spectrometry (AIMS Lab, Department of

Chemistry, University of Toronto).

6.6 SDS-PAGE analysis

Protein molecular weights were determined by polyacrylamide gel electrophoresis

(PAGE). Unless otherwise noted, 12% separating gels (pH 8.8) and 5% stacking gels (pH 6.8)

were used, each with 10% sodium dodecyl sulfate (SDS). Protein were denatured by heating at

100 °C for 10 min. Finished gels were stained with Coomassie Brilliant Blue. Further protocol

details can be found in the literature.90

6.7 Native PAGE analysis

The 2-Dimensional Tris-HCl polyacrylamide gels contained 6/12% separating gel (pH 8.8)

and 4% stacking gel (pH 8.8). Sample buffer was adjusted to pH 6.8 and running buffer to pH 8.3.

The finished gels were stained with Coomassie Brilliant Blue. A comprehensive native

polyacrylamide gel electrophoresis (PAGE) procedure is described in the literature.90

6.8 Oxygen binding analysis

The oxygen pressure at half-saturation (P50) and the Hill’s coefficient of cooperativity at

half-saturation (n50) were determined using a Hemox Analyzer. Carbon monoxide-bound samples

(0.013 M, 5 mL in sodium phosphate buffer (0.01 M, pH 7.4)) were oxygenated by stirring under

a stream of oxygen at 4 °C for 1.5 h prior to analysis. The sample was then contained in a cell

connected to the Hemox Analyzer and equilibrated to 27 °C prior to acquisition of the desaturation

curve. The protein was converted to the deoxygenated state by flushing the cell with nitrogen. The

data was fitted to the Adair equation using computation of a best fit by the method of non-linear

least squares unless otherwise indicated. Oxygen binding studies were also conducted in the

presence of IHP where indicated: to the oxygenated protein sample, 2.3 eq. IHP per heme (7.5 μL

of a 0.08 M solution in sodium phosphate buffer (0.01 M, pH 7.4)) was added to give 0.1 mM IHP.

91

Chapter 7

Conclusions and Future Work

‘Click’ chemistry is exalted as the premier methodology for bioorthogonal protein

modification, despite the harsh realities we faced in personalizing this chemistry for Hb coupling.

Site-specific introduction of an azide was addressed by a unique double cross-linking strategy,

whereby a fumaryl bridge simultaneously protects α-subunits and influences oxygen affinity. The

roadblock in the stepwise approach of protect, functionalize and click is the non-quantitative

CuAAC coupling. In addition to the inherently flawed phase-directed approach, free radicals

rampant in solution elicit rapid heme oxidation.

Exploration of SPAAC as a potential alternative was initially restricted by the difficulty of

synthesizing cyclooctyne-equipped cross-linkers. That is, until we optimized in situ amine addition

to β-subunit cross-linked Hb, as originally reported by Yang et al.41, to the near perfect efficiency

required to justify this path. The metal-free approach is ideal because it is both high yielding and

gentle with respect to the heme. Eventually we would like to develop a purification strategy based

on avidin affinity column to circumvent laborious gel purification. Optimizing the bioorthogonal

structures, the octyne in particular, is the key to overcoming sluggish kinetics. This chemistry can

easily be extended to the preparation of a Hb-superoxide dismutase conjugate. A variety of cargo

for HBOC visualization (e.g. for observing accumulation in leaky tumors) can theoretically be

conveyed with ease, including green fluorescent protein (GFP) or MRI agents.

The success of metal-free click chemistry placed us in a highly strategic position to develop

the next breakthrough HBOC with SPAAC clustering technology. Cross-linker installation

requires the conversion of COHb into deoxyHb, by a procedure operationally complex for large

scale synthesis. Hb-albumin clusters were prepared by SPAAC working exclusively with the

carbon monoxide-bound form of the central scaffold. The result is a transfusion-ready material

rapidly assembled from raw materials that requires minimal purification.

Self-assembly of oppositely charged proteins Hb and avidin is an exciting starting point for

new materials stabilized by electrostatic interactions. The high affinity biotin-avidin interaction

provides directed self-assembly of the triple protein HbAvHb. Scavenging of free tetramer by

avidin is analogous to the action of haptoglobin in living systems. The preparation is instantaneous,

92

easy to execute and, assuming avidin is in excess, the end product does not require purification.

Eventually we would like to determine the exact geometry required to cross-link Hb through

avidin’s paired binding sites. For now, the vacant avidin binding sites may be occupied by

biotinylated drugs or other agents.

In my studies, HBOC design was refined from several angles, with SPAAC and Hb-avidin

self-assembly being remarkably successful as compared to CuAAC (Table 7.1). The rationale

behind CuAAC is supported by the convenient synthetic methods that are available to prepare

terminal alkynes and organic azides. However, these superficial advantages are superseded by the

poor yields that accompany the copper-mediated transformations. The significant challenges of

working with a metal catalyst to produce Hb bis-tetramers inspired the exploration of strained

cyclooctynes as an alternative. The conversion from cross-linked tetramers to bis-tetramers by

SPAAC is undeniably efficient and structural features of previously studied bis-tetramers are

retained in the final product. Development of improved syntheses for cyclooctynes and a rapid

protein purification protocol to remove undermodified species should enable large scale

production of these entities. Meanwhile, a sequence that is already finely tuned for industrial

scalability was developed by adapting SPAAC for the preparation of Hb-albumin clusters. Fully

functional oxygen carriers are assembled by uncomplicated reactions carried out exclusively in the

carbon monoxide-bound state of the protein. The end result does not require purification from

potentially vasoactive species because the Hb tetramers are thoroughly modified by the virtually

quantitative conjugation process. Hb-avidin assembly provides an equally productive

methodology to generating well-defined assemblies of coupled proteins with the added advantage

of an economically superior biotin-based linker. Avidin’s compatibility in the context of a

circulating oxygen carrier is unexplored territory but we would expect that the larger size of the

overall entity would be beneficial in terms of minimizing extravasation behavior.

93

Approach Scalability

Properties in vivo Yield Complexity

CuAAC

(bis-tetramers)

✗Low ✓Reagents functionalized with

terminal alkynes and azides are

cheap and easy to synthesize

✓Positive

SPAAC

(bis-tetramers)

✓High ✓Opportunities to optimize the

protein purification strategy

✗Cyclooctynes are synthetically

challenging

✓Expect positive

SPAAC

(clusters)

✓Quantitative

(no waste Hb)

✓Minimal purification required

✓Entire preparation in stable COHb

state

✗Cyclooctynes are synthetically

challenging

✓Expect positive

Hb-Avidin-Hb

✓Quantitative

(no waste Hb)

✓Minimal purification required

✓Biotin is cheap and readily

available

✗Unknown

✓Expect larger size

to be advantageous

Table 7.1: Comparative overview of the Hb-based products designed.

Despite all the attention the design of HBOCs has received, it is not clear if one material

alone can serve as a replacement for the red blood cell. Perhaps, a combination of materials will

be required. Hb is likely to have evolved within a protective and dynamic cell. It remains an

important challenge to provide effective protection for that protein to remain functional in

circulation through chemical modification and conjugation.

94

References

1. Schechter, A. N. (2008) Hemoglobin research and the origins of molecular medicine,

Blood 112, 3927-3938.

2. Thomas, C., and Lumb, A. B. (2012) Physiology of haemoglobin, Continuing Education

in Anaesthesia, Critical Care & Pain.

3. Ralston, S. L., Lieberthal, A. S., Meissner, H. C., Alverson, B. K., Baley, J. E.,

Gadomski, A. M., Johnson, D. W., Light, M. J., Maraqa, N. F., Mendonca, E. A., Phelan,

K. J., Zorc, J. J., Stanko-Lopp, D., Brown, M. A., Nathanson, I., Rosenblum, E., Sayles,

S., Hernandez-Cancio, S., Ralston, S. L., Lieberthal, A. S., Meissner, H. C., Alverson, B.

K., Baley, J. E., Gadomski, A. M., Johnson, D. W., Light, M. J., Maraqa, N. F.,

Mendonca, E. A., Phelan, K. J., Zorc, J. J., Stanko-Lopp, D., Brown, M. A., Nathanson,

I., Rosenblum, E., Sayles, S., and Hernandez-Cancio, S. (2014) Clinical Practice

Guideline: The Diagnosis, Management, and Prevention of Bronchiolitis, Pediatrics 134,

e1474-e1502.

4. Perutz, M. F. (1970) Stereochemistry of Cooperative Effects in Haemoglobin: Haem-

Haem Interaction and the Problem of Allostery, Nature 228, 726-734.

5. Kluger, R., Foot, J. S., and Vandersteen, A. A. (2010) Protein-protein coupling and its

application to functional red cell substitutes, Chemical Communications 46, 1194-1202.

6. Schumacher, M. A., Dixon, M. M., Kluger, R., Jones, R. T., and Brennan, R. G. (1995)

Allosteric transition intermediates modelled by crosslinked haemoglobins, Nature 375,

84-87.

7. Chen, J.-Y., Scerbo, M., and Kramer, G. (2009) A Review of Blood Substitutes:

Examining The History, Clinical Trial Results, and Ethics of Hemoglobin-Based Oxygen

Carriers, Clinics (Sao Paulo, Brazil) 64, 803-813.

8. Kim-Shapiro, D. B., Schechter, A. N., and Gladwin, M. T. (2006) Unraveling the

Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics,

Arteriosclerosis, Thrombosis, and Vascular Biology 26, 697-705.

9. Silverman, M. D. Toby A., and Weiskopf, M. D. Richard B. (2009) Hemoglobin-based

Oxygen CarriersCurrent Status and Future Directions, Anesthesiology 111, 946-963.

10. Gladwin, M. T., and Kim-Shapiro, D. B. (2008) The functional nitrite reductase activity

of the heme-globins, Blood 112, 2636-2647.

11. Ulatowski, J. A., Nishikawa, T., Matheson-Urbaitis, B., Bucci, E., Traystman, R. J., and

Koehler, R. C. (1996) Regional blood flow alterations after bovine fumaryl beta beta-

crosslinked hemoglobin transfusion and nitric oxide synthase inhibition, Critical Care

Medicine 24, 558-565.

95

12. Nakai, K., Sakuma, I., Ohta, T., Ando, J., Kitabatake, A., Nakazato, Y., and Takahashi,

T. A. (1998) Permeability characteristics of hemoglobin derivatives across cultured

endothelial cell monolayers, Journal of Laboratory and Clinical Medicine 132, 313-319.

13. Sarin, H. (2010) Physiologic upper limits of pore size of different blood capillary types

and another perspective on the dual pore theory of microvascular permeability, Journal of

Angiogenesis Research 2, 14-14.

14. Toy, R., Hayden, E., Shoup, C., Baskaran, H., and Karathanasis, E. (2011) Effect of

Particle Size, Density and Shape on Margination of Nanoparticles in Microcirculation,

Nanotechnology 22, 115101-115101.

15. Serruys, P. W., Vranckx, P., Slagboom, T., Regar, E., Meliga, E., de Winter, R. J.,

Heyndrickx, G. R., Schuler, G., van Remortel, E., Dubé, G. P., and Symons, J. (2008)

Haemodynamic effects, safety, and tolerability of haemoglobin-based oxygen carrier-201

in patients undergoing PCI for CAD, EuroIntervention 3, 600-609 - DOI610.

16. Olofsson, C., Nygårds, E. B., Ponzer, S., Fagrell, B., Przybelski, R., Keipert, P. E.,

Winslow, N., and Winslow, R. M. (2008) A randomized, single-blind, increasing dose

safety trial of an oxygen-carrying plasma expander (Hemospan®) administered to

orthopaedic surgery patients with spinal anaesthesia, Transfusion Medicine 18, 28-39.

17. Vandegriff, K. D., Young, M. A., Lohman, J., Bellelli, A., Samaja, M., Malavalli, A., and

Winslow, R. M. (2008) CO-MP4, a polyethylene glycol-conjugated haemoglobin

derivative and carbon monoxide carrier that reduces myocardial infarct size in rats,

British Journal of Pharmacology 154, 1649-1661.

18. Belcher, J. D., Young, M., Chen, C., Nguyen, J., Burhop, K., Tran, P., and Vercellotti, G.

M. (2013) MP4CO, a pegylated hemoglobin saturated with carbon monoxide, is a

modulator of HO-1, inflammation, and vaso-occlusion in transgenic sickle mice, Blood

122, 2757-2764.

19. Marks, P. W., Epstein, J. S., and Borio, L. (2016) Maintaining a safe blood supply in an

era of emerging pathogens, Journal of Infectious Diseases.

20. Lui, F. E., Yu, B., Baron, D. M., Lei, C., Zapol, W. M., and Kluger, R. (2012)

Hemodynamic responses to a hemoglobin bis-tetramer and its polyethylene glycol

conjugate, Transfusion 52, 974-982.

21. Haruki, R., Kimura, T., Iwasaki, H., Yamada, K., Kamiyama, I., Kohno, M., Taguchi, K.,

Nagao, S., Maruyama, T., Otagiri, M., and Komatsu, T. (2015) Safety Evaluation of

Hemoglobin-Albumin Cluster “HemoAct” as a Red Blood Cell Substitute, Scientific

Reports 5, 12778.

22. Siren, E. M. J., Singh, S., and Kluger, R. (2015) Bioorthogonal phase-directed copper-

catalyzed azide-alkyne cycloaddition (PDCuAAC) coupling of selectively cross-linked

superoxide dismutase dimers produces a fully active bis-dimer, Organic & Biomolecular

Chemistry 13, 10244-10249.

96

23. Lui, F. E., and Kluger, R. (2009) Enhancing Nitrite Reductase Activity of Modified

Hemoglobin: Bis-tetramers and Their PEGylated Derivatives, Biochemistry 48, 11912-

11919.

24. Hosaka, H., Haruki, R., Yamada, K., Böttcher, C., and Komatsu, T. (2014) Hemoglobin

Albumin Cluster Incorporating a Pt Nanoparticle: Artificial Oxygen Carrier with

Antioxidant Activities, PLoS ONE 9, e110541.

25. Brooks, J. (1937) The Action of Nitrite on Haemoglobin in the Absence of Oxygen,

Proceedings of the Royal Society of London B: Biological Sciences 123, 368-382.

26. Huang, K. T., Keszler, A., Patel, N., Patel, R. P., Gladwin, M. T., Kim-Shapiro, D. B.,

and Hogg, N. (2005) The Reaction between Nitrite and Deoxyhemoglobin: Reassessment

of Reaction Kinetics and Stoichiometry, Journal of Biological Chemistry 280, 31126-

31131.

27. Sletten, E. M., and Bertozzi, C. R. (2011) From Mechanism to Mouse: A Tale of Two

Bioorthogonal Reactions, Accounts of Chemical Research 44, 666-676.

28. Saxon, E., Armstrong, J. I., and Bertozzi, C. R. (2000) A “Traceless” Staudinger Ligation

for the Chemoselective Synthesis of Amide Bonds, Organic Letters 2, 2141-2143.

29. Fan, Y., Deng, C., Cheng, R., Meng, F., and Zhong, Z. (2013) In Situ Forming Hydrogels

via Catalyst-Free and Bioorthogonal “Tetrazole–Alkene” Photo-Click Chemistry,

Biomacromolecules 14, 2814-2821.

30. Blackman, M. L., Royzen, M., and Fox, J. M. (2008) Tetrazine Ligation: Fast

Bioconjugation Based on Inverse-Electron-Demand Diels−Alder Reactivity, Journal of

the American Chemical Society 130, 13518-13519.

31. Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B. (2002) A Stepwise

Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of

Azides and Terminal Alkynes, Angewandte Chemie International Edition 41, 2596-2599.

32. Tornøe, C. W., Christensen, C., and Meldal, M. (2002) Peptidotriazoles on Solid Phase:

[1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of

Terminal Alkynes to Azides, The Journal of Organic Chemistry 67, 3057-3064.

33. Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click Chemistry: Diverse Chemical

Function from a Few Good Reactions, Angewandte Chemie International Edition 40,

2004-2021.

34. Huisgen, R., Szeimies, G., and Möbius, L. (1967) 1.3-Dipolare Cycloadditionen, XXXII.

Kinetik der Additionen organischer Azide an CC-Mehrfachbindungen, Chemische

Berichte 100, 2494-2507.

35. Meldal, M., and Tornøe, C. W. (2008) Cu-Catalyzed Azide−Alkyne Cycloaddition,

Chemical Reviews 108, 2952-3015.

97

36. Kislukhin, A. A., Hong, V. P., Breitenkamp, K. E., and Finn, M. G. (2013) Relative

Performance of Alkynes in Copper-Catalyzed Azide-Alkyne Cycloaddition, Bioconjugate

chemistry 24, 684-689.

37. Presolski, S. I., Hong, V. P., and Finn, M. G. (2011) Copper-Catalyzed Azide–Alkyne

Click Chemistry for Bioconjugation, Current Protocols in Chemical Biology 3, 153-162.

38. Carr, A., and Frei, B. (1999) Does vitamin C act as a pro-oxidant under physiological

conditions?, The FASEB Journal 13, 1007-1024.

39. Mir, S. A., Bhat, A. S., and Ahangar, A. A. (2014) An in vitro demonstration of Pro-

oxidant effect of Ascorbic acid in sheep erythrocyte hemolysate and purified hemoglobin

preparations, International Journal of PharmTech Research 6, 743-750.

40. Foot, J. S., Lui, F. E., and Kluger, R. (2009) Hemoglobin bis-tetramers via cooperative

azide-alkyne coupling, Chemical Communications 47, 7315-7317.

41. Yang, Y., and Kluger, R. (2010) Efficient CuAAC click formation of functional

hemoglobin bis-tetramers, Chemical Communications 46, 7557-7559.

42. Glaser, C. (1869) Beiträge zur Kenntniss des Acetenylbenzols, Berichte der deutschen

chemischen Gesellschaft 2, 422-424.

43. Hay, A. S. (1962) Oxidative Coupling of Acetylenes., The Journal of Organic Chemistry

27, 3320-3321.

44. Eglinton, G., and Galbraith, A. R. (1959) 182. Macrocyclic acetylenic compounds. Part I.

Cyclotetradeca-1 :3-diyne and related compounds, Journal of the Chemical Society

(Resumed), 889-896.

45. Berg, R., and Straub, B. F. (2013) Advancements in the mechanistic understanding of the

copper-catalyzed azide–alkyne cycloaddition, Beilstein Journal of Organic Chemistry 9,

2715-2750.

46. Lampkowski, J. S., Villa, J. K., Young, T. S., and Young, D. D. (2015) Development and

Optimization of Glaser–Hay Bioconjugations, Angewandte Chemie International Edition

54, 9343-9346.

47. Agard, N. J., Prescher, J. A., and Bertozzi, C. R. (2004) A Strain-Promoted [3 + 2]

Azide−Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living

Systems, Journal of the American Chemical Society 126, 15046-15047.

48. Haugland, R. P., and Bhalgat, M. K. (2008) Preparation of Avidin Conjugates, In Avidin-

Biotin Interactions: Methods and Applications (McMahon, R. J., Ed.), pp 1-12, Humana

Press, Totowa, NJ.

49. Petronzelli, F., Pelliccia, A., Anastasi, A. M., Lindstedt, R., Manganello, S., Ferrari, L.

E., Albertoni, C., Leoni, B., Rosi, A., D'Alessio, V., Deiana, K., Paganelli, G., and De

98

Santis, R. (2010) Therapeutic Use of Avidin Is Not Hampered by Antiavidin Antibodies

in Humans, Cancer Biotherapy and Radiopharmaceuticals 25, 563-570.

50. Lai, Y.-T., Sato, M., Ohta, S., Akamatsu, K., Nakao, S.-i., Sakai, Y., and Ito, T. (2015)

Preparation of uniform-sized hemoglobin–albumin microspheres as oxygen carriers by

Shirasu porous glass membrane emulsification technique, Colloids and Surfaces B:

Biointerfaces 127, 1-7.

51. Desfougères, Y., Croguennec, T., Lechevalier, V., Bouhallab, S., and Nau, F. (2010)

Charge and Size Drive Spontaneous Self-Assembly of Oppositely Charged Globular

Proteins into Microspheres, The Journal of Physical Chemistry B 114, 4138-4144.

52. Jones, R. T., Shih, D. T., Fujita, T. S., Song, Y., Xiao, H., Head, C., and Kluger, R.

(1996) A Doubly Cross-linked Human Hemoglobin: Effects of Cross-links Between

Different Subunits, Journal of Biological Chemistry 271, 675-680.

53. Singh, S., Dubinsky-Davidchik, I. S., Yang, Y., and Kluger, R. (2015) Subunit-directed

click coupling via doubly cross-linked hemoglobin efficiently produces readily purified

functional bis-tetrameric oxygen carriers, Organic & Biomolecular Chemistry 13, 11118-

11128.

54. Chatterjee, R., Welty, E. V., Walder, R. Y., Pruitt, S. L., Rogers, P. H., Arnone, A., and

Walder, J. A. (1986) Isolation and characterization of a new hemoglobin derivative cross-

linked between the alpha chains, Journal of Biological Chemistry 261, 9929-9937.

55. Safo, M. K., Burnett, J. C., Musayev, F. N., Nokuri, S., and Abraham, D. J. (2002)

Structure of human carbonmonoxyhemoglobin at 2.16 A: a snapshot of the allosteric

transition, Acta Crystallographica Section D 58, 2031-2037.

56. Adamson, J., and Finch, C. (1975) Hemoglobin Function, Oxygen Affinity, and

Erythropoietin, Annual Review of Physiology 37, 351-369.

57. Bruno, S., Bonaccio, M., Bettati, S., Rivetti, C., Viappiani, C., Abbruzzetti, S., and

Mozzarelli, A. (2001) High and low oxygen affinity conformations of T state

hemoglobin, Protein Science 10, 2401-2407.

58. Li, R., Nagai, Y., and Nagai, M. (2000) Changes of tyrosine and tryptophan residues in

human hemoglobin by oxygen binding: near- and far-UV circular dichroism of isolated

chains and recombined hemoglobin, Journal of Inorganic Biochemistry 82, 93-101.

59. Dragan, S. A., Olsen, K. W., Moore, E. G., and Fitch, A. (2008) Spectroelectrochemical

study of hemoglobin A, alpha- and beta-fumarate crosslinked hemoglobins; implications

to autoxidation reaction, Bioelectrochemistry 73, 55-63.

60. Cantu-Medellin, N., Vitturi, D. A., Rodriguez, C., Murphy, S., Dorman, S., Shiva, S.,

Zhou, Y., Jia, Y., Palmer, A. F., and Patel, R. P. (2011) Effects of T-state and R-state

stabilization on deoxyhemoglobin-nitrite reactions and stimulation of nitric oxide

signaling, Nitric Oxide: Biology and Chemistry 25, 59-69.

99

61. Yu, P. D. B., Shahid, P. D. M., Egorina, M. D. P. D. Elena M., Sovershaev, M. D. P. D.

Mikhail A., Raher, B. S. Michael J., Lei, M. D. C., Wu, P. D. Mei X., Bloch, M. D.

Kenneth D., and Zapol, M. D. Warren M. (2010) Endothelial Dysfunction Enhances

Vasoconstriction Due to Scavenging of Nitric Oxide by a Hemoglobin-based Oxygen

Carrier, Anesthesiology 112, 586-594.

62. Magenta, A., Greco, S., Capogrossi, M. C., Gaetano, C., and Martelli, F. (2014) Nitric

Oxide, Oxidative Stress, and Interplay in Diabetic Endothelial Dysfunction, BioMed

Research International 2014, 16.

63. Rohlfs, R. J., Bruner, E., Chiu, A., Gonzales, A., Gonzales, M. L., Magde, D., Magde, M.

D., Vandegriff, K. D., and Winslow, R. M. (1998) Arterial Blood Pressure Responses to

Cell-free Hemoglobin Solutions and the Reaction with Nitric Oxide, Journal of

Biological Chemistry 273, 12128-12134.

64. Ulatowski, J. A., Koehler, R. C., Nishikawa, T., Traystman, R. J., Razynska, A., Kwansa,

H., Urbaitis, B., and Bucci, E. (1995) Role of nitric oxide scavenging in peripheral

vasoconstrictor response to beta-beta cross-linked hemoglobin, Artificial Cells Blood

Substitutes & Immobilization Biotechnology 23, 263-269.

65. Alayash, A. I. (2014) Blood substitutes: why haven't we been more successful?, Trends in

Biotechnology 32, 177-185.

66. Winslow, R. M. (2003) Current status of blood substitute research: towards a new

paradigm, Journal of Internal Medicine 253, 508-517.

67. Olson, J. S., Foley, E. W., Rogge, C., Tsai, A.-L., Doyle, M. P., and Lemon, D. D. (2004)

No scavenging and the hypertensive effect of hemoglobin-based blood substitutes, Free

Radical Biology and Medicine 36, 685-697.

68. Sakai, H., Hara, H., Yuasa, M., Tsai, A. G., Takeoka, S., Tsuchida, E., and Intaglietta, M.

(2000) Molecular dimensions of Hb-based O2 carriers determine constriction of

resistance arteries and hypertension, American Journal of Physiology - Heart and

Circulatory Physiology 279, H908-H915.

69. Chow, E. C. Y., Liu, L., Ship, N., Kluger, R. H., and Pang, K. S. (2008) Role of

Haptoglobin on the Uptake of Native and β-Chain [Trimesoyl-(Lys82)β-(Lys82)β] Cross-

Linked Human Hemoglobins in Isolated Perfused Rat Livers, Drug Metabolism and

Disposition 36, 937-945.

70. Snyder, S. R., Welty, E. V., Walder, R. Y., Williams, L. A., and Walder, J. A. (1987)

HbXL99 alpha: a hemoglobin derivative that is cross-linked between the alpha subunits is

useful as a blood substitute, Proceedings of the National Academy of Sciences of the

United States of America 84, 7280-7284.

71. Dorman, S. C., Kenny, C. F., Miller, L., Hirsch, R. E., and Harrington, J. P. (2002) Role

of redox potential of hemoglobin-based oxygen carriers on methemoglobin reduction by

plasma components, Artificial Cells, Blood Substitutes, and Biotechnology 30, 39-51.

100

72. Abutarboush, R., Pappas, G., Arnaud, F., Auker, C., McCarron, R., Scultetus, A., and F.

Moon-Massat, P. (2013) Effects of N-Acetyl-L-Cysteine and Hyaluronic Acid on HBOC-

201-Induced Systemic and Cerebral Vasoconstriction in the Rat, Current Drug Discovery

Technologies 10, 315-324.

73. den Boer, P. J., Bleeker, W. K., Rigter, G., Agterberg, J., Stekkinger, P., Kannegieter, L.

M., de Nijs, I. M. M., and Bakker, J. C. (1992) Intravascular Reduction of

Methemoglobin in Plasma of the Rat in Vivo, Biomaterials, Artificial Cells and

Immobilization Biotechnology 20, 647-650.

74. Hopmann, K. H., Cardey, B., Gladwin, M. T., Kim-Shapiro, D. B., and Ghosh, A. (2011)

Hemoglobin as a Nitrite Anhydrase: Modeling Methemoglobin-Mediated N(2)O(3)

Formation, Chemistry (Weinheim an der Bergstrasse, Germany) 17, 6348-6358.

75. Gow, A. J., Luchsinger, B. P., Pawloski, J. R., Singel, D. J., and Stamler, J. S. (1999) The

oxyhemoglobin reaction of nitric oxide, Proceedings of the National Academy of

Sciences of the United States of America 96, 9027-9032.

76. Rifkind, J. M., Lauer, L. D., Chiang, S. C., and Li, N. C. (1976) Copper and oxidation of

hemoglobin: a comparison of horse and human hemoglobins, Biochemistry 15, 5337-

5343.

77. Jewett, J. C., Sletten, E. M., and Bertozzi, C. R. (2010) Rapid Cu-Free Click Chemistry

with Readily Synthesized Biarylazacyclooctynones, Journal of the American Chemical

Society 132, 3688-3690.

78. Dockal, M., Carter, D. C., and Rüker, F. (1999) The Three Recombinant Domains of

Human Serum Albumin: Structural Characterization and Ligand Binding Properties,

Journal of Biological Chemistry 274, 29303-29310.

79. Kleinova, M., Belgacem, O., Pock, K., Rizzi, A., Buchacher, A., and Allmaier, G. (2005)

Characterization of cysteinylation of pharmaceutical-grade human serum albumin by

electrospray ionization mass spectrometry and low-energy collision-induced dissociation

tandem mass spectrometry, Rapid Communications in Mass Spectrometry 19, 2965-2973.

80. Sengupta, S., Wehbe, C., Majors, A. K., Ketterer, M. E., DiBello, P. M., and Jacobsen, D.

W. (2001) Relative Roles of Albumin and Ceruloplasmin in the Formation of

Homocystine, Homocysteine-Cysteine-mixed Disulfide, and Cystine in Circulation,

Journal of Biological Chemistry 276, 46896-46904.

81. Li, Y., Zhou, H., E, Y., Wan, L., Huang, F., and Du, L. (2013) Synthesis and

characterization of a new series of rigid polytriazole resins, Designed Monomers and

Polymers 16, 556-563.

82. Singh, S., and Kluger, R. (2016) Self-Assembly of a Functional Triple Protein:

Hemoglobin-Avidin-Hemoglobin via Biotin–Avidin Interactions, Biochemistry 55, 2875-

2882.

101

83. Hossain, K. M. Z., Patel, U., and Ahmed, I. (2014) Development of microspheres for

biomedical applications: a review, Progress in Biomaterials 4, 1-19.

84. Guillochon, D., Vijayalakshmi, M. W., Thiam-Sow, A., Thomas, D., and Chevalier, A.

(1986) Effect of glutaraldehyde on hemoglobin: functional aspects and Mössbauer

parameters, Biochemistry and Cell Biology 64, 29-37.

85. Caccia, D., Ronda, L., Frassi, R., Perrella, M., Del Favero, E., Bruno, S., Pioselli, B.,

Abbruzzetti, S., Viappiani, C., and Mozzarelli, A. (2009) PEGylation Promotes

Hemoglobin Tetramer Dissociation, Bioconjugate Chemistry 20, 1356-1366.

86. Livnah, O., Bayer, E. A., Wilchek, M., and Sussman, J. L. (1993) Three-dimensional

structures of avidin and the avidin-biotin complex, Proceedings of the National Academy

of Sciences of the United States of America 90, 5076-5080.

87. Repo, S., Paldanius, T. A., Hytönen, Vesa P., Nyholm, T. K. M., Halling, Katrin K.,

Huuskonen, J., Pentikäinen, Olli T., Rissanen, K., Slotte, J. P., Airenne, T. T., Salminen,

T. A., Kulomaa, Markku S., and Johnson, Mark S. (2006) Binding Properties of HABA-

Type Azo Derivatives to Avidin and Avidin-Related Protein, Chemistry & Biology 13,

1029-1039.

88. Wang, Z.-X., Ravi Kumar, N., and Srivastava, D. K. (1992) A novel spectroscopic

titration method for determining the dissociation constant and stoichiometry of protein-

ligand complex, Analytical Biochemistry 206, 376-381.

89. Kluger, R., Song, Y., Wodzinska, J., Head, C., Fujita, T. S., and Jones, R. T. (1992)

Trimesoyltris(3,5-dibromosalicylate): specificity of reactions of a trifunctional acylating

agent with hemoglobin, Journal of the American Chemical Society 114, 9275-9279.

90. Srinivas, P. R. (2012) Introduction to Protein Electrophoresis, In Protein Electrophoresis:

Methods and Protocols (Kurien, T. B., and Scofield, H. R., Eds.), pp 23-28, Humana

Press, Totowa, NJ.

improved approaches to protein-protein coupling and the

Documents