enzyme reaction profile

Review of Analytical MethodsPart 1: Spectrophotometry

Roger L. Bertholf, Ph.D.Associate Professor of Pathology

Chief of Clinical Chemistry & Toxicology

University of Florida Health Science Center/Jacksonville

Analytical methods used in clinical chemistry

• Spectrophotometry

• Electrochemistry

• Immunochemistry

• Other– Osmometry– Chromatography– Electrophoresis

Introduction to spectrophotometry• Involves interaction of electromagnetic

radiation with matter• For laboratory application, typically

involves radiation in the ultraviolet and visible regions of the spectrum.

• Absorbance of electromagnetic radiation is quantitative.

Electromagnetic radiation

E

H

A Velocity = c

Wavelength ()

Wavelength, frequency, and energy

hc

hE E = energyh = Plank’s constant = frequencyc = speed of light = wavelength

The Electromagnetic Spectrum

x-ray UV visible IR Rf

10-11 10-9 10-6 10-5 10-4 10-2 102

Wavelength (, cm)

Frequency (, Hz)

108101210141015101610191021

NuclearInner shellelectrons

Outer shellelectrons

Molecularvibrations

Molecularrotation

NuclearSpin

Visible spectrum

390 780450 520 590 620

Wavelength (nm)

IR UV Increasing Energy

Increasing Wavelength

“Red-Orange-Yellow-Green-Blue”

Absorption of EM radiation

I0 (radiant intensity) I (transmitted intensity)

abcAbckTkNI

Idnk

I

dIkI

dn

dI I

I

N

;log;ln;;00

0

0

Manipulation of Beer’s Law

)2(10%,

)log(%2

)log(%2)log(%)100log(%

100log

%

100log

1loglog

ATand

TA

TTT

TTTabcA

Hence, 50% transmittance results in an absorbance of 0.301, andan absorbance of 2.0 corresponds to 1% transmittance

Absorbance

Err

or (

dA/A

)

0.0 2.0

Beer’s Law error in measurement

0.434

Design of spectrometric methods

• The analyte absorbs at a unique wavelength (not very common)

• The analyte reacts with a reagent to produce an adduct that absorbs at a unique wavelength (a chromophore)

• The analyte is involved in a reaction that produces a chromophore

Measuring total protein

• All proteins are composed of 20 (or so) amino acids.

• There are several analytical methods for measuring proteins:– Kjeldahl’s method (reference)– Direct photometry– Folin-Ciocalteu (Lowery) method– Dye-binding methods (Amido black; Coomassie

Brilliant Blue; Silver)– Precipitation with sulfosalicylic acid or

trichloracetic acid (TCA)– Biuret method

Kjeldahl’s method

SpecimenHot H2SO4 digestionCorrection for non-protein nitrogen

NH4+

Titration or Nessler’sreagent (KI/HgCl2/KOH)

Protein nitrogen

Total protein

Multiply by 6.25 (100%/16%)

Direct photometry

• Absorption at 200–225 nm can also be used (max for peptide bonds)

• Free Tyr and Trp, uric acid, and bilirubin interfere at 280 nm

C

NH2

H2C COOH

H

OH

C

NH2

H2C COOH

H

HN CH

Tyrosine Tryptophan

max= 280 nm

Folin-Ciocalteu (Lowry) method

• Sometimes used in combination with biuret method

• 100 times more sensitive than biuret alone• Typically requires some purification, due

to interferences

Reduced form (blue)Phosphotungstic/phosphomolybdic acidProtein

(Tyr, Trp)

Biuret method

• Sodium potassium tartrate is added to complex and stabilize the Cu++ (cupric) ions

• Iodide is added as an antioxidant

H2NHN NH2

OO

HC

C NH

C

O

HN

O

or . . .Cu++

OH-Blue adduct ( = 540 nm)

Measuring albumin• Albumin is the most abundant protein in serum (40-

60% of total protein)• Albumin is an anionic protein (pI=4.0-5.8)

– Enriched in Asp, Glu

Albumin reacts with anionic dyes– BCG (max= 628 nm), BCP (max= 603 nm)

• Binding of BCG and BCP is not specific, since other proteins have Asp and Glu residues– Reading absorbance within 30 s improves specificity

Specificity of bromocresol dyes

AlbuminBCG (pH 4.2)

BCP (pH 5.2)green or purple adduct

Abs

orba

nce

Time 30 s

Measuring glucose

• Glucose is highly specific for -D-Glucose• The peroxidase step is subject to interferences

from several endogeneous substances – Uric acid in urine can produce falsely low results– Potassium ferrocyanide reduces bilirubin interference

• About a fourth of clinical laboratories use the glucose oxidase method

Glucose + O2 Gluconic acid + H2O2

Glucoseoxidase Peroxidase

o-DianisideOxidized o-dianiside

max= 400–540 (pH-dependant)

Glucose isomers

• The interconversion of the and isomers of glucose is spontaneous, but slow

• Mutorotase is added to glucose oxidase reagent systems to accelerate the interconversion

OH

OH

H

OH

H

OHH

OH

CH2OH

OH

OH

OH

H

H

OHH

OH

CH2OH

-D-glucose (36%) -D-glucose (64%)

Measuring creatinine

• The reaction of creatinine and alkaline picrate was described in 1886 by Max Eduard Jaffe

• Many other compounds also react with picrate

NH

NH

H3C

O

O-

NO2O2N

NO2

OH-

-O

O2N

O2N

NO2

HN

NH

CH3

-O

+

Creatinine Picric acidJanovski complex

max= 485 nm

Modifications of the Jaffe method

• Fuller’s Earth (aluminum silicate, Lloyd’s reagent)– adsorbs creatinine to eliminate protein interference

• Acid blanking– after color development; dissociates Janovsky complex

• Pre-oxidation– addition of ferricyanide oxidizes bilirubin

• Kinetic methods

Kinetic Jaffe methodA

bsor

banc

e (

= 5

20 n

m)

Time (sec) 0 8020

Fast

-rea

ctin

g(p

yruv

ate,

glu

cose

,as

corb

ate)

Slow

-rea

ctin

g(p

rote

in)

t

A

ratet

A

creatinine (and -keto acids)

Enzymatic creatinine method

NH

N

O

CH3

NH

NH

N

O

CH3

O

H3C

HN COOH

H2O H2O2

NH

C

COOH

O

HN

CH3

H2NCH2

COOH

NH

C

COOH

O

HN

CH3

+ CH2O

Creatinine N-Methylhydantoin N-Carbamoylsarcosine

Sarcosine Glycine

Creatinineiminohydrolase

N-Methylhydantoinamidohydrolase

N-Carbamoylsarcosineamidohydrolase

Sarcosineoxidase

NH3 + CO2N-Carbamoylsarcosine

• H2O2 is measured by conventional peroxidase/dye methods


Enzymatic creatinine method


NH

N

O

CH3

NHN

CH3

NH

NH2COOH

H3C

HN COOH

O2 H2O2

H2NCH2

COOH+ CH2O

H3C

HN COOH

Creatinine

Creatinineamidohydrolase

CreatineUrea

Sarcosine

Sarcosine

Sarcosineoxidase

Glycine

Creatineamidohydrolase

Measuring urea (direct method)

• Direct methods measure a chromagen produced directly from urea

• Indirect methods measure ammonia, produced from urea

H3CCH3

O

NOH

H+

H3CCH3

O

OH2N NH2

O

N N

H3C CH3

O

+H+,

Diacetyl monoxime Diacetyl Urea Diazone

max= 540 nm

Measuring urea (indirect method)

• The second step is called the Berthelot reaction• In the U.S., urea is customarily reported as “Blood

Urea Nitrogen” (BUN), even though . . .– It is not measured in blood (it is measured in serum)– Urea is measured, not nitrogen

H2N NH2

O

Urease2 NH4

+ +

OH

OH-N

-O O

Urea Phenol Indophenol

max = 560 nm

Conversion of urea/BUN

dLLureammolNmg

mmolureamgdLmgBUNLmgUrea

LdLmmolureamg

ureammolNmgLmgureadLmgBUN

/10/28

/60)/(/

/1.0/60

/28)/(/

Measuring uric acid

• Tungsten blue absorbs at = 650-700 nm• Uricase enzyme catalyzes the same reaction, and is

more specific– Absorbance of uric acid at 585 nm is monitored

• Methods based on measurement of H2O2 are common

HN

NH

NH

N

O

O

O-

O2 H2O2NH

HN

NH

H2N

O

O

O

Phosphotungstic acid Tungsten blue

Uric Acid Allantoin

Measuring total calcium

• More than 90% of laboratories use one or the other of these methods.

• Specimens are acidified to release Ca++ from protein, but the CPC-Ca++ complex forms at alkaline pH

NN N

N

AsO3H2OH OH

H2O3As

SO3--O3S

O

CH3

HO

CH3

OH

N

O

-O

N

-O O O-O

O

O-

O

Arsenazo III

max= 650 nm

o-Cresolphthalein complexone

max= 570 - 580 nm

Measuring phosphate

• Phosphate in serum occurs in two forms:– H2PO4

- and HPO4-2

• Only inorganic phosphate is measured by this method. Organic phosphate is primarily intracellular.

H3PO4 + (NH4)6Mo7O24

H+

(NH4)3[PO4(MoO3)12]

max= 340 nm

Molybdenum blue

max= 600-700 nm

Red.

Measuring magnesium

• Formazan dye and Xylidyl blue (Magnon) are also used to complex magnesium

• 27Mg neutron activation is the definitive method, but atomic absorption is used as a reference method

N

N

H3C

OH

HO

SO3-

SO3-

CH3

HO

H3C CH3 H3C CH3

O

N

O

O-

O-OCH3

N

O

-O

O O-

Calmagite

max= 530 - 550 nm

Methylthymol blue

max= 600 nm

Measuring iron

• The specimen is acidified to release iron from transferrin and reduce Fe3+ to Fe2+ (ferrous ion)

N N N N

SO3H

SO3NaBathophenanthroline Ferrozine

Fe++

max= 534 nm

Fe++

max= 562 nm

Measuring bilirubin

• Diazo reaction with bilirubin was first described by Erlich in 1883

• Azobilirubin isomers absorb at 600 nm

NH

O NH

HO

O

NH

O

OH

NH

HO3S N N+Cl-NH

O

OH

NH

HO3S N N

NH

ONH

HO

O

SO3HNN

Diazotized sulfanilic acid

Bilirubin (unconjugated)

Azobilirubin (Isomer II)

Azobilirubin (Isomer I)

Evolution of the diazo method

• 1916: van den Bergh and Muller discover that alcohol accelerates the reaction, and coined the terms “direct” and “indirect” bilirubin

• 1938: Jendrassik and Grof add caffeine and sodium benzoate as accelerators – Presumably, the caffeine and benzoate displace un-

conjugated bilirubin from albumin

• The Jendrassik/Grof method was later modified by Doumas, and is in common use today

Bilirubin sub-forms

• HPLC analysis has demonstrated at least 4 distinct forms of bilirubin in serum: -bilirubin is the un-conjugated form (27% of total

bilirubin) -bilirubin is mono-conjugated with glucuronic acid

(24%) -bilirubin is di-conjugated with glucuronic acid (13%) -bilirubin is irreversibly bound to protein (37%)

• Only the , , and fractions are soluble in water, and therefore correspond to the direct fraction

-bilirubin is solubilized by alcohols, and is present, along with all of the other sub-forms, in the indirect fraction

Measuring cholesterol by L-B

• The Liebermann-Burchard method is used by the CDC to establish reference materials

• Cholesterol esters are hydrolyzed and extracted into hexane prior to the L-B reaction

HO

H2SO4/HOAc

HOO2S

Cholesterol Cholestahexaene sulfonic acid

max = 620 nm

L-B reagent

Enzymatic cholesterol methods

• Enzymatic methods are most commonly adapted to automated chemistry analyzers

• The reaction is not entirely specific for cholesterol, but interferences in serum are minimal

Cholesterol esters

Cholesterol

Cholesterylester

hydroxylase

Choles-4-en-3-one + H2O2

Cholesteroloxidase

Quinoneimine dye (max500 nm)

Phenol4-aminoantipyrinePeroxidase

Measuring HDL cholesterol• Ultracentrifugation is the most accurate method

– HDL has density 1.063 – 1.21 g/mL

• Routine methods precipitate Apo-B-100 lipoprotein with a polyanion/divalent cation– Includes VLDL, IDL, Lp(a), LDL, and chylomicrons

HDL, IDL, LDL, VLDL HDL + (IDL, LDL, VLDL)Dextran sulfate

Mg++

• Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol

• Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol

Measuring triglycerides

• LDL is often estimated based on triglyceride concentration, using the Friedwald Equation:

[LDL chol] = [Total chol] – [HDL chol] – [Triglyceride]/5

Triglycerides

Glycerol + FFAsLipase

Glycerophosphate + ADPGlycerokinase

ATP

Dihydroxyacetone + H2O2

Glycerophasphateoxidase

PeroxidaseQuinoneimine dye (max 500 nm)

Spectrophotometric methods involving enzymes

• Often, enzymes are used to facilitate a direct measurement (cholesterol, triglycerides)

• Enzymes may be used to indirectly measure the concentration of a substrate (glucose, uric acid, creatinine)

• Some analytical methods are designed to measure clinically important enzymes

Enzyme kinetics

E + S ES E + Pk1

k-1

k2

1

1

k

k

ES

SESEK

ESEE

ES

SEK

totm

total

m

The Km (Michaelis constant) for an enzyme reaction is a measure of the affinity of substrate for the enzyme.

Km is a thermodynamic quantity, and has nothing to do with the rate of the enzyme-catalyzed reaction.

Enzyme kinetics

E + S ES E + Pk1

k-1

k2

SK

SVvso

VvandESEsaturatedisenzymewhen

SK

SEkvESforngsubstituti

ESkv

m

tot

m

tot

max

max

2

2

,

,,

,

The Michaelis-Menton equation

)(111

,

maxmax

max

max

max

BurkLineweaverVSV

K

vgetwe

SK

SVvofreciprocalthetakingor

MentonMichaelisS

K

v

vVgetwe

SK

SVvgrearrangin

m

m

m

m

The Lineweaver-Burk equation is of the form y = ax + b, so a plot of 1/v versus 1/[S] gives a straight line, from which Km and Vmax can be derived.

v

[S]

The Michaelis-Menton curve

Vmax

½Vmax

Km

SKV

vwhen

SK

SVv

m

m

,2max

max

The Lineweaver-Burk plot

1/[S]

1/v

1/Vmax

-1/Km

maxmax

111

VSV

K

vm

Enzyme inhibition

• Competitive inhibitors compete with the substrate for the enzyme active site (Km)

• Non-competitive inhibitors alter the ability of the enzyme to convert substrate to product (Vmax)

• Un-competitive inhibitors affect both the enzyme substrate complex and conversion of substrate to product (both Km and Vmax)

M-M analysis of an enzyme inhibitor

v

[S]

Vmax

Km

Vmax(i)

Non-competitive

Km(i)

Competitive

L-B analysis of an enzyme inhibitor

1/[S]

1/v

1/Vmax

-1/Km

CompetitiveNon-competitive

Measuring enzyme-catalyzed reactions

• The progress of an enzyme-catalyzed reaction can be followed by measuring:– The disappearance of substrate– The appearance of product– The conversion of a cofactor

Substrate ProductEnzyme

Cofactor Cofactor*

Measuring enzyme-catalyzed reactions

• When the substrate is in excess, the rate of the reaction depends on the enzyme activity

• When the enzyme is in excess, the rate of the reaction depends on the substrate concentration

Substrate ProductEnzyme

Cofactor Cofactor*

Enzyme cofactors

N+CH2

HHOH OH

H HO

OP

-O

O

O

NH2

O

P

O

-OO

H2C

N

N

N

N

NH2

H

OH OHO

H

Nicotinamide adenine dinucleotide (NAD+, oxidized form)

NAD UV absorption spectraA

bsor

banc

e

250 300 350 400

(nm)

NAD+

NADHmax= 340 nm

Lag

pha

se

Enzyme reaction profileP

rodu

ct

Time Mix

Sub

stra

te d

eple

tion

Linear phase

ESt

A

Measuring glucose by hexokinase

• The hexokinase method is used in about half of all clinical laboratories

• Some hexokinase methods use NADP, depending on the source of G-6-PD enzyme

• A reference method has been developed for glucose based on the hexokinase reaction

ATP ADP NAD+ NADH

Glucose Glucose-6-phosphate 6-PhosphogluconateHexokinase

Glucose-6-phosphatedehydrogenase

Measuring bicarbonate

• The specimen is alkalinized to convert all forms of CO2 to HCO3

-, so the method actually measures total CO2

• Enzymatic methods for total CO2 are most common, followed by electrode methods

C

O

O-HOC

O

COO-H2C

P

O-

-O O

H2C

CCOO-O

COO-NADH NAD+

H2C

CHCOO-HO

COO-

+

Malatedehydrogenase

Bicarbonate

Phosphoenolpyruvate

Oxaloacetate Malate

PEPcarboxylase

Measuring lactate dehydrogenase

• Both PL and LP methods are available– At physiological pH, PL reaction if favored– LP reaction requires pH of 8.8-9.8

• LD (sometimes designated LDH) activity will vary, depending on which method is used

H3CO-

O

O NADH NAD+

H3CO-

OH

O

Pyruvate Lactate

Lactatedehydrogenase

Measuring creatine kinase (CK)

• Both creatine and phosphocreatine spontaneously hydrolyze to creatinine

• The reverse (PCrCr) reaction is favorable, although the reagents are more expensive

• All methods involve measurement of ATP or ADP

N

HN NH2

CH2H3C

COO-

ATP ADP

N

HNHN

CH2H3C

COO-

P

O

O

O-

Creatine kinase

PhosphocreatineCreatine

Measuring creatine kinase

• Potential sources of interferences include:– Glutathione (Glutathione reductase also uses

NADPH as a cofactor)– Adenosine kinase phosphorylates ADP to ATP

(fluoride ion inhibits AK activity– Calcium ion may inhibit CK activity, since the

enzyme is Mg++-dependent.

ADP ATP ADP

NADP+ NADPH

Creatine phosphate Creatine

CKpH 6.7

Glucose Glucose-6-phosphateG-6-PDH

6-PhosphogluconateHK

Measuring creatine kinase

• Since the forward (Cr PCr) reaction is slower, the method is not sensitive

• Luminescent methods have been developed, linking ATP to luciferin activation

ATP ADP

PK

ATP

NADH NAD+

Creatine Creatine phosphate

CKpH 9.0

Phosphoenolpyruvate PyruvateLD

Lactate

Measuring alkaline phosphatase

• The natural substrate for ALKP is not known

N+O O-

O

P

O-

OO H2O PI

N+O O-

O-

N+

-O O-

O

p-Nitrophenolphosphate

Alkaline phosphatase

pH 10.3, Mg++

p-Nitrophenoxide

Benzoid(colorless)

Quinonoid

(max= 404 nm)

Measuring transaminase enzymes

• Pyridoxyl-5-phosphate is a required cofactor• Oxaloacetate and pyruvate are measured with their

corresponding dehydrogenase enzymes, MD and LD

H2N CH C

CH3

OH

O

H2N CH C

CH2

OH

O

C

OH

O

COO-

C O

CH2

CH2

COO-

COO-

C O

CH2

COO-

COO-

C O

CH3

COO-

HC NH2

CH2

CH2

COO-

+ +

L-Aspartate

L-Alanine

2-OxyglutaratePyruvate

Oxaloacetate

L-Glutamate

Aspartatetransaminase

Alaninetransaminase

Measuring gamma glutamyl transferase

• Method described by Szasz in 1969, and modified by Rosalki and Tarlow

C

CH2

CH2

HC

COOH

NH2

HNO

NO2

COOH

CH2

NH

C

CH2

O

NH2

NO2

NH2 COOH

CH2

NH

C

CH2

O

HNCO

CH2

CH2

HC

COOH

NH2

-glutamyl-p-nitroanalide Glycylglycine p-Nitroanaline

max= 405 nm

-Glutamylglycylglycine

+ +

-Glutamyltransferase

pH 8.2

Measuring amylase

• Hydrolysis of both (14) and (1 6) linkages occur, but at different rates.

• Hence, the amylase activity measured will depend on the selected substrate

• There are more approaches to measuring amylase than virtually any other common clinical analyte

O

OH

OH

CH2OH

O

OH

OH

CH2OH

O

-Amylose

-Amylase

Ca++Glucose, Maltose

(14)

Amyloclastic amylase method

• The rate of disappearance of the blue complex is proportional to amylase activity

• Starch also can be measured turbidimetrically

• Starch-based methods for amylase measurement are not very common any more

Starch + I2 Blue complexAmylase

Red complex

Saccharogenic amylase method

• Several methods can be used to quantify the reducing sugars liberated from starch

• Somogyi described a saccharogenic amylase method, and defined the units of activity in terms of “reducing equivalents of glucose”

• Alternatively, glucose or maltose can be measured by conventional enzymatic methods

StarchAmylase

Glucose + Maltose Reduced substrate

Chromogenic amylase method

• J&J Vitros application allows small dye-labeled fragments to diffuse through a filter layer

• Abbott FP method uses fluorescein-labeled starch

Dye-labeled starchAmylase

Small dye-labeled fragments

Photometric measurement of dyeSeparation

step

Defined-substrate amylase method

-Glucosidase does not react with oligosaccharides containing more than 4 glucose residues

• A modification of this approach uses -2-chloro-4-NP, which has a higher molar absorptivity than 4-NP

4-NP-(Glucose)7

Amylase4-NP-(Glucose)4,3,2

-Glucosidase

4-NP-(Glucose)4 + Glucose + NPmax= 405 nm

Measuring lipase (direct)

• The Cherry/Crandall procedure involves lipase degradation of olive oil and measurement of liberated fatty acids by titration

• Alternatively, the decrease in turbidity of a triglyceride emulsion can be monitored

• For full activity and specificity, addition of the coenzyme colipase is required

H2C OFA

HC

H2C

OFA

OFA

H2C OH

HC

H2C

OFA

OFAFA FA

H2C OH

HC

H2C

OH

OFAFA

H2C OH

HC

H2C

OH

OH

Lipase Lipase Lipase

Triglyceride ,-Diglyceride -Monoglyceride Glycerol

Measuring lipase (indirect)

• Indirect methods for lipase measurement focus on:– Enzymatic phosphorylation (Glycerol kinase)

and oxidation (L--Glycerophosphate oxidase) of glycerol, and measurement of liberated H2O2

– Dye-labeled diglyceride that releases a chromophore when hydrolyzed by lipase

• Several non-triglyceride substrates have been proposed, as well

Post-test

• Folin-Wu• Jendrassik-Grof• Somogyi-Nelson• Kjeldahl• Lieberman-Bourchard• Rosalki-Tarlow• Jaffe• Bertholet• Fisk-Subbarrow

GlucoseBilirubinGlucose/AmylaseTotal proteinCholesterolGGTCreatinineUreaPhosphate

Identify the methods proposed by the following:

enzyme reaction profile

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