Contact inhibition within hemoglobin S polymer by thiol reagents

82

Biochimica et Biophvsica Acta 874 (1986) 82-89

Elsevier BBA32636

Contact inhibition within hemoglobin S polymer by thiol reagents J. Caburi-Martin, M.C. Garel, C. Domenget, C. Prehu and Y. Beuzard INSERM U-91, Hg~pitalttenri Mondor, 94010 Cr~teil (France)

(Received 11 June 1986) Key words: Sicklecell: HemoglobinS: Anti-sickling activity: Thiol reagent: Chemical modification: (Human) N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted l]93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + H b S and HbA + H b S NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid aA/3A/3S, aA/3A'NEM/3S or ¢][A/3A/3S,NEM respectively and 25% parental molecules as confirmed by ion-exchange H P L C performed in anaerobic conditions. Within the hybrid molecule, /3A or /3A,NEM chain has to be present in the a/3 dimer located in trans to the dimer which contains the only 136 valine residue participating in intermolecular contacts (dimer in cis), while/3s or /3S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + H b S and 20% for HbA + l-IbS NEM mixtures compared to HbA + H b S mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of H b S + Hb Cr6teii/389 Ser -~ Asn. The/389 residue in trans next to the cysteine/393 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the a chains.

Introduction Formation of fibers in the red cells of individuals with sickle cell disease depends upon the single amino acid substitution, /36 Glu ---, Val, but many portions of the surface of the hemoglobin S (HbS) molecule participate in the intermolecular contacts of the fibers. The multiplicity of surface residues involved in contacts is related to the complexity of the fiber structure which places HbS molecules in unequivalent positions. Several models have been proposed for the structure of the fibers of deoxyHbS [1,2]. Of these, the 14-stranded structure proposed by Dykes et al. Correspondence: Dr. Y. Beuzard, INSERM U.91, H&pital Henri Mondor, 94010 Crfiteil, France.

[3] from direct image reconstruction of the fiber itself is the most probable, and was composed of a basic unit, the double-stranded structure as described for HbS crystals by Wishner et al. [4]. In such a multi-stranded helix at least three classes of intermolecular contacts can be distinguished: (1) the axial contacts between molecules within a single strand; (2) the lateral contacts between filaments within a double strand, including the contacts formed by one of the two /36 Val residues per tetramer; (3) a much more diverse group of contacts, those holding the double strands together to form the multi-stranded helix. The two first types of contacts, the 'internal contacts', are well documented by Wishner et al. [4] in the crystals of HbS. The third class of contacts, the 'external contacts', between double

0167-4838/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

83 strands are numerous and less well characterized because they must be different in the crystal, in which the double strands are packed linearly, from those present in the fibers, in which double strands are twisted [5]. As reported by Wishner et al. [4], only one of the two /36 Val residues participates within the double strand in lateral contacts and is called 'donor site'. Consequently the effect of an antisickling agent will depend on its position relative to this primary contact. When a reagent or a structural change is located on the aft dimer which provides the/36 Val for a contact, its position is said to be 'cis' to the dimer bearing the/36 Val donor site. When the compound or the structural change is located on the opposite dimer, its position is 'trans' to the dimer bearing the f16 Val donor site. The trans /3 chain bears the complementary, 'receptor' site of the/36 Val, formed by a hydrophobic pocket at the EF corner of the fl chain including Phe fl85 and Leu fl88. The/36 Val donor site complementary to the receptor site is provided by a tetramer of the other strand of the double filament. In an attempt to prevent polymerization of deoxyHbS various chemicals have been tested [6]. They can be classified into four classes: (1) those that inhibit contacts in the fiber; (2) those that increase the oxygen affinity of hemoglobin and consequently decrease the proportion of HbS in the 'T' conformation, which is the structure able to polymerize; (3) those that act on the membrane function and prevent the dehydration of sickle cells, and (4) those that act at the gene level and increase fetal hemoglobin and the production of HbF-containing cells. Recently, results obtained in our laboratory have shown that the antisickling effect of numerous thiol reagents varied widely from one compound to the other and was related to the extent of the increased oxygen affinity and to a direct inhibition of intermolecular contacts [7]. An increased solubility of modified deoxyHbS was shown [7] as well as an increase in the delay time before polymerization [8]. The purpose of the study reported here was to define the relative contribution of the two fl chains modified by N-ethylmaleimide, in cis or in trans, in the inhibition of HbS polymerization. The fl93

cysteine residue in cis is far away from internal contacts and this residue in trans is close to the receptor site. Hemoglobin modified by N-ethylmaleimide has a high oxygen affinity related to a destabilization of the T conformation and especially of the Cterminal portion of the fl chain, as reported in the crystallographic studies of Perutz et al. [9]. As a comparison, the high oxygen affinity variant, Hb Cr~teil f189(Fs) Ser ~ Asn, has been studied in its interaction with HbS because the high oxygen affinity of this variant is related, as in the case of Hb modified by N-ethylmaleimide, to a destabilization of the C-terminal portion of the fi chain [10], and the residue Ser fl89, close to the cysteine fl93 residue, has been suggested by Wishner et al. [4] to be an intermolecular contact within the double strand in crystals. Materials and Methods

Materials N-Ethylmaleimide and iodoacetamide were purchased from Sigma. Carrier ampholytes (ampholines pH 6-8 and pH 7-9) for isoelectric focusing were obtained from LKB. Acrylamide and bisacrylamide were from Fluka; ammonium persulfate was from Merck; N,N,N ",N '-tetramethylethylenediamine was from Sigma. All chemicals were of analytical grade. Preparation and chemical modification of hemoglobin Hemoglobin A and HbS used for these studies were purified by chromatography on DEAE-cellulose as described by Abraham et al. [11]. Purified H b was chemically modified as follows: hemoglobin (0.2 mM) was subsequently reacted in 0.1 M sodium phosphate buffer, pH 7.4, with N-ethylmaleimide or iodoacetamide with a molar ratio c o m p o u n d / H b (SH) of 2 for 1 h at 37°C in a rotary shaker. At the end of the incubation the mixture was concentrated under vacuum and dialyzed against 0.15 M potassium phosphate buffer, pH 7.35, in order to eliminate excess thiol reagent. The complete modification of hemoglobin by the reagents was checked by isoelectric focusing on slabs of acrylamide gels performed as previously described [12]. Hemoglobin Cr6teil fl89 Ser --* Asn

84

was separated from HbA by DEAE-Sephadex chromatography using 0.05 M Tris-HC1 buffer, pH 7.75 [13]. Polymerization studies

Solubility measurements (C,a,) were performed by the ultracentrifuge assay under conditions used by Benesch et al. [14]. For mixtures of HbS and non-HbS, the hemoglobins were first mixed in air, in the proportion about of 50% HbS and 50% non-HbS. Then, the mixtures were deoxygenated and ultracentrifuged after a 2 h incubation at 30°C. After centrifugation the total hemoglobin concentrations of the supernatant (C~at) and the pellet were determined as cyanmethemoglobin [15]. The proportion of HbS and non-HbS in the supernatant was determined by isoelectric focusing on slabs of acrylamide gels, as previously described [16]. Hybrid study

The proportion of the asymmetrical hybrids and O/2/3A'NEM/3 S w a s determined by ion-exchange HPLC according to the method described by Anbari et al. [17], modified as follows: Aquapore 10 tt CX300 Brownlee Column 4.6 × 100 mm equilibrated in 0.084 M Bistris buffer, pH 6.58, containing sodium dithionite (3 raM). A linear sodium chloride gradient was used to separate hemoglobin fractions. The flow rate was 1.5 m l / m i n . Buffers were deoxygenated with argon before the addition of sodium dithionite and were maintained under argon during chromatography. Concentrated solutions of mixtures of hemoglobins ( ~ 10 g / d l ) were deoxygenated and maintained under argon for various periods of time, up to 5 h, before chromatographic separation. Dilutions were made anaerobically just prior to each experiment and an aliquot ( = 10 ttg Hb) was anaerobically transferred to the column. O{2/3A/3 S, O~2/~A/3S'NEM

Results

Effect of N-ethylmaleimide and iodoacetamide on the polymerization of H b S

In our preliminary experiments [7] we indicated that two thiol reagents, N-ethylmaleirnide and iodoacetamide, increased the solubility of deoxyHbS when linked to cysteine fl93 residues

TABLE I EFFECT OF N-ETHYLMALEIMIDE AND IODOACETAMIDE ON THE SOLUBILITY OF HbS Hemoglobin

Hemoglobin concentration (g/dl) initial

supernatant

pellet

(C,~t) HbS HbS NEM HbS IAA

26.4 25.9 26.5

15.9 21.3 18.3

49.1 50.1 49

(Table I). This observed increase in the solubility indicates that thiol reagents inhibit directly intermolecular contacts in the fiber. Only N-ethylmaleimide was studied in its cis or trans effect because of its higher inhibitory effect on the solubility of deoxyHbS. When the two hemoglobins HbA and HbS are mixed in the presence of oxygen, the rapid dissociation of oxyHb tetramers into dimers and the random reassociation into tetramers give rise to the presence in the mixture of 25% HbA, 25% HbS and 50% asymmetrical hybrid tetramer ~2fiAfi s [18}'. In the mixture of HbA modified by N-ethylmaleimide (HbA NEM) + HbS the hybrid tetramer OI2/3A'NEM/3S contains N-ethylmaleimide trans to the/36 Val contact. In the mixture of HbA + HbS modified by N-ethylmaleimide (HbSNEM), the hybrid t e t r a m e r 0~2/3A/3S'NEM contains N-ethylmaleimide cis to the /36 Val residue and the HbS NEM tetramer contains the reagent cis + trans to the fi6 Val contact. Results reported in Table II show that when N-ethylmaleimide is located trans to the /36 Val, i.e. in the mixture of HbA N~M + HbS, only a slight increase in the solubility can be observed compared to the effect of unmodified HbA in the mixture of HbA + HbS. In contrast, when N-ethylmaleimide is located cis and cis + trans in the mixture of HbA + HbS NEM, a marked increase in the solubility is observed. These results show that the presence of N-ethylmaleimide in the trans position has little effect on the polymerization of HbS. So, the presence of N-ethylmaleimide in the cis position must be responsible for most of the solubilizing effect observed when the two/3 chains are modified.

85

T A B L E II SOLUBILITY OF HbS IN M I X T U R E S O F HbS A N D HbA, M O D I F I E D OR N O T W I T H N - E T H Y L M A L E I M I D E Hemoglobins are mixed in the presence of oxygen in order to produce asymmetrical hemoglobin tetramers. Hemoglobin mixtures

Hybrids present

Hemoglobin concentration (g/dl) initial

supernatant

(c~a,) A+S

o~2a/3A,Ss

31.4 31.6

25.6 25.6

A NEM + S

Ot2A/~A'NEM/es

31.5 31.6

26.4 26.7

A + S NEM

a~/9 A,8s'NEM

34.0 34.1

30.6 30.7

Analysis of Hb distribution in the supernatant after centrifugation Isoelectric focusing of mixtures of HbA and HbS, modified or not with N-ethylmaleimide, allowed quantification of each hemoglobin fraction in the supernatant after centrifugation. Table III summarizes the proportions of each hemoglobin fraction in the different mixtures. In various mixtures of unmodified or modified HbA + HbS there is a reduced proportion of HbS in the supernatant in comparison to the proportion of HbS in the initial solution. The small differences in the proportions of hemoglobins may be related to the accuracy of densitometry of the gel.

Study of asymmetrical hybrid hemoglobins To determine whether the binding of N-ethylmaleimide to/3A or to ,ss chain could change the proportion of the hybrid hemoglobin, the separation of hybrid hemoglobins from parental molecular species was obtained by HPLC of hemoglobin mixtures in anaerobic conditions. The hemoglobin molecules were mixed in the presence of Oz before deoxygenation. The results shown in Fig. 1 indicate that in the absence of oxygen the O~2/3A,8s hybrid formed 50% of the hemoglobin species, HbA being 25% and HbS 25%. A similar result was obtained with hybrids a2,sA,8s'NEM (45%) and a2,SA'NEM,8s (48%). These results indicated that the hybrid molecule in the conditions used was the main hemoglobin species in all mixtures studied and was present in similar proportions. Preincubation of deoxygenated mixtures for up to 5 h did not change the proportion of hybrids. Interaction of Hb Crbteil ,889 Ser --* Asn with HbS In order to study the interaction of Hb Cr6teil with HbS, mixtures of hemoglobins were made in the oxy form and then deoxygenated to allow the formation of hybrid molecules a~,8creteil/3s. In such a hybrid the mutation /389 Ser ~ Asn is located trans to the ,86 Val contact. Results of solubility studies of mixtures composed of 50% Hb Cr6teil + 50% HbS compared to mixtures made of 50% HbA + 50% HbS show that HbA and Hb Cr~teil interact similarly with HbS (Table IV).

g T A B L E Ill D I S T R I B U T I O N OF H E M O G L O B I N S IN T H E SUPERNATANT AFTER CENTRIFUGATION

HYBRIDS

w

~2l~ A 8,s

Hemoglobin

Initial mixture (%)

Supernatant (%)

mixtures

HbA

HbS

HbA

HbS

A+S

54 55

46 45

60 60

40 40

A NEM + S

50 52

50 48

63 64

37 36

A + S NEM

48 47

52 53

57 55

43 45

~2flA~ s~M

m,, Q

o

2'o

;o ELUTION TIME

30

(minutes)

Fig. 1. Anaerobic HPLC separation of molecular species present in a mixture of HbA and HbS ( ) and of HbA and HbS NEM ( . . . . . . ).

86 TABLE IV EFFECT OF fl89 MUTATION ON THE SOLUBILITY OF HbS Hemoglobin mixtures were made in the presence of oxygen in order to produce asymmetricalhybrid molecules. Hemoglobin mixtures

Hybrids present

Hemoglobinconcentration (g/dl) initial super- pellet natant

S

none

29.4 29.3

16.4 16.5

50,2 50.1

A +S

aaB ABs

33.3 33.0

24.1 23.9

50,3 49.8

Hb Cr6teil+ S

a~BCr~teiIBs

33.9 34.0

24.8 25.0

49.3 48,7

Discussion When N-ethylmaleimide is linked to cysteine /393, the increased solubility of HbS [7] and the increased delay time [8] suggest a direct inhibition of intermolecular contacts in the fibers. In order to define further the contacts implicated in this inhibitory effect, one approach consists of studying inhibition of polymerization of HbS when the reagent is located in the a/3 dimer present in cis or trans to the/36 Val residue. The results presented in this paper provide evidence that N-ethylmaleimide linked to cysteine /393 of Hb inhibits polymerization of HbS when located cis to the/36 Val. This assumption is correct only if the proportion of the asymmetrical hybrid remains similar in the various mixtures of hemoglobins we have studied. As shown by HPLC studies performed in anaerobic conditions, asymmetrical hybrid tetramers represent close to 50% of the total Hb molecules in various mixtures: A + S (50%), A + S NE~ (45%) and ANEM+ S (48%). These values suggest that dissociation of hybrid molecules containing one N-ethylmaleimide-reacted fl-chain into dimers and their preferential reassociation into parental tetramers was probably slight in the experimental conditions used for Csat determination

performed at high Hb concentration (30-34 g/dl), in spite of an increased dissociation constant of N-ethylmaleimide-reacted Hb [19]. Residue cysteine/393 does not participate itself directly in intermolecular contacts, as reported by Wishner et al. [4] for crystals and by Edelstein for fibers [5] of deoxy HbS. Nevertheless, cysteine /393 is located not far from residues which are implicated either in lateral contacts between two/3 subunits within double strands (internal contacts) or in contacts between double strands (external contacts) involving essentially a chain residues (Fig. 2). Residues close to Cys/393 which are implicated in lateral contacts within the double strand of the HbS polymer are located t r a n s to the /~6 Val. They involve residues of the EF corner and the F helix of the/3 chain. In the F helix, two residues, Phe /385 and Leu fl88, are of particular interest because they participate in the primary hydrophobic contacts complementary of the f16 Val residue (Fig. 2). Because of the slight inhibitory effect of N-ethylmaleimide on the polymerization of Hb S when located t r a n s to the/36 Val residue, it can be postulated that these hydrophobic contacts are little perturbed. In the vicinity of this acceptor pocket other residues such as /387, /389, /390 and /391, located not far from cysteine/393, have also been reported to be implicated in t r a n s contacts in the crystals of deoxyHbS. By using naturally occurring mutant hemoglobins having a single amino acid change of one of these residues, Nagel et al. have shown that altering residue fl87 in HbD lbadan /387 T h r - ~ Lys decreases the tendency toward gel formation [20], while altering residue f190 in Hb Agenoni f190 Glu --. Lys does not modify the gelation of HbS [21]. In the same manner and in order to confirm or not the possible contact involving Ser fl89 residue we have studied the interaction of Hb Cr6teil fl89 Ser Ash with HbS. As reported by Arnone et al. [10], crystallographic studies of Hb Cr~teil show that replacement of serine /389 by asparagine causes severe disordering of the COOH-terminal tetrapeptide of the/3 chain and of Cys fl93 in the deoxy conformation. The two salt bridges associated with His /3146, i.e. the intrasubunit ionic bond between the imidazole group of His/3146 and Asp/394 and the

87

"'Trans" CO Corner or

"1

!

['Cis" 893 !

Cis ~6 Val "Trans receptor

j

IlUNI

D~I

I

#

" Cis" dimer

Fig. 2. Amino acid residues implicated in contacts in HbS polymer (white circles with black numbers) and suggested to be modified by the binding of N-ethylmaleimideto cysteine fl93. This diagram is a modification of that published by Dickerson and Geis [27].

intersubunit contact between the COOH-terminal carboxyl group of His /3146 and Lys a40, are broken. Therefore it was of interest to test this variant in its interaction with HbS because the location of the substitution was proposed by Wishner to be a direct contact in the crystals and the perturbation of the ionic bonds involving His B146 is comparable to the perturbation observed by Perutz in Hb modified by N-ethylmaleimide [9l. The results reported in Table IV show that Hb Cr&eil does not change the solubility of HbS in the mixture more than does HbA. In spite of the difference in structure between a serine residue and an asparagine residue it can be postulated that residue fl89 does not participate in direct contact in fibers of deoxyHbS. In addition perturbation of the terminal tetrapeptide of the /3 chain does not inhibit contacts in the polymer

when located trans to the fl6 Val. These results can be compared to those obtained by Bookchin and Nagel [22] on gelation of HbS in a mixture of HbS + half liganded hybrid tetramers of H b A in which/3 chains were fixed in the cyanmet (CNmet) liganded state. Hybrids formed in the mixture (a2A/3A-CNmet/3S) contain one liganded /3 chain in the trans position which has an ' r ' tertiary structure even in the absence of oxygen. Because of identical gelation properties of this mixture and an H b A + HbS mixture, Bookchin and Nagel have postulated that polymerization of HbS is not affected when the trans fl chain is in an ' r ' conformation. Such results led us to suppose that the hydrophobic contact between fl6 Val and Phe fl85 and Leu fl88 is possible even if the trans/3 chain is in the ' r ' or ' t ' tertiary structure. Such results agree with the small inhibitory effect of N-ethylmaleimide when located trans to the /36 Val.

88

According to the nuclear magnetic resonance studies of Craescu et al. [23] this thiol reagent, when linked to cysteine/~93, disturbs the F helix as in the case of the T ~ R transition. Consequently small changes in the receptor site induced by the t ~ r modification do not modify the Phe 85 and Leu 88 contacts. The absence of an inhibitory effect of Hb Cr6teil on the polymerization of HbS can be compared to the non-inhibitory effect obtained with Hb Agenoni by Nagel et al. [21]. These two results show that neither the /389 residue nor the fi90 residue seems to be implicated in contacts in the fibers as predicted from crystals. In contrast, Lys /395, also located near cysteine 1~93, did not appear to be a contact point in the crystal structure, but its replacement by either an Asn in Hb Detroit or a Glu in H b N Baltimore reduces gelation of HbS [21]. It can be deduced from these results that the/~95 location and the two others, /~89 and /~90, represent true differences in this contact region between crystals and fibers. When we examine mixtures of HbA + HbS NEM, N-ethylmaleimide is located either in the cis position in the hybrid molecule a2A/~A/~ S'NEM or in the ^ At2 S,NEM cis and trans positions in the tetramer t~2p 2 The observed increase in the solubility of HbS in these mixtures suggests that the inhibitory effect of N-ethylmaleimide on the polymerization of HbS is essentially obtained when N-ethylmaleimide is located cis to the f16 Val as there is only a slight inhibition when located trans. As cysteine/~93 is located too far from Val f16, it was difficult to explain this cis inhibitory effect by a modification of the /~6 contact region. Analysing the contacts described by Wishner et al. [4] and those described by Edelstein [5] it appeared that a possible explanation of this cis inhibitory effect would be a modification of the CD corner of the opposite a chains located in the trans aft dimer (Fig. 2). This region of the a chains is presumed to be involved in contacts between double strands, especially residues a45, a46 and a47. As in the case of Hb Cr~teil, the breaking of the salt bridge between His/~146 and Asp/~94 when N-ethylmaleimide is linked to cysteine/~93 must be accompanied by a rupture or a destabilization of the salt bridge between His /~146 and Lys a40. Under these

conditions residues located in the same region as Lys a40 must be perturbed, especially the CD corner region of the a chain. Consequently with such a hypothesis the inhibitory effect of N-ethylmaleimide located cis to the f16 Val residue could be due to a destabilization of contacts between double strands involving mainly a chain residues. According to the nuclear magnetic resonance studies of Miura and Chien Ho [24] on cross-linked asymmetrically modified hemoglobin A, the effect of N-ethylmaleimide reacted with the /~ subunit on the a subunit proximal histidine located in the same dimer (cis) is less marked than on the a subunit located in the other dimer (trans). Such a result may be attributed to different interactions between the two different types of dimers and agrees with our hypothesis of modifications at the C D corner of the opposite a chains (trans) when N-ethylmaleimide is linked to cysteine /~93 of HbS. Recently a refined version of the crystal structure of deoxyHbS has been reported by Padlan and Love [25,26]. Their results confirm those obtained by Wishner [4] but with some modifications. In this refined model residue Ser/?89 is not implicated in contacts in the crystals of deoxyHbS. Such a result is in agreement with the data obtained in this study with Hb Cr~teil. In addition, Lys 144/~ forms a contact within the polymer when located cis to the /~6 Val residue. Such a contact could also be inhibited when Nethylmaleimide is located cis to the/~6 Val donor site. In conclusion, the results presented in this paper show that inhibition of polymerization of HbS by a thiol reagent linked to cysteine /~93 is mainly due to a cis inhibitory effect.

Acknowledgments The authors thank Professor S. Edelstein for helpful discussion, and Mrs. A.M. Dulac and M. Tassier for preparation of the manuscript. This work was supported by research grants from the Institut National de la Sant6 et de la Recherche M~dicale (PRC 121038), the Delegation G~n~rale /~ la Recherche Scientifique et technique (80E0873) and I N S E R M / S A N O F I Recherche (81003).

89

References l Finch, J.T., Perutz, M.F., Bertles, J.F. and Dobler, J. (1973) Proc. Natl. Acad. Sci. USA 70, 718-722 2 Josephs, R., Jarosch, H.S. and Edelstein, S.J. (1976) J. Mol. Biol. 102, 409-426 3 Dykes, G.E., Crepeau, R.H. and Edelstein, S.J. (1979) J. Mok Biol. 130, 451-472 4 Wishner, B.C., Hanson, J.C,, Ringle, W.M. and Love, W.E. (1975) in Proceedings of the Symposium on Molecular and Cellular Aspects of Sickle Cell Disease; Department of Health, Education and Welfare, Publication No. 76-1007, pp. 1-29, Dallas 5 Edelstein, S.J. (1981) J. Mol. Biol. 150, 557-575 6 Noguchi. C.T. and Schechter, A.N. (1985) Annu. Rev, Biophys. Biophys. Chem. 14, 239-263 7 Garek M.C., Domenget, C., Galacteros, F., Caburi-Martin, J. and Beuzard, Y. (1984) Mol, Pharmacol. 26, 559-565 8 Domenget, C,, Garel, M.C., Rhoda, M.D., Caburi-Martin, J., Galacteros, F. and Beuzard, Y. (1985) Biochim. Biophys. Acta 830, 71-79 9 Perutz, M.F., Muirhead, H., Mazzarella, L., Crowther, R.A., Greer, J. and Kilmartin, J.V. (1969) Nature 222, 1240-1243 10 Arnone, A., Thillet, J. and Rosa, J. (1981) J, Biol. Chem. 256, 8545-8552 11 Abraham, E.C., Reese, A., Stallings, M, and Huisman, T.H.J. (1976-1977) Hemoglobin 1, 27-44 12 Basset, P., Beuzard, Y., Garel, M.C. and Rosa, J. (1978) Blood 51,971-982 13 Thillet, J., Blouquit, Y., Garel, M.C., Dreyfus, B., Reyes, F., Cohen-Solal, M., Beuzard, Y. and Rosa, J. (1976) J. Mol. Med. 1, 135-150

14 Benesch, R.E., Kwong, S., Edalji, R. and Benesch, R. (1979) J. Biol. Chem, 254, 8169-8172 15 Drabkin, D.L, (1949) Arch. Biochem. 21,224-234 16 Dubart, A., Goossens, M., Beuzard, Y,, Monplaisir, N,, Testa, U., Basset, P. and Rosa, J. (1980) Blood 56, 1092-1099 17 Anbari, M., Adachi, K., Yuip, C. and Asakura, T. (1985) J. Biol. Chem. 260, 15522-15525 18 Park, C.M. (1973) Ann. N.Y. Acad. Sci. 209, 237-257 19 Turner, B.W., Pettigrew, D.W. and Ackers, G K . (1982) Methods Enzymol. 76, 596-628 20 Nagel, R.L., Bookchin, R.M., Johnson, J., Labie, D., Wajcman, H., Isaac-Sodeye, W.A., Honig, G.R., Schiliro, G., Crookston, J.H. and Matsumoto, K. (1979) Proc. Natl, Acad. Sci. USA 76, 670-672 21 Nagel, R.L., Johnson, J., Bookchin, R.M., Garel, M.C., Rosa, J., Schiliro. G., Wajcman, H., Labie, D., Moo-Penn, W. and Castro, O. (1980) Nature 283, 832-834 22 Bookchin, R.M. and Nagel, R.L. (1973) J. Mol. Biol. 76, 233-239 23 Craescu, C.T., Mispelter, J., Schaeffer, C. and Beuzard, Y. (1985) J. Biol. Chem. 260, 15616-15622 24 Miura, S. and Ho, C. (1984) Biochemistry 23, 2492-2499 25 Padlan, E.A. and Love, W.E. (1985) J. Biol. Chem. 260, 8272-8279 26 Padlan, E.A. and Love, W.E. (1985) J. Biol. Chem. 260, 8280-8291 27 Dickerson, R.E. and Geis, I. (1983) Hemoglobin: Structure, Function, Evolution and Pathology, The Benjamin/ Cummings Publishing Company, Menlo Park, CA