Bimane fluorescent labels

201

Biochimica et Biophysica Acta, 6 2 2 ( 1 9 8 0 ) 2 0 1 - - 2 0 9 © Elsevier/North-Holland Biomedical Press

BBA 38399

BIMANE FLUORESCENT LABELS CHARACTERIZATION OF THE BIMANE LABELING OF HUMAN HEMOGLOBIN

N E C H A M A S. K O S O W E R a , . , G E R A L D L. N E W T O N a, E D W A R D M. K O S O W E R b , * * a n d H E L E N M. R A N N E Y a

a Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093 and b Department of Chemistry, University of California, San Diego, La Jolla, CA 92093 (U.S.A.) (Received July 20th, 1979)

Key words: Hemoglobin; Bimane labeling; Oxygen affinity; Electrophoretic mobility

Summary The products of the bimane labeling (using a monobromobimane, a dibromobimane and a quaternary bromobimane) of hemoglobin are characterized. Peptide mapping identifies cysteine-~93 as the reactive thiol site. Electrophoretic mobility of hemoglobin varies with the label used, that of monobromobimane-labeled hemoglobin being unaltered, while dibromobimane- and trimethylammoniobromobimane-labeled hemoglobin exhibit changes. The oxygen affinity of labeled hemoglobin is changed from that of hemoglobin. Deoxyhemoglobin is subtantially less reactive towards monobromobimane than oxyhemoglobin. Bimane-labeled hemoglobin is more easily denatured on heating than unlabeled hemoglobin. Possible uses for bimane labels in the study of protein properties are pointed out. Bimane labeling agents are derivatives of 3,4,6,7-tetramethyl-l,5-diazabicyclo[ 3.3.0 ]-octa-3,6-diene-2,8-dione 9,10-

dioxa-syn-(methyl,methyl)bimane). Introduction We have described a useful new class of fluorescent labeling agents, the bromobimanes, and their application to human red cells [1]. The highly fluores* Permanent address: Department of Human Genetics, Sackler S c h o o l of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel. * * Permanent addresses: D e p a r t m e n t of Chemistry, Tel-Aviv University, Ramat-Aviv, Tel-Aviv, Israel and D e p a r t m e n t of Chemistry, State University of New York, S t o n y Brook, N.Y. 11794, U.S.A. Abbreviations: Hb, hemoglobin; SDS, s o d i u m dodecyl sulfate.

202 cent globin derived from the hemoglobin of such treated cells was shown to carry the label only on the fl-chain. In the present report, we show that cysteine-~93 is the labeled thiol group, a concomitant being that labeled protein may be subjected to the usual degradative and mapping procedures. Certain aspects of hemoglobin behavior are examined: (1) the reactivity of the thiol groups of deoxyhemoglobin in comparison to those of oxyhemoglobin; (2) the reaction of bimane-labeled hemoglobin with oxygen; (3) the electrophoretic behavior of bimane-labeled hemoglobin; (4) the heat stability of bimane-labeled hemoglobin, and (5) variations in product properties arising from the chemical differences among the bimane labels. These studies extend the utility of 9.10-dioxabimane labeling (briefly: bimane labeling) of cells and proteins and point to some interesting and useful ways in which the properties of the bimanes may be exploited. The nomenclature and structures for bimanes and their derivatives are given elsewhere [2,3]. Materials and Methods

Hemoglobin. Blood was obtained from normal individuals and heparin added to prevent coagulation. After centrifugation, the buffy coat was removed and the red cells washed twice with 0.15 M NaC1. The packed red cells were mixed with 2 vols. of water and 0.5 vol. toluene, shaken for several minutes, then centrifuged. The hemoglobin solution (the middle layer) was siphoned off, recentrifuged at 22 000 X g for 20 min, and then dialyzed against various buffers. Deoxyhemoglobin. Hemoglobin was deoxygenated by passing nitrogen over swirled solutions of the protein, the process being followed spectroscopically in a cell attached to the gas-exchange apparatus. The deoxygenation was adjudged complete when the spectrum in the visible range corresponded to the well-known spectrum for deoxyhemoglobin [4]. Bromobimanes and labeling procedure. The monobromo-, the dibromo- and the m o n o b r o m o quaternary bimanes were disolved and utilized as previously described [ 1 ] (the compounds are available from Calbiochem-Behring, La Jolla, CA, U.S.A.). Usually, 10--20 pl of m o n o b r o m o b i m a n e or dibromobimane solution (50 mM) in acetonitrile, or 100--200 pl of aqueous 5 mM quaternary bromobimane, were used per ml of 0.25 mM hemoglobin solution. Electrophoresis. Starch gel or cellulose acetate strips were utilized to examine the behavior of hemoglobin solutions [ 5]. SDS-acrylamide gels were used for examination of labeled globins [6]. Globin preparation and peptide analysis. Globin was isolated from hemoglobin by precipitation with HC1/acetone at --20°C [7]. The a- and H-chains were separated by fractionation of the globin on a CM-cellulose column [8]. The separated chains were passed through a desalting column, lyophilized, aminoethylated with ethyleneimine, and treated with trypsin for 12--24 h. Peptide separations were carried out on paper [9] or silica gel [10]. Electrophoresis was performed on two plates in parallel at pH 4.7 at 4°C for 8 h at 500 V in one dimension, followed by chromatography in the second dimension. Peptide maps were photographed under ultraviolet illumination to record the fluorescent peptides, then stained with ninhydrin and photographed under

203 ordinary light. Peptide spots could be readily located under ultraviolet illumination. The peptides were extracted, hydrolyzed with 6 N HC1 at 110°C for 22 h, and amino acid analysis was carried out on a Beckman amino acid analyser, Model 119 (although bimanes are stable at room temperature in strong acid, they are converted to smaller molecules by heating under these conditions) (Kosower, E.M'. and Pazhenchevsky, B., unpublished results). Oxygen binding curves. These were determined as previously described [4]. Thermal stability o f hemoglobins. This was measured by the method of Carrel a n d K a y [ 11 ]. Light absorption and emission measurements. Experiments were done as previously described [1], including the removal of traces of heme from globin solutions by treatment with H202 and subsequent addition of ascorbic acid. Heme interferes with the accuracy of the absorbance measurements of the bimane, and could reduce fluorescence quantum yields by energy transfer. Thiol analysis. Hemoglobin SH groups were measured as described elsewhere [12]. Results

After isolation of bimane-labeled globin from labeled hemoglobin, and separation into a- and /]-chains, the fluorescent ~-chains were degraded with trypsin and the resulting fluorescent peptide mixture mapped by a combination of electrophoresis and chromatography. Only five significantly fluorescent spots were seen under ultraviolet illumination. Two of the spots could be assigned to tryptophan-containing peptides. One spot proved to be non-peptide, yielding only ammonia in the amino acid analysis. Two spots which exhibited the characteristic bimane fluorescence, proved to be tryptic peptide 10(T/~10) and tryptic peptide 10--11 (Tl310--11) by amino acid analysis. Peptide maps of trypsin-degraded fl-chain of globin derived from labeling at pH 9.8 were identical to those found after labeling at pH 7.4. There were only minor differences between maps made from peptides from monobromobimanelabeled Hb and those from peptides made from dibromobimane-labeled Hb. The fluorescent peptide map (derived from monobromobimane-labeled Hb) is compared to the map after staining in Fig. 1A and B. The electrophoretic mobility of monobromobimane-labeled hemoglobin was similar to that of unlabeled hemoglobin, but the mobility of trimethylammoniobromobimane-labeled hemoglobin was lower, with gels exhibiting two bands. The faster moving band corresponded to Hb A and the slower moving band to a hemoglobin with more positive charge. The Hb A2 band was diminished, and there was an ill-defined slower moving band behind Hb A2. The dibromobimanelabeled hemoglobin presents a more complex picture after electrophoresis, with three bands: one slower, one faster, and one (the major one) similar in mobility to that of Hb A. The globins derived from all three bands are fluorescent. The Hb A2 band is also diminished after dibromobimane labeling. The electrophoretic patterns are illustrated in Fig. 2. Electrophoresis of bimane-labeled globin derived from bimane-labeled hemoglobin on SDS-acrylamide gel shows that no augmentation in the quantity of 'dimer' (32 000) seen in control gels for globin from Hb A is produced by

204

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labeling with either monobromo- or dibromobimane. The major band seen in all samples appears at 16 000 daltons (Fig. 3). The rate of reaction of oxyhemoglobin with monobromobimane is illustrated in Fig. 4. The fluorescence intensity of the isolated globin increases as the number of SH groups decrease. Reaction is substantially complete within 40 min. The rate of reaction of deoxyhemoglobin with monobromobimane is shown in Fig. 5, along with a parallel series of measurements for the reaction of mono-

205

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Fig. 2. P a t t e r n s o b t a i n e d a f t e r e l e c t r o p h o r e s i s o f b i m a n e - l a b e l e d p r o t e i n s . H e m o l y s a t e s w e r e r e a c t e d w i t h m o n o b r o m o b i m a n e ( m B B r ) , d i b r o m o b i m a n e ( b B B r ) o r q u a t e r n a r y b r o m o b i m a n e ( q B B r ) (2 m o l / m o l H b in cell l y s a t e ) f o r 6 0 m i n a t 2 3 ° C . E l e c t r o p h o r e s i s w a s c a r r i e d o u t in v e r t i c a l s t a r c h gel a t p H 8 . 6 . T h e labels o n t h e p h o t o g r a p h i n d i c a t e t h e r e a g e n t u s e d f o r r e a c t i o n w i t h h e m o g l o b i n . O t h e r n o t a t i o n s : H b A , n o n - t r e a t e d , n o r m a l h e m o g l o b i n ; C o n t r o l , h e m o g l o b i n s o l u t i o n t r e a t e d w i t h 1% a c e t o n i t r i l e ; N H P , n o n hemoglobin protein.

bromobimane with oxyhemoglobin. The rate of reaction of the deoxy form is clearly much lower than that of the oxygenated protein, with only 30% reacted after 60 min. Thic] ~ o u p analysis of the deoxyhemoglobin after 60 min reaction showed that 0.50--0.60 SH group per hemoglobin molecule had reacted.

Fig. 3. P r o t e i n p a t t e r n s a f t e r e l e c t r o p h o r e s i s o f g l o b i n s i s o l a t e d f r o m b i m a n e - l a b e l e d h e m o g l o b i n . H e m o globin was reacted with monobromobimane (mBBr) or dibromobimane (bBBr) (2--4 tool/tool Hb), the g l o b i n i s o l a t e d , d i s s o l v e d in 1% S D S s o l u t i o n , p H 7 . 0 , a n d e l e c t r o p h o r e s i s p e r f o r m e d o n 1 2 % a c r y l a m i d e gel. F l u o r e s c e n c e o f g l o b i n b a n d s is s h o w n o n t h e l e f t side; p r o t e i n b a n d s s t a i n e d w i t h C o o m a s s i e b l u e a r e s h o w n o n t h e r i g h t side. B a n d l a b e l s r e p r e s e n t g l o b i n s i s o l a t e d f r o m h e m o g l o b i n : 1, c o n t r o l ; 2, m B B r t r e a t e d H b (4 t o o l / t o o l Hb); 3, b B B r - t r e a t e d H b (2 t o o l / t o o l H b ) ; 4, b B B r - t r e a t e d H b (4 t o o l / t o o l H b ) .

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TIME OF REACTION (min) Fig. 4. Parallelism in a p p e a r a n c e of f l u o r e s c e n c e in globin a n d d i s a p p e a r a n c e of thiol grot~ps of h e m o g l o b i n on r e a c t i o n of h e m o g l o b i n w i t h m o n o b r o m o b i m a n e ( m B B r ) . H e m o g l o b i n s o l u t i o n w a s m i x e d w i t h m B B r , yielding a r e a c t i o n s o l u t i o n c o n t a i n i n g 0,5 m M H b , 1.5 m M m B B r , a n d 0.01 M p h o s p h a t e b u f f e r a t p H 7.4. T h e s o l u t i o n s w e r e i n c u b a t e d at 2 3 ° C ; a l i q u o t s w e r e r e m o v e d at intervals, m i x e d w i t h 0.2 vol. o f 10 m M g l U t a t h i o n e ( G S H ) , a n d d i a l y z e d t o r e m o v e excess G S H a n d t h e b i m a n e - l a b e l e d G S H . T h e h e m o g l o b i n w a s a s s a y e d f o r SH g r o u p s a n d t h e globin i s o l a t e d f o r f l u o r e s c e n c e m e a s u r e m e n t s . ( T h e r e a c t i o n w i t h G S H is fast, b u t n o t i n s t a n t a n e o u s , so t h a t t h e c u r v e i l l u s t r a t e d s h o w s a r a t e w h i c h is s o m e w h a t h i g h e r t h a n t h e a c t u a l r a t e . T h e t e c h n i q u e used for t h e e x p e r i m e n t i l l u s t r a t e d in Fig. 5 is m o r e accUrate w i t h r e s p e c t t o r a t e , b u t d o e s n o t p e r m i t t h e analysis of SH I~OUpS in h e m o g l o b i n b e f o r e precipit a t i o n o f globin), o, SH g r o u p s r e a c t e d ; e, f l u o r e s c e n c e i n t e n s i t y of globin i s o l a t e d f r o m r e a c t i o n of h e m o globin w i t h m B B r .

Fig. 5. R a t e o f r e a c t i o n o f d e o x y h e m o g l o b i n w i t h m o n o b r o m o b i m a n e ( m B B r ) . H e m o g l o b i n s o l u t i o n was d e o x y g e n a t e d in a t o n o m e t e r (visible s p e c t r u m ) . A d e o x y g e n a t e d s o l u t i o n of m B B r w a s a d d e d (3 tool m B B r / m o l H b ) w i t h a n air-tight s y r i n g e (50 m M m B B r in a c e t o n i t r i l e was d i l u t e d to 1 m M w i t h 0 . 0 1 M p h o s p h a t e b u f f e r ( p H 7.4) a n d d e o x y g e n a t e d w i t h N 2. Final p r o p o r t i o n of a c e t o n i t r i l e , 1.3%). T h e react i o n s o l u t i o n was i n c u b a t e d at 2 3 ° C ; a f t e r 60 rain, a s p e c t r u m s h o w e d only d e o x y h e m o g l o b i n . A l i q u o t s w e r e w i t h d r a w n t h r o u g h a syringe cap using a n i t r o g e n - f l u s h e d air-tight syringe, a n d a d d e d to HCI/ a c e t o n e a t - - 2 0 ° C , t h e p r e c i p i t a t e d globin w a s h e d t h r e e t i m e s w i t h cold a c e t o n e , dried, b l e a c h e d w i t h H 2 0 2 , a n d e x a m i n e d . T h e u p p e r c u r v e r e p r e s e n t s a r e a c t i o n of H b w i t h m B B r in t h e p r e s e n c e of air. T h e results are e x p r e s s e d in t e r m s of t h e p e r c e n t o f t h e m a x i m u m a b s o r b a n c e o r f l u o r e s c e n c e w h i c h is a t t a i n e d in t h e r e a c t i o n w i t h o x y h e m o g l o b i n a f t e r 4 0 - - 6 0 m i n . 0, A b s o r b a n c e of globin i s o l a t e d f r o m r e a c t i o n o f o x y h e m o g l o b i n ; e, f l u o r e s c e n c e i n t e n s i t y of globin isolated f r o m r e a c t i o n of o x y h e m o g l o b i n ; A, a b s o r b a n c e of globin i s o l a t e d f r o m r e a c t i o n of d e o x y h e m o g l o b i n ; &, f l u o r e s c e n c e i n t e n s i t y of globin isolated f r o m r e a c t i o n o f d e o x y h e m o g l o b i n .

The oxygen binding curves were measured for monobromobimane- and dibromobimane-labeled hemoglobin and compared to that of unlabeled hemoglobin. The bimane labeling increases the affinity of the hemoglobin for oxygen, the oxygen pressure for half-saturation (Ps0) in 0.1 M sodium phosphate buffer (pH 7.0) decreasing from 9.3 Torr in the control to 4.8 Tort in monobromobimane-labeled hemoglobin to 3.0 Torr in dibromobimane-labeled hemoglobin. The Hill coefficient, n, which measures the cooperativity of the oxygen binding or dissociation process, does not change for monobromobimane-labeled hemoglobin and decreases only moderately for dibromobimanelabeled hemoglobin (n = 2.9 in control, 2.8 in monobromobimane-labeled hemoglobin and 2.2 in dibromobimane-labeled hemoglobin).

207 The ease of denaturation ('heat stability') of control and bimane-labeled hemoglobins was estimated by measuring the hemoglobin remaining in solution in isopropyl alcohol/Tris-HC1 buffer (pH 7.4) [11] after heating at 37°C for 15--60 min. Both monobromobimane- and dibromobimane-labeled hemoglobins were more unstable (50% in solution after 60 min) than the control hemoglobin (80~ in solution after 60 min). Discussion

The site of bimane labeling of hemoglobin is now definitely established as cysteine-f193 by peptide mapping and amino acid analysis, utilizing trypsindegraded fl-chain prepared from bimane-labeled hemoglobin. Tryptic peptide /310, which contains the cysteine-fi93 is strongly fluorescent on a peptide map, and amino acid analysis confirms that the labeled peptide is indeed peptide 10. Another strongly labeled peptide proved to be Till0--11 on amino acid analysis. Thus, bimane labeling of cysteine-~93 produces a fi-chain which is partially resistant to trypsin degradation, a phenomenon observed for ~-chains from certain abnormal hemoglobins. In hemoglobin N Memphis, the lysine at fi95 is replaced by glutamic acid and yields a peptide 10--11. We may infer that the lysine-/395, necessary for tryptic cleavage at 95--96, is blocked, either sterically through the presence of the bimane moiety or both sterically and chemically through covalent bond formation with the second reactive site of dibromobimane (Fig. 6). The electrophoretic mobility of bimane-labeled hemoglobin varies with the particular bromobimane used for the labeling reaction. The monobromobimane-labeled hemoglobin exhibits a mobility similar to that of the untreated hemoglobin. The trimethylammoniobromobimane-labeled hemoglobin, which bears the two positive charges resident in the bimane label, moves more slowly than unlabeled hemoglobin. The dibromobimane-labeled hemoglobin shows the most interesting behavior, with one c o m p o n e n t moving faster, the major fraction moving at the same rate, and another c o m p o n e n t moving slower than untreated hemoglobin. SDS gel electrophoresis shows that no dimer has formed, excluding cross-linking as a source of protein with altered electrophoretic mobility. (Some membrane proteins are cross-linked by dibromobimane, showing that such reactions are feasible [1].) Since the stoichiom-

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208 etry of the dibromobimane-labeling reaction involves the consumption of one reactive thiol group for each dibromobimane, the second bromine present in the labeling agent must be removed readily (see below). The relatively mild change in the cooperativity of oxygen binding for dibromobimane-labeled hemoglobin suggests that the resulting conformational change must be minor. The formation of the slow moving component involves the consumption of an anion, probably a carboxylate group of a glutamic acid. The formation of a fast moving component in electrophoresis implies the consumption of a positively charged group, presumably an amino group which normally exists under these conditions in the ammonium form. The unchanged, major component probably involves the reaction of water with the bromide, with the formation of a hydroxy derivative. Catalysis by the protein may affect the rate of the hydrolysis of the second bromide (the imidazole of the histidine-fi97 could be responsible), or reaction may occur via the intervention of a sulfonium ion intermediate (Kosower, E.M. and Radkowski, A., unpublished results). It seems likely from the stoichiometry of the dibromobimane-labeling reaction and the lack of dimer formation that the hydrolysis takes place soon after the first reaction with the thiol group. What is certain is that the second bromine is gone by the time the globin is isolated since the globin is highly fluorescent and the bromobimanes have essentially no fluorescence (see scheme, Fig. 6). A number of reagents (N-ethylmaleimide, iodoacetamide, 4-hydroxymercuribenzoate, etc.) have been reacted with the cysteine-fi93 thiol group of hemoglobin [13]. The conformational changes associated with the passage from the relaxed (R) quaternary structure of oxyhemoglobin to the tense (T) quaternary structure of deoxyhemoglobin lead to a diminished reactivity of the cysteine~93 groups towards all reagents for which rates can be measured. The nature of the reagent determines the difference in reactivity and the character of the group added affects the degree to which particular properties of the altered hemoglobin change [14--18]. The subject has been thoroughly reviewed by Shulman et al. [19]. The bromobimanes should be very useful in probing conformational isomers of proteins via differences in the rates of reaction because of the ease with which the formation of a fluorescent derivative may be followed. Certain hemoglobin mutants and some chemically modified hemoglobins have a higher affinity for oxygen than unmodified hemoglobin [20]. The higher affinity for oxygen exhibited by the bimane-labeled hemoglobins could be interpreted as a modest increase in the energy required for the approach of the fl-chain F region to the s-chain C region brought about by the presence of the bimane group. Bimane labeling might be useful in labeling proteins without affecting biological activity and regulatory properties provided that the labeled thiol group is not directly involved in the function under study. The heat stability of the bimane-labeled hemoglobins is lower than that of unmodified hemoglobins, the difference being similar to that found for chemically modified and mutant hemoglobins [20]. The present observations extend the usefulness of the bimane-labeling agents as summarized in the previous article [1]. Bimane labels are valuable for (1) locating reactive thiol groups by peptide mapping; (2) modifying the electrophoretic behavior of proteins with an agent which perturbs their biological pro-

209

perties relatively little and provides a convenient fluorescent tag, and (3) local mapping (with dibromobimane) of nucleophilic groups in proteins with potential for modest and useful modifications of biological activity. Acknowledgements Wayne Foran provided valuable and essential technical assistance. The National Institutes of Health are thanked for support through USPHS Grant No. AM17348 (to H.M.R.). Support from the John Simon Guggenheim Memorial Foundation for a Fellowship during 1977-78 is appreciated (E.M.K.). References 1 K o s o w e r , N.S., K o s o w e r , E.M., N e w t o n , G.L. a n d R a n n e y , H.M. ( 1 9 7 9 ) Proc. Natl. Acad. Sci. U.S.A. 76, 3 3 8 2 - - 3 3 8 6 2 K o s o w e r , E.M., P a z h e n c h e v s k y , B. a n d H e r s h k o w i t z , E. ( 1 9 7 8 ) J. Am. Chem. Soc. 100, 6 5 1 6 - - 6 5 1 8 3 K o s o w e r , E.M., Bernstein, J., G o l d b e r g , I., P a z h e n c h e v s k y , B. a n d Goldstein, E. ( 1 9 7 9 ) J. Am. Chem. Soc. 1 0 1 , 1 6 2 0 - - 1 6 2 1 4 R a n n e y , H.M., Briehl, R.W. a n d J a c o b s , A.S. ( 1 9 6 5 ) J. Biol. Chem. 2 4 0 , 2 4 4 2 - - 2 4 4 7 5 Smithies, O. ( 1 9 6 5 ) V o x Sang. 10, 3 5 9 - - 3 6 2 6 Reid, M.S. a n d Bielski, R.L. ( 1 9 6 8 ) Anal. B i o c h e m . 2 2 , 3 7 4 - - 3 8 1 7 Rossi-Fanelli, A., A n t o n i n i , E. a n d C a p u t o , E. ( 1 9 5 8 ) Biochim, Biophys. A c t a 30, 6 0 8 - - 6 1 5 8 Clegg, J.B., N a u g h t o n , M.A. a n d Weatherall, D.J. ( 1 9 6 8 ) N a t u r e , 2 1 9 , 6 9 - - 7 0 9 Clegg, J.B., N a u g h t o n , M.A. a n d Weatherall, D.J. ( 1 9 6 6 ) J. Mol. Biol. 19, 9 1 - - 1 0 8 10 Jensen, M., Oski, F.A., N a t h a n , D.G. a n d Bunn, H.F. ( 1 9 7 5 ) J. Clin. Invest. 55, 4 6 9 - - 4 7 7 11 Carrel, R.W. a n d K a y , R. ( 1 9 7 2 ) Br. J. H e m a t o l . 2 3 , 6 1 5 - - 6 1 9 12 K o s o w e r , N.S., K o s o w e r , E.M. a n d K o p p e l , R.L. ( 1 9 7 7 ) Eur. J. Biochem. 77, 5 2 9 - - 5 3 4 13 Benesch, R.E., R a n n e y , H.M., Benesch, R. a n d S m i t h , G.M. ( 1 9 6 1 ) J. Biol. Chem. 2 3 6 , 2 9 2 6 - - 2 9 2 9 14 Benesch, R. a n d Benesch, R.E. ( 1 9 6 1 ) J. Biol. C h e m . 2 3 6 , 4 0 5 - - 4 1 0 15 Riggs, A. ( 1 9 6 1 ) J. Biol. C h e m . 2 3 6 , 1 9 4 8 - - 1 9 5 4 16 Benesch, R.E. a n d Benesch, R. ( 1 9 6 2 ) B i o c h e m i s t r y 1, 7 3 5 - - 7 3 8 17 G u i d o t t i , G. ( 1 9 6 5 ) J. Biol. Chem. 2 4 0 , 3 9 2 4 - - 3 9 2 7 18 G u i d o t t i , G. ( 1 9 6 7 ) J. Biol. C h e m . 2 4 2 , 3 6 7 3 - - 3 6 8 4 19 S h u l m a n , R.G., H o p f i e l d , J.J. a n d Ogawa, S. ( 1 9 7 5 ) Q. Rev. Biophys. 8, 3 2 5 - - 4 2 0 20 B u n n , H.F., F o r g e t , B.G. a n d R a n n e y , H.M. ( 1 9 7 7 ) H u m a n H e m o g l o b i n s , W.B. S a u n d e r s Co., Philadelphia