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Vadim T. Ivanov,

Andrei A. Karelin,Hemoglobin as a Source

Marina M. Philippova,

of Endogenous BioactiveIgor V. Nazimov,Vladimir Z. Pletnev Peptides: The Concept ofShemyakin-Ovchinnikov

Institute of Bioorganic Tissue-Specific Peptide PoolChemistry

Russian Academy of

Sciences,

Ul. Miklukho-Maklaya 16/10,

117871 Moscow, Russia

Abstract: Scattered literature data on biologically active hemoglobin-derived peptides are

collected in the form of tables. Respective structurefunctional correlations are analyzed and

the general conclusion is reached that hemoglobin fragments must have a profound physiologi-

cal function. Evidence is presented that generation of hemoglobin fragments starts inside the

erythrocytes. At that stage a- andb-globin chains of hemoglobin predominantly give rise to

relatively long peptides containing ca. 30 amino acid residues. The primary proteolysis is

followed by the next degradation step coupled with excretion of newly formed shorter peptides

form red blood cells. Both the primary and the secondary proteolysis products are subjected

to further stepwise C- and N-terminal chain shortening, giving rise to families of closely

related peptides that are actually found in animal tissue extracts. The possible sites of primary

proteolysis are compared with the positions of the exposed secondary structure elements within

the monomeric a- andb-globins as well as the tetrameric hemoglobin. Two tentative schemes

are proposed for hemoglobin degradation, one of which starts at the globin loops exposed on

the surface of the tetramer and the other, at monomeric globins where more sites are available

for the action of proteases.

The concept of a tissue-specific peptide pool is formulated, describing a novel system of

peptidergic regulation, complementary to the conventional hormonal and neuromodulatory

systems. According to that description, hemoglobin is only a single example, although an

important one, of a vast number of functional proteins providing their proteolytically derived

fragments for maintaining the tissue homeostasis. 1997 John Wiley & Sons, Inc. Biopoly

43: 171188, 1997

Keywords: hemoglobin-derived peptides; hemoglobin proteolysis in vivo; peptide excretion

from erythrocytes; tissue homeostasis; tissue specific peptide pool

INTRODUCTION tide faded as soon as it turned out to be a 110

fragment of the b-chain of hemoglobin. Moreover,other 416 membered peptides isolated later fromAs early as 1971, Schally et al. in their pursuit of

novel hypothalamic factors discovered in pig hypo- the same source were also considered artefacts

solely because they reproduced the amino acid se-thalamus a decapeptide with growth hormone re-

leasing activity.1 However, the interest in that pep- quences ofa- or b-globin chains.2,3 The apparent

Correspondence to: Vadim T. Ivanov

1997 John Wiley & Sons, Inc. CCC 0006-3525/97/020171-18

171

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172 Ivanov et al.

Table I Hemoglobin Fragments Isolated by Schally et al. (19711980) from Pig Hypothalamus

Sequence Fragment Activity

FLGFPTTKTYFPHFNL2 a 3348

FLGFPTTKTYFPHF2 a 3346 Corticotropin release in vitro2

VHLSAEEKEA1 b 110 Growth hormone release in vivo and in vitro1

GKVN3 b 1619 Prolactin release in vitro3

LVVYPWTQRF3

b 3241 VVYPWTQRF3 b 3341

although never explicitly formulated reason for such laboratories (Table IV) , including that of Nyberg

et al., as described in his contribution to this issue.36conclusion was the belief that since the function

of hemoglobin as an oxygen carrier is firmly estab- The list of endogenous peptides with hemoglobin

amino acid sequences was extended by a group oflished, one should not expect other significant func-

tions from the same protein. The subsequent dis- coronaro-constrictory peptides from bovine hypo-

thalamus (Table V) . Five of them, positionedcovery in bovine brain in 1979 and in 1982 of

kyotorphin 4 and neokyotorphin5 reproducing corre- within the b-chain (3241) segment, apparently

belong to the hemorphin group, although their opi-spondingly the 140141 and 137141 C-terminal

sequences ofa-globin, also were not interpreted as oid properties have not been studied.The family of myelopeptides, i.e., peptides se-an indication of the second function of hemoglobin.

In part that was due to the short length of the above- creted by pig red bone marrow cell primary culture

contains b-chain (3237) peptide anda-chain (33mentioned peptides, which therefore could originate

from another protein. 38) peptide fragments.38 Both these peptides were

studied in a variety of immunological test systems.In 1986 Brantl et al. found that proteolytic treat-

ment of hemoglobin gives rise to a series of pep- The results reviewed in this issue add another di-

mension to the spectrum of bioactivities of hemo-tides, called hemorphins, with opioid-like activity.6

That pioneering work was followed by a series of globin peptide fragments.39 Although the above-

mentioned myelopeptides induce naloxon-supressedcommunications dealing with proteolytic degrada-

tion of hemoglobin as well as with bioactive prod- analgesia in vivo, the a-chain (3338) peptide only

weakly inhibited binding ofm- and d-opiate ligandsucts resulting from such treatment.710 Since these

experiments were done in vitro, it remained uncer- to their receptors, and the b-chain (3237) pep-

tide was completely inactive in that test (IC50tain whether similar processes occur in the livingorganism. 1004M) .40 Therefore one should also not expect

any significant opioid activity from the above-men-Only in 1990s did it become clear that hemoglo-

bin serves in vivo as a powerful source of bioactive tioned coronaro-constrictoryb-chain (3337) pep-

tide shown in Table V. We believe the minimalpeptides that must have a profound role in homeo-

stasis. The present paper reviews the available data hemoglobin-derived unit displaying genuine opioid

activity is the tetrapeptide Tyr-Pro-Trp-Thr, i.e.,b-on the structure and properties of these peptides,

and describes new results related to the mechanism chain (3538) fragment or hemorphin-4. In other

words, not all biological properties of peptides fromof their biosynthesis.

the strategic b-chain (3241) zone should be

ascribed opioid receptor binding.

Systematic study of peptide composition of vari-STRUCTURE AND BIOLOGICALACTIVITY OF HEMOGLOBIN-DERIVED ous tissue extracts attempted several years ago in

our laboratory resulted in an avalanche of new pep-PEPTIDES FROM TISSUE EXTRACTStide structures.16,30 As seen from Tables VIVIII,

many of these peptides originate from a- or b-glo-Data on peptides mentioned in the introduction are

summarized in Tables IIII. The search for opioid- bin chains.

Neokyotorphin and its fragment (1 4 ) werelike hemoglobin fragments (hemorphin-containing

peptides) obtained by in vitro proteolysis was con- found in the brain of ground squirrels as well as in

the brain, lung, and heart of rat (Tables II and VI).tinued in publications of Piot et al. reviewed in this

issue of Peptide Science.8 Moreover, hemorphin- Neokyotorphin was shown to terminate the hiberna-

tion state in ground squirrels and to enhance thecontaining peptides were found in vivo in several

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Hemoglobin as a Source of Peptides 173

Table II Neokyotorphin Family of Hemoglobin Fragments

Peptide Fragment Source Activity

YR Kyotorphin a 140 141 Bovine brain4 Analgesic4

Rat spinal cord11

Rat brain11

TSKYR Neokyotorphin a 137 141 Bovine brain5 Analgesic5

Hibernating ground squirrel12

Antihibernatic12

Rat brain13 Ca2//K/ current regulation12

Rat lung13

Rat heart13

Human lung carcinoma14

Human erythrocytes15

TSKY a 137 140 Hibernating ground squirrel16 Ca2//K/ current regulation16

Rat brain13 Cytotoxicity13

Rat lung13

Rat heart13

Human erythrocytes15

SKYR a 138 141 Hibernating ground squirrel16

inward potential dependent Ca2/ current in cardiac ERYTHROCYTES AS A PRIMARYSOURCE OF HEMOGLOBIN-DERIVEDmyocytes of rat42 ; it was not cytotoxic in human

erythroid leukemia (K562) and murine-transformed PEPTIDESfibroblast (L929) cell lines.13 In contrast to neokyo-

torphin, the shortened by one C-terminal amino acid The abundance of hemoglobin fragments in variousfragment inhibited the Ca2/ current42 and showed tissues raises a question of the mechanism of theirpronounced cytotoxic effects in the above-men- formation and delivery. In principle, the globintioned transformed cell lines.13 genes can be expressed not only in red blood cells

Peptides belonging to the hemorphin family [i.e., and therefore respective peptides might form di-with sequences spanning the b-globin (3241) se- rectly within any cell.46 However, the huge amountquence] were amply represented in the bovine brain of hemoglobin circulating inside the erythrocytes

extracts ( Table VII) . suggest itself as the most probable precursor of pep-Several of the peptides from bovine red bone tides listed in Tables I, II, and IVVIII.

marrow were able to restore the hemopoietic func- Chromatographic analysis of the peptide contenttion of mice subjected to radiation or treated with 5- in human erythrocytes (Figures 2A and 3A) demon-fluorouracil (Table VIII) .30,41 It is worth mentioning strated that intensive proteolytic degradation of he-that one of these hemoregulatory peptides, a-globin moglobin takes place under normal conditions, giv-(116), spans three quarters of the sequence of the ing rise to peptides shown in Table X. In additionpeptide a-globin (121) from Table V. to the earlier published sequences, the latter also

A number of hemoglobin-derived peptides were shows the relative concentrations of isolated pep-isolated from brain extracts by Sleemon et al. (Table tides. The overall amount of these peptides com-IX). Although the structural approach applied in prised ca. 0.5% of the hemoglobin content. With thethat work did not allow the determination of the exception of neokyotorphin and its des-C-terminuscomplete amino acid sequences, it was shown that derivative found in very minor amounts, the intraer-

some of these peptides can serve as markers of such ythrocytic peptides have notably longer sequencespathologies as Alzheimers disease and brain isch- than most of the above-discussed endogenous he-emia.4345 moglobin fragments. Of them, the longest a-chain

In summary, hemoglobin fragments isolated (133) peptide is by far the most abundant frag-from various sources cover almost the entire se- ment present inside the erythrocytes.quences ofa- and b-globins. In turn, a considerable The reason for the above-mentioned discrepancypart of these sequences display distinct bioactivity. was found in our recent study of peptides released

The current state of the above-mentioned relation- by erythrocyte primary culture. The supernatant of

erythrocytes incubated in the phosphate buffer (pHship is illustrated in Figure 1.

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174 Ivanov et al.

Table III Biologically Active Hemoglobin Fragments Obtained by In Vitro Proteolysisa

Peptide Fragment Proteolitic Treatment Activity

ASHLPSDFTPAVHASL a 110 125 Bovine hemoglobin treated Bradykinin potentiation7

with pepsin7

LANVST a 129 134 Bovine hemoglobin treated Bradykinin potentiation8

with pepsin8

LVVYPWTQRF b 3241 Bovine hemoglobin treated Specific opiate receptor binding17

with pepsin9 Inhibition of electrically induced

contractions of GPI9

Inhibition of ACE18

Coronaro-constrictory19

VVYPWTQRF b 3341 Bovine hemoglobin treated Specific opiate receptor binding17

with pepsin9 Inhibition of electrically induced


Bitter peptide20

Inhibition of ACE18

YPWT Hemorphin-4 b 3538 Bovine hemoglobin treated Naloxon-depending analgesia21

with pepsin/cathepsin6 Specific opiate receptor binding22

Inhibition of electrically induced


YPWTQ Hemorphin-5 b 3539 Bovine hemoglobin treated Naloxon-depending analgesia21

with pepsin/cathepsin6 Inhibition of electrically induced


GKKVLQ b 6469 Pig hemoglobin treated Inhibition of ACE10

with trypsin10

FQKVVA b 130 135 Pig hemoglobin treated Inhibition of ACE10

with trypsin10

FQKVVAG b 130 136 Pig hemoglobin treated Inhibition of ACE10

with trypsin10

a In Table III and the following tables the numbering of bovine b-globin chain starts with No. 2 in order to allow correct alignment

with sequences from other animal species.

7.2) was fractionated on size-exclusion (Figure 2B) chains as peptides found earlier in bovine extracts:a-(112), b-(211), b-( 2 9) (Table VIII) andand reverse phase high performance liquid chroma-

tography ( RP-HPLC; Figure 3B) columns. As with b-(133146) (Table V). As a result, these pep-

tides, as well as most of the other peptides fromthe erythrocytes lysates, the chromatographic pro-

files obtained with the supernatant did not show Table XI [with the exception ofa-chain (8488)

and b-chain (8791) fragments], show a very con-any significant individual differences, i.e., peptide

content of both, the erythrolysate and the superna- siderable overlap with respective peptides in Tables

I, IV, VII, and VIII. Accordingly, the b-chain (34tant did not depend upon the blood group, sex, or

age of the donor. 39) peptide is expected to display opioid properties

(Table IV) ; b-chain (1 11 ) peptidegrowth hor-The obtained fractions were rechromatographed

and substances corresponding to main peaks were mone release activity1 ; b-chain (34 37) peptide

coronaro-constrictory28 and/or immunomodula-analyzed by Edman technique. As a result, 15 amino

acid sequences of isolated peptides were estab- tory38 activities; a-chain (117), a-chain (112),

a-chain ( 1 11), b-chain (1 10) , b-chain (1 6 ),lished. Thirteen peptides were identified as hemo-globin fragments, (Table XI) the remaining two andb-chain (7284) peptideshemopoietic activ-

ity 41 (Table VIII).(NDKVQPLE and KEATQE, not shown in Fig. 3B

and Table XI) are derived from other proteins. The A striking feature of the obtained result is the

fact that not a single peptide found inside erythro-overall content of peptides in the supernatant after

4.5 h incubation was ca. 25% of the peptide con- cytes was present in the supernatant. From that ob-

servation we conclude that release of peptides fromtent inside erythrocytes.

Four of the peptides present in the supernatant erythrocytes is accompanied by proteolytic degrada-

tion of initially formed long peptide sequences, pos-occupy the same positions in the a- and b-globin

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Table IV Hemorphins and Related Peptide Fragments ofb-Globin

Sequence Position Source Activity

LVVYPWTQRF 32 41 Pig hypothalamus3 Specific opiate receptor binding17

Bovine hypothalamus19 Inhibition of electrically induced


Bovine brain23 Coronaro-constrictory activity19

Human liquor24

Inhibition of ACE18

Human gingival crevicular fluid25

LVVYPWTQR 32 40 Human pituitary gland26 Specific opiate receptor binding26



Inhibition of ACE27

LVVYPWTQ 32 39 Bovine brain23

LVVYPWT 32 38 Bovine brain23 Coronaro-constrictory activity28

Bovine hypothalamus28 Inhibition of enkephalin-degrading enzyme29

Bovine spinal cord29 Convulsion in vivo30

VVYPWTQRF 33 41 Pig hypothalamus3 Specific opiate receptor binding17

Bovine brain23 Inhibition of electrically induced


Mice peritoneal macrophages31 Inhibition of ACE18

Bitter peptide20

VVYPWTQR 33 40 Bovine hypothalamus32 Coronaro-constrictory activity32

VVYPWTQ 33 39 Bovine hypothalamus33 Specific opiate receptor binding33

Bovine brain23 Inhibition of electrically induced


Cytotoxicity34

VVYPWT 33 38 Bovine hypothalamus28 Coronaro-constrictory activity28

YPWTQRF 35 41 Human plasma35 Specific opiate receptor binding9



sibly by membrane-associated proteases. The partic- There are all grounds to assume that release of

short peptides from erythrocytes makes an im-ular type of protease as well as the mechanism oftransmembrane passage (direct transport or secre- portant contribution to the overall peptide content

of various tissues. At the same time the array oftion involving vesicle formation, energy, and carrier

requirement, etc.) require further investigation. concrete peptides found in three thus far studied

tissues (bovine bone marrow, bovine brain, and hu-

man cerebellum, see Tables VII, VIII, and IX ) dis-Table V Hemoglobin Fragments Isolated from plays considerable differences. For instance, hemor-Bovine Hypothalamus by Galoyan et al.

Sequence Fragment Table VI Hemoglobin Fragments Isolated from

Brain of Hibernating Yakut Ground SquirrelsVLSAADKGNVKAAWGKVGGHA37 a 121 Citellus undulatus16

ASHLPSDFTAPAVAS37 a 110124

SHLPSDFTPAV32 a 111121 Sequence FragmentHLPSDFTPAVHASLD32 a 112126LVVYPWTQRF19,a b 3241 VLSPA a 15LVVYPWT28,a b 3238 TSKYR a 137141VVYPWTQR32,a b 3340 TSKY a 137140VVYPWT28,a b 3338 SKYR a 138141VVYPW28,a b 3337 VHLSDGEKNAISTAWG b 116FQKVVAGVANALAHRYH32 b 130146 VHLSDGEKNAISTA b 114VVAGVANALAHRYH37 b 133146 IVIVMA b 110115

VVAGVANA b 133140a These peptides display coronaro-constrictory properties.

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Table VII Hemoglobin Fragments Isolated from Bovine Brain30

Content

Sequence Fragment (nmol/g Tissue)

VLSAADKGNVKAAWGKVGGHAAEYGAEALERM a 132 0.11.0

VLSAADKGNVKAAWGKVGGHAAEYGAEALE a 130 0.11.0

VLSAADKGNVKAAWGKVGGHAAEY a 124 0.11.0

DKGNV a 610 0.11.0LSHSL a 101105 0.11.0

ASHLPSDFTPAVHASLDKFLANV a 110132 1.03.0

ASHLPSDFTPAVHASLDK a 110127 0.11.0

FLANVSTVL a 128136 0.11.0

MLTAEEKAAVTAFWGKVKVDEVGGEALGRL b 231 0.11.0

MLTAEEKAAVTAFWGKVKVDEVGGEALG b 229 0.11.0

MLTAEEKAAVTAF b 214 0.11.0

MLTAEEKA b 29 0.11.0

MLT b 24 0.11.0

WGKVKVDEVGGEA b 1527 0.11.0

WGKVKVDEVG b 1524 0.11.0

EVGGEALG b 2229 0.11.0

EVGGEAL b 2228 0.1

GGE b 2426 0.11.0LVVYPWTQRF b 3241 1.03.0

LVVYPWTQ b 3239 1.03.0

LVVYPWT b 3238 0.11.0

LVVYP b 3236 0.11.0

VVYPWTQRF b 3341 1.03.0

VVYPWTQ b 3339 0.11.0

VVVLARNFGKFFTPVLQADFQKVVAGVAN-? b 111139? 0.11.0

VVVLARNFGKFFTPVLQ b 111127 0.11.0

VVVL b 111114 0.11.0

ARNFGKFFTPVLQ b 115127 0.11.0

VLQ b 125127 0.11.0

FQKVVAGVANALAHRYH b 130146 0.11.0

phins were not found in bovine bone marrow ex- taining ca. 30 amino acid residues and found in

tissue extracts come from the intraerythrocyte pooltracts and are well represented in bovine brain (1

nmol/ g of tissue) . In other words, in spite of argu- of the peptides where they are represented in high

amounts (Table X) . The shorter peptides are genu-ments favoring the role of erythrocytes as a common

primary source of hemoglobin fragments, each tis- ine tissue specific peptides and are either absorbed

from the blood stream or result from proteolyticsue has its own, specific set of hemoglobin-derived

peptide components. degradation of the absorbed peptides.

The available quantitative, data although rather

fragmentary on the content of hemoglobin-derived

peptides in various samples, also speak of tissue PROTEOLYTIC DEGRADATIONOF HEMOGLOBINspecificity. For instance, the level of neokyotorphin

in rat lung is 45 times higher than in erythro-cytes.13 We believe that peptides released from the With more that 150 established amino acid se-

quences of endogenous hemoglobin fragmentserythrocytes either accumulate in the tissue sur-

rounding the blood vessel or are further degraded available for comparison, it is tempting to analyze

the pathways of hemoglobin proteolysis in moreby tissue-specific proteases. The observed level of

peptides in the tissue extract is a sum of the contri- detail. For that purpose we collected in Figures 4

and 5 all peptides from Tables IXI and alignedbutions from the erythrocytes always present in

blood vessels and from the rest of the tissue. them under the a- and b-globin sequences of re-

spective species.Accordingly, we assume that long peptides con-

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Table VIII Hemoglobin Fragments Isolated from Bovine Red Bone Marrow30

Content

Sequence Fragment (nmol/g Tissue)

VLSAADKGNVKAAWGKa a 116 0.01

VLSAADKGNVKAAWG a 115 0.01

VLSAADKGNVKAAa a 113 0.010.1

VLSAADKGNVKA a 112 0.01LSAADKGNVKAA a 213 0.010.1

SAADKGNVa a 310 0.01

KVGGHAAEYGAEAa a 1628 0.010.1

KVGGHAAEYGAE a 1627 0.010.1

KVGGHAAEYGA a 1626 0.010.1

VGGHAAEYGAEALa a 1729 0.01

VGGHAAEYGAEAa a 1728 0.010.1

AEYGAELa a 2229 0.01

AEYGAE a 2228 0.10.3

GAEALERa a 2531 0.010.1

AEALERM a 2632 0.010.1

EALERM a 2732 0.010.1

EALEa a 2730 0.010.1

LSFPTTK a 3440 0.01DLSHGSAQV a 4755 0.11.0

ALTKA a 6569 0.11.0

LPGALSELS a 7684 0.11.0

LPGALSEa a 7682 0.11.0

LPGA a 7679 0.010.1

LASHLPSDFTPAV a 109121 0.010.1

ASHLPSDFTPAVHA a 110123 0.10.3

ASHLPSDFTPAVH a 110122 0.010.1

ASHLPSDFTPAV a 110121 0.010.1

ASHLPSDFTPA a 110120 0.010.1

ASHLPSDF a 110117 0.010.1

ASHLPS a 110115 0.10.3

ASHLP a 110114 0.010.1

LPSDFTPAVH a 113122 0.010.1LPSDF a 113117 0.10.3

LDKFLA a 125130 0.010.1

MLTAEEKAAVTa b 212 0.010.1

MLTAEEKAAVa b 211 0.010.1

MLTAEEKAAa b 210 0.010.1

MLTAEEKA b 29 0.010.1

MLTAE b 26 0.010.1

LTAEEKA b 39 0.010.1

AEEKAA b 510 0.010.1

GKVKVDEVGGEALGRLa b 1631 0.010.1

VKVDEVGGEALGRL b 1831 0.010.1

DEVGGEALGR b 2130 0.010.1

EVGGEALGRLa b 2231 0.10.3

EVGGEALGR b 2230 0.10.3EALG b 2629 0.010.1

ALG b 2729 0.10.3

SNGMKGLDDLK b 7282 0.010.1

KLHVDPEa b 95101 0.010.1

ARNFGKFFa b 115122 0.010.1

NFGKEFTPV b 117125 0.010.1

VLQA b 125128 0.010.1

a These peptides display hemopoietic activity.41

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178 Ivanov et al.

Table IX Hemoglobin Fragments as Peptide Markers of Brain Pathologies (Sleemon et al. 19921996)

Sequence Fragment Source Pathology

VLSPADKTNVKAAWGKVGAHAGEYGA-? a 126? Human cerebellum Alzheimers disease43

VLSPADK-? a 17? Human cerebellum Healthy donor44

?-MFAAFPTTK-? a (32 40)? Rat hippocampus Ischemia45

FLSFPTTKTYFPHFDLSHGSAQV-? a 3355? Human cerebellum Alzheimers disease43

FLSFPTTKTYFPHFDLSH-? a 3350? Human cerebellum Alzheimers disease43

FLSFPTTK-? a 3340? Human cerebellum Alzheimers disease43

?-TYFNHIDVSP-? a (41 50)? Rat hippocampus Ischemia45

LVTLAAHLP-? a 106114? Human cerebellum Alzheimers disease43

AAHLPAEFTP-? a 110119? Human cerebellum Alzheimers disease43

?-LASVSTVLT-? a (129 137)? Rat hippocampus Ischemia45

VHLTPEEKSAVTAL-? b 114? Human prefrontal cortex Alzheimers disease45

?-VVYPWTQRY-? b (33 41)? Rat hippocampus Ischemia45

FESFGDLSTPDAV-? b 4254? Human cerebellum Alzheimers disease43

(AV)MGNPK-? b 53 59? Human postcentral gyrus Alzheimers disease45

SELHCDKLHVEFTPPVQAAYQK-? b 89132? Human cerebellum Alzheimers disease43

VCVLAHHFGKEFTPPVQAAY-? b 111130? Human cerebellum Alzheimers disease43

The obtained overall picture clearly speaks of a several smaller pieces, e.g., at sites (13) (Ala/ Cys) -

Trp-(Gly/ Glu) -Lys-( Val/ Ile)( 17) , (127)Lys-Phe-nonrandom, stepwise manner of hemoglobin degra-

dation, regardless of the chosen species and tissue. Leu-(Ala/Ser) -(Asn/Ser)-Val-Ser( 133) , or (135)

Val-Leu-Thr-Ser(138) in the a-globin, and (13)As seen from the positions of the nonoverlapping

sequences, the primary splitting might occur at the (Ala/ Gly/ Thr)-(Phe/Leu/ Ala) -Trp-Gly-Lys-Val

(18), (30)Arg-Leu-Leu-Val-Val(34), (108)Asn-sites marked in Figures 4 and 5 by arrows, giving

rise to 2 4 fragments 3060 amino acid resi- (Val /Met) -( Leu/Ile )-Val-( Val/ Cys/Met) -Val-

(Leu/Met)- (Ala/Gly)( 115) , or (127)Gln-Ala-dues long. These positions for a-globin are

(33) Met-Phe-(Leu/Ala)(35), (55)Val-rrr-Ala(65), (Asp/ Ala) -( Phe/ Tyr) -Gln( 131) in the b-globin.

The products of both primary and secondary proteo-(69)Ala-rrr-( Leu/Met/ Val) (76) , (88) Ala-rrr-

Leu(101) and (105)Leu-Leu(106). For b-globin lytic attack are degraded by carboxy- and aminopep-

tidases, giving rise to ladders of stepwise C- andthe respective sites are (41)(Phe/Tyr)-Phe(42),

(54)(Val/Ile)-rrr-(Ser/Asn)( 72), (85)Phe-rrr N-terminally shortened peptides. The products ofcarboxypeptidase action are more frequently repre-(Ala/Thr/Lys/Ser/His)(87).

The obtained polypeptides are further cut into sented in Figures 4 and 5. However, part of that

FIGURE 1 Hemoglobin sequences covered by peptides isolated from animal tissues and

biological activities associated with respective sequences. Sequences covered by isolated pep-

tides are marked by dark shading. Types of biological activities: HPhemopoietic; HR

hormone releasing; CCcoronaro-constrictory; NKpeptides from neokyotorphin group;

IMimmunomodulatory; AGantigonadotropic; OPopioid.

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(Ala/Leu/Phe)-Trp, Trp-Gly, and Gly-Lys sites.

Such diffuse specificity might be also associated

with conformational features of the products of pri-

mary splitting. Certainly, more work should be done

to confirm the above speculation since possible par-

ticipation of more than one enzyme in degradation

as well as inclusion into Figures 4 and 5 of peptides

from various tissues and various species can serveas alternative explanations.

We tried to correlate the putative positions of

primary cutting with the secondary structure of glo-

bin chains around those positions as well as their

availability to intermolecular interactions with pro-

teases. Figure 6 presents the space filling model of

the tetrameric hemoglobin globule in which helix-

connecting loops of the polypeptide chains are

marked with different colors and labeled with num-

bers, as shown in the figure caption. It is clear from

a brief glance at the picture that all loops, although

FIGURE 2 Size-exclusion chromatography of peptide

material from the lysate of human erythrocytes (A) and

the supernatant of the primary culture of human erythro-

cytes (B). The collected fractions are underlined.

abundance might be due to increasing uncertainty

in identification of the last C-terminal residues in

the course of automatic sequence analysis by theEdman procedure.

Following the arguments given in previous sec-

tion, we assume that primary proteolysis of hemo-

globin takes place exclusively inside the erythro-

cytes while all the following steps predominantly

occur on crossing the erythrocyte membrane and

later within the tissue.

There are no obvious regularities in amino acid

sequences around the primary or secondary splitting

sites. It is only clear that trypsin-like proteases do

not participate in the processing of hemoglobin,

since there are no arginines and very few lysines in

those sequences. All splitting sites contain hy-drophobic residues.

As for the secondary splitting, it is worth men-FIGURE 3 RP-HPLC of the fractions obtained after

tioning that it takes place at several adjacent peptidesize-exclusion separation. (A) The lysate of human eryth-

bonds rather than at strictly defined single pairs of rocytes; (B) the supernatant of primary culture of humanamino acid residues. For instance, the a-chain (32 erythrocytes. Absorbance range in mV (1800 mV 2.5634) segment (Met-Phe-Leu) is cut at Met-Phe and AU) . The peaks containing peptides subjected to seque-Phe-Leu sites and the b-chain (1417) segment nation are marked with numbers. The obtained sequences

are given in Tables X and XI at respective numbers.[( Ala/Leu/Phe) -Trp-Gly-Lys] is apparently cut at

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180 Ivanov et al.

Table X Peptides Isolated from Human Erythrolysate15

Content

No. Sequence Fragment (nmol/mL cells)

6 VLSPADAKTNAVKAWGKVGAHAGEYGAEALERMF a 133 140160

5 VLSPADAKTNAVKAWGKVGAHAGEYGAEALERMa a 132 612

3 VLSPADAKTNAVKAWGKVGAHAGEYGAEALERa a 131 35

4 VLSPADAKTNAVKAWGKVGAHAGEYGAEALE a 130 487 VTLAAHLPAEGFTPAVHASLDKFLASVSTVL a 107136 2550

9 AAHLPAEGFTPAVHASLDKFLASVSTVLTSKYR a 110141 2040

1 TSKYR a 137141 24

2 TSKY a 137140 24

8 AHHFGKEFTPPVQAAYQKVVAGVANALAHRYH b 115146 2550

a The content of these peptides was 515 times higher in the blood samples of patients with Hodgkins disease than those of healthy

donors.47

to a varying degree, are represented at the surface of to loops 2 and 7, as well as the adjacent a-helical

amino acid residues [in particular, a-chain (3334)the globule. However, more detailed analysis proves

that the above-mentioned sites a-chain (3335), and b-chain (4142)] become a plausible targetfor the protease. Such splitting explains the forma-a-chain (88101), and b-chain (4142) are not

sterically accessible. Of the remaining sites, the tion of the peptides a-chain (133)15 and b-chain

(141).26symmetric a-chain (6976) and b-chain (5672)

ones, belonging respectively to loops 3 and 8, seem It is too early to give a preference to either of the

proposed degradation pathways. One should alsoto satisfy the steric requirements. The remaining a-

chain (59 65), a-chain (105107), and b-chain consider that conformational rearrangement might

accompany the nicking of both the tetramer and the(8587) sites fall into the middle of respective heli-

cal segments. dissociated forms, leading to formation of additional

degradation sites. In other words, if general princi-Besides the tetramer, monomeric a- and b-glo-

bins whose content in the equilibrium is ca. 0.001 ples of hemoglobin proteolysis leading to formation

of peptide families are reasonably established, the0.01% of the total protein 48 can also be a subject

of enzymatic degradation. Secondary structures of concrete sites of primary splitting require identifi-

cation of longer peptides containing ca. 60 aminothese chains are shown in Figure 7. In that case sitesa-chain (3652) and b-chain (35 57 ) , referring acid residues.

Table XI Hemoglobin Fragments Released from Human Erythrocytes in Primary Culture

Content

No. Sequence Fragment (nmol/mL Cells)

13 VLSPADKTNVKAAWGKV a 117 0.040.06

6 VLSPADKTNVKA a 112 0.030.05

5 VLSPADKTNV a 110 0.030.05

3 VLSPADKTN a 19 0.030.05

4 SDLHA a 8488 0.050.07

8 VHLTPEEKSAV b 111 0.100.12

2 VHLTPEEKSA b 110 0.060.08

1 VHLTPEEK b 18 0.030.05

10 VYPWTQ b 3439 0.030.05

9 VYPW b 3437 0.010.02

11 SDGLAHLDNLKGTF b 7284 0.060.08

7 TLSEL b 8791 0.030.05

12 VVAGVANALAHRYH b 133146 0.100.12

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FIGURE

4

a-G

lobin

fragmentsisolatedfrom

differentsour

ces.Sequencesofa-g

lobins:(A)bovi

ne,

(B)human,

(C)pig,

(D)Yakutg

roundsquirrelsCitellus

undulatus,

and(E)rat.

Theprimarysplittingsitesaremarkedbyarrows.Sequencesofbiologicallyactivepeptidesareprintedinbolditalic.

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182 Ivanov et al.

FIGURE5

b-G

lobinfra

gmentsisolatedfrom

differentsources.S

equencesofb-g

lobins:(A)bovine,

(B)

human,

(C)pig,

(D)YakutgroundsquirrelsCitellusundulatus,

and(E)rat.Theprimarysplittingsitesaremarkedbyarrows.Sequencesofbiologicallyactivepeptidesa

reprintedinbolditalic.

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FIGURE 6 Space-filling model of human deoxyhemoglobin tetrameric structure. (A) front

view; (B) and (C) views of the model rotated by 90 around X and Y axes, respectively.

Helices of two a- and b-chains are shown in white and magenta, respectively. Loops of two

a-chains are shown in light and dark blue [position numbers: (1 ) fragment 1819; (2) fragment

3652; (3) fragment 7475; (4) fragment 9294; (5) fragment 113118] and those of two

b-chainsin red and brown [position numbers: (6) fragment 1819; (7) fragment 3550;

(8) fragment 7780; (9) fragment 9699; (5) fragment 119123].

HEMOGLOBIN FRAGMENTS AS globin carries out an important in vivo regulatoryfunction. In other words, we believe participationCOMPONENTS OF TISSUE-SPECIFIC

PEPTIDE POOL of hemoglobin fragments in homeostasis providesan example of a novel peptide-mediated regulatory

pathway complementary to traditional hormonal orThe broad spectrum of biological effects exhibited

by endogenous hemoglobin-derived peptides forces neuropeptide mechanisms. That concept originally

formulated in 1992 was further supported by thea conclusion that proteolytic degradation of hemo-

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184 Ivanov et al.

FIGURE 7 A view of the overall structure fold of human deoxyhemoglobin. (A) a-chain;

(B) b-chain. The a-helices and loops are shown in magenta and yellow, respectively. The

numbers indicate the beginning and the end of the a-helical secondary structure elements.

above-described data on intensive formation in and It is generally accepted that the proteins normally

present in the tissue are digested by proteolytic en-release from the erythrocytes of active peptides sub-

sequently found in various tissues.30 zymes after fulfilling their function. We suggest that

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Table XII Distinctive Features of Peptidergic Regulatory Systems

Peptidergic Regulatory System

Characteristic Nervous Endocrine and Paracrine Tissue-Specific Peptide Pool

Peptides Neurotransmitters Hormones Fragments of functional proteins

Precursor Specific protein precursor Functional protein

Type of processing Discrete site specific processing Action of tissue proteinasesLevel (pM/g tissue) 0.001 1.0 0.001 1.0 1010,000

Type of regulation Synaptic secretion Extracellular secretion Alteration of the level in the tissue

Mechanism of action Binding to receptors Binding to receptors in Binding to receptors of homologous

in postsynaptic cellular membranes hormones

membrane

Receptor binding 1 1000 0.110 10010,000

constants (Kd, nM)

Time range of action Secondsminutes Minuteshours Hoursdays

Biological role Transmission of nerve Regulation of physiological Maintenance of tissue homeostasis

impulse processes in the tissue or

the whole organism

protein elimination does not occur by random hy- of proteolytic processes depends on such a relatively

conservative parameter as the metabolic state of thedrolysis directly leading to amino acids, which are

further utilized in metabolic reactions. Instead, it organism, we suggest that the tissue-specific peptide

pool predominantly controls long-term processes,represents a specific process regulated by the level

of tissue-specific proteases and the availability of i.e., is responsible for tissue homeostasis. The latter

includes cell proliferation, differentiation, and celltheir substrates. As a result of that process, a large

number of peptides is formed that can be defined death. Prevention of cell transformation and lysis

of tumor cells also fall into that category. The recentas a peptide buffer, peptide background, or

more accurately as tissue-specific peptide pool. finding that hemoglobin fragments induce death of

transformed cells13,34 and regulate proliferation andThe properties of that pool depend upon the con-

crete set of peptide components as well on individ- differentiation of normal cells38,41 provides a con-

vincing argument favoring our view.ual levels of those components. Characteristic prop-

erties of peptides from the tissue-specific pool in Components of the tissue specific peptide poolshave several features in common with the so calledcomparison with the classical regulatory peptides

are summarized in Table XII. In contrast to peptides cytomedines, a term proposed in early 80-ies for

the components with low molecular mass fractionsderived from functional proteins, the signal peptide

molecules of the nervous tissue (neurotransmitters of the total tissue extracts. Similarly to the peptide

pool, cytomedines were ascribed a homeostatic roleand/or neurohormones) or endocrine system (hor-

mones and parahormones) are released from a nar- for the given tissue (see the review49 and the refer-

ences therein). However, most of the porperties ofrow circle of specific precursors for which no other

than having a precursor function is known. Compo- cytomedines were studied on complex, poorly char-

acterized mixtures containing all types of regulatorynents of the peptide pool bind to the same receptors

as neurotransmitters or hormones modulating peptides presented in Table XII.

There is a growing evidence of changes in tissuethereby the availability of the receptors to their

true ligands. The binding affinities of the pool composition of hemoglobin-derived peptides ac-

companying various pathologies, such as humanpeptides are by several orders of magnitude lowerthan for hormones or neurotransmitters. However, lung adenocarcinoma14 (Tables II ) , Alzheimers

disease,43,45 brain ischemia45 (Table IX), and Hodg-these peptides are found in higher amounts and typi-

cally occur as families of related molecules rather kins disease47 (Table X) . Notwithstanding the clin-

ical and biochemical differences, all the above-men-than single representatives. Hemorphins and closely

related peptides with their opioid receptor binding tioned pathologies have a common principle fea-

ture they involve changes in tissue homeostasisability consistently found in most of the investigated

sources ( see Tables I, IV, V, and VII ) provide a or metabolic state of the cells, whatever the origin of

the disease: cell transformation ( adenocarcinoma) ,good example of that tendency. Since the intensity

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186 Ivanov et al.

tissue atrophy ( Alzheimers disease and ischemia) , Destruction of Erythrocytesor impaired lymphoproliferation ( Hodgkins dis-

The washed clean cells were subjected to two cycles ofease). Although it is not known whether the above- freezing (070C) and unfreezing (5C), homogenized inmentioned changes in peptide composition are the Potter S (B. Braun, Germany) (900 rev 1 5 min) andcause or the consequence of the disease, these centrifuged (10000 rev. 1 15 min) at Biofuge B (Her-changes are in full accord with our views of the aeus, Germany). The amount of 0.25 mL of supernatant

was dissolved in 10 mL 0.1M acetic acid and injectedbiological role of tissue-specific peptide pool.into the size-exclusion column.

More data should be accumulated before the con-cept of tissue-specific peptide pool finds its final

shape. However, regardless of these developments Supernatant of the Primary Culture ofthere is little doubt that besides the oxygen transport Erythrocytes(and possibly the transport of nitrogen oxide50) he-

Ten milliliters of washed cells were incubated with 40moglobin serves as a rich source of bioactive pep-mL of buffer A in culture flacks for 4 h at 37C. Aftertides. In that case the function of erythrocytes mightincubation, the cells were pelleted by centrifugation inbe compared with the function of endocrine gland.conditions described above. The obtained supernatants

Endogenous fragmentation of the hemoglobinwere collected and lyophilized.

and the properties of respective peptides are studiedA portion of lyophilized supernatant obtained from 50

much better than analogous processes with other mL of blood cells was dissolved in 8 mL 0.1M aceticfunctional proteins. Still, identification of active acid and subjected to centrifugation at 11000 rpm for 5peptides derived in vivo from cytochrome c oxi- min. Supernatant was immediately injected into the size-

dase,51

immunoglobulins,30,5254

albumins,55

fi- exclusion column.brinogen,56 and other functional proteins30,57 allows

us to conclude that the regulatory role of hemoglo-Size-Exclusion Chromatography

bin-derived peptides should not be considered aThe samples were separated on Sephadex G-25 sf (Phar-unique, isolated phenomenon. On the contrary, it ismacia, Sweden) column (2.5 1 85 cm) equilibrated withonly one of the components (although an important0.1Macetic acid. Elution was carried out at 120 mL/h,one) of the general system of peptidergic regulationwith detection at wavelengths 206/280 nm. The fractionsof tissue homeostasis. One can even suggest thatcorresponding to the zone of elution of substances withthis system is phylogenetically more ancient than0.54.0 kDa molecular masses about (Figure 2) were

the endocrine and the nervous system since the lattercollected and lyophilized.

deal with higher level of regulation, involving sev-

eral types of tissues, several organs, and the wholeRP-HPLC Separationorganism.

The material obtained after size-exclusion chromatogra-

phy was dissolved in 250 mL of 0.1% solution of triflour-

oacetic acid (TFA) in water and subjected to separation

on Nucleosil 120/5m C8 cartridge (4.0 1 250 mm; Mash-MATERIALS AND METHODSerey-Nagel, Germany), equilibrated with buffer A (0.1%

TFA in water). The column was washed for 5 min by

buffer A, then the elution was performed for 60 min byPreparation of Human Erythrocyteslinear gradient of acetonitril from 0 to 60% of buffer B

(0.1% TFA, 80% acetonitril solution in water) at 0.75Peripheral venous blood was obtained from 10 healthy

mL/min. The detection was carried out at 226 nm.volunteers [7 men and 3 women with different blood

groups A(II), B(III), AB(IV); aged 2035 years] by

veinpuncture after confirmation of their health status by Isolation of the PeptidesResearch Haematological Centre, Russian Academy of

The peaks corresponding to homogeneous substancesMedical Sciences.were collected as shown in Figure 3, lyophilized andBlood samples (25 mL) were placed into the tubessubjected to separation on Nucleosil 120/5m C18 cartridgecontaining citrate buffer (to final citrate concentration(4.0 1 250 mm) in linear acetonitril gradient from 8of 0.25%). The cells were separated from plasma byto 40%.centrifugation at 1000 rpm for 15 min at 0C. The ob-

tained pellet (1012 billions of cells, 99% of erythro-

cytes) was washed four times by buffer A (0.025M Amino Acid SequencesNaH2PO4 containing 0.1M NaCl, pH 7.2). Both lysate

and supernatant were prepared from the same batch of Amino acid sequences of isolated peptides were deter-

mined in the gas-phase sequencer Applied Biosystemscells.

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447A (Foster City, USA). Estimation of the peptide con- Mikhaleva, I. I., Ivanov, V. T., Kokoz, Yu. M., Alek-

seev, A. E., Korystova, A. F., Sukhova, G. S., Eme-tent was made from the sequencing data.

lyanova, T. G. & Usenko, A. B. (1994) Bioorgan.

Khim. 20, 677702 (in Russian).Molecular Graphic 17. Garreau, I., Zhao, Q., Pejoan, C., Cupo, A. & Piot,

J.-M. (1995) Neuropeptides 28, 243250.The x-ray coordinates of the human deoxyhemoglobin18. Zhao, Q., Sannier, F., Garreau, I., Guillochon, D. &(Brookhaven PDP code 4hhb) solved at 1.74 A resolu-

Piot, J.-M. (1994) Biochem. Biophys. Res. Commun.tion 58 have been used for the analysis using CHAIN pro-

204, 216223.gram.59 Figures 6 and 7 were prepared by SETOR pro-19. Barkhudaryan, N. A., Kellermann, J., Galoyan,gram.60

A. A. & Lottspeich, F. (1993) FEBS Lett. 329, 215

218.

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