The Reaction of N-a-(Bromoacetoxymethyl)maleimide with Hemoglobin*

SUMMARY The reaction of N-cu-(bromoacetoxymethyl)maleimide (AM) with human and horse hemoglobin was observed over a range of pH and ligand conditions. In the case of human hemoglobin, cys F9(93)/? (PERUTZ, M. F., J. Mol. Biol., 13, 646 (1965)) was alkylated by the maleimide ring. This was followed by rapid hydrolysis of the ester bond in the reagent. With horse oxyhemoglobin, a transient covalent bridge between p chains was formed by reaction of the maleimide ring with cys F9(93)/3 followed by reaction of the bromoacetyl portion of the reagent with val NAl(1)/3 of the other /3 chain in the tetramer. After hydrolysis of the ester bond, which occurred spontaneously, the resulting hemoglobin derivative showed no cooperative interactions. A comparison of the dissociation behavior and rates of carboxypeptidase A digestion of horse oxy- and deoxy-AM-hemoglobin showed that the derivative resembled the oxy form of horse hemoglobin even in the absence of ligands. The alkylation of horse oxyhemoglobin could be reaction, of the physical and functional properties also discuss the po-tential of AM as a general bifunctional cross-linking reagent for proteins based on studies of its reactivity with various amino acids.


SUMMARY
The reaction of N-cu-(bromoacetoxymethyl)maleimide (AM) with human and horse hemoglobin was observed over a range of pH and ligand conditions.
In the case of human hemoglobin, cys F9(93)/? (PERUTZ, M. F., J. Mol. Biol., 13, 646 (1965)) was alkylated by the maleimide ring. This was followed by rapid hydrolysis of the ester bond in the reagent. With horse oxyhemoglobin, a transient covalent bridge between p chains was formed by reaction of the maleimide ring with cys F9(93)/3 followed by reaction of the bromoacetyl portion of the reagent with val NAl(1)/3 of the other /3 chain in the tetramer.
After hydrolysis of the ester bond, which occurred spontaneously, the resulting hemoglobin derivative showed no cooperative interactions. A comparison of the dissociation behavior and rates of carboxypeptidase A digestion of horse oxy-and deoxy-AM-hemoglobin showed that the derivative resembled the oxy form of horse hemoglobin even in the absence of ligands.
The alkylation of horse oxyhemoglobin could be carried out in two steps.
By first reacting the AM reagent with cyanomethemoglobin at pH 5.7, the cys F9(93)/3 sulfhydryl group was alkylated.
Addition of excess carbonmonoxyhemoglobin and raising the pH to 7 resulted in alkylation of the NH2 termini of the fl chains by the bromoacetyl portion of the reagent. Separation of these hemoglobin derivatives was achieved.
The two-step reaction sequence was also used to examine one feature of the conformation of a mixed cyanomet-AM-deoxyhemoglobin tetramer.
In an accompanying paper we have discussed the chemical characterization and functional properties of horse hemoglobin modified with various N-substituted maleimides (1). In this report we will describe experiments with N-a-(bromoacetoxymethyl)maleimide and hemoglobin that have led to more complete understanding of this reaction. We also present data on Our interest in hifunctional reagents that could cross-link amino acid residues in hemoglobin was stimulated by the observation that reaction of bis(N-maleimidomethyl)ether with hemoglobin eliminated cooperative oxygen binding without introducing drastic alterations in the tertiary or quaternary structure of the molecule (2). Thus, BME-hemoglobin could be crystallized in a form isomorphous with native horse oxyhemorlobin.
Furthermore, in contrast to crystals of native horse oxyhemoglobin, removal of ligand did not result in fracture of the crystals thus implying that the extensive conformational changes that occur with the native hemoglobin do not take place with BME-hemoglobin (3). The BME reagent has certain limitations as a general cross-linking reagent for proteins which we attempted to overcome by synthesis of an unsymmetrical reagent having the structure shown in Fig. 1. The choice of this reagent was based on three considerations. First, maleimides react faster than oc-haloacetyl derivatives with the cys F9(93)P (4) sulfhydryl group at pH values below 6 (5), permitting the alkylation reactions to be carried out in two steps.
Second, the ester linkage makes it possible to sever any cross-link introduced into the molecule. Thus the effect of cross-linkage or monosubstitution on the functional behavior of the hemoglobin can be examined. Third, a-haloacetates react with various amino acid residues producing derivatives that can be easily identified by amino acid analysis (6). In the previous paper it was shown that the reaction of horse hemoglobin with AM resulted in a derivative in which all functional interactions are eliminated (1). The AM-hemoglobin binds ligands reversibly, crystallizes in a form isomorphous with horse oxyhemoglobin and retains the oxy conformation even after removal of ligand (1). This paper describes the course of the reaction, as well as some of the physical and functional properties of ANI-hemoglobin. We shall also discuss the potential of AM as a general bifunctional cross-linking reagent for proteins based on studies of its reactivity with various amino acids.

Materials
The "slow" component of horse hemoglobin was prepared from the blood of a single horse as previously described (I). Human blood obtained from the blood bank of the Yale-New Haven Hospital was used to prepare hemoglobin according to the method of Drabkin (7).

Synthesis of Piode Compounds
Reaction of N-a-(Bromoacetoxymethyl)maleimide with N-AcetyLcysleine-A 1 M solution of AM in 0.2 M sodium phosphate, pH 7.2, was reacted with a 5-fold molar excess of N-acetyl-n-cysteine for 10 hours at 25" in a nitrogen atmosphere. The reaction mixture was filtered through Sephadex G-10.
The major product was characterized by paper electrophoresis at pH 6.5 and amino acid analysis after hydrolysis with 6 N HCI at 100" for 22 hours.
The rates of reaction of N-acetyl-t-cysteine with AM were studied at pH 5.5 in 0.2 M sodium acetate buffer and at pH 7.2 in 0.2 M sodium phosphate buffer. Aliquots of these reaction mixtures after separation on Sephadex G-10 were hydrolyzed with 6 N HCl, and the content of succinylcysteine and S-carboxymethylcysteine was determined by amino acid analysis. Preparation oj N-Acetylcysteine-N-cr-(Bromoacetoxymethyl)maleimide-N-a-Acetylhistidine Adduct-The reaction product of N-acetylcysteine and AM at pH 5.5 was treated with a 10% molar excess of cY-N-acetyl-n-histidine at pH 7.2 for 2 hours at 25". The product was isolated by chromatography on Sephadex G-10 followed by paper electrophoresis.
Its identity was confirmed by amino acid analysis.

Amino Acid AnaZysis
A Beckman-Spinco 120B amino acid analyzer was used with the usual buffers (8) except for the separation of succinylcysteine and S-carboxymethylcysteine where the pH 3.26 sodium citrate buffer was adjusted to pH 2.86 with 6 N HCI.

Reaction of Human and Horse Oxyhemoglobin with N-ol-(Bromoacetoxymethyl)maleimide
The conditions for the reaction of human hemoglobin with AM were identical with those described previously for horse hemoglobin (1). The rate of alkylation by the maleimide portion of the reagent was measured by the methods given in the accompanying paper (1). They were stored at 4" in 20% EtOH and were equilibrated before use by washing in water followed by heating to 60" in buffer. The buffer used for the exl:eriments was 0.4 M MgC12, 0.05 M Tris-HCl, pH 7.0, containing 100 ~1 of Brij-35 per 100 ml.
The solutions used for the measurements contained 0.1 to 1.5% hemoglobin. The attainment of equilibrium across the membrane was monitored by the rate of change of the osmotic pressure.

Acrylamide
Gel Electrophoresis Aliquots of the reaction mixture of AM and hemoglobin were incubated in 0.1 xr sodium phosphate buffer, pH 7.1, containing I y0 sodium dodecyl sulfate and 1% P-mercaptoethanol and 25% glycerol.
Aliquots containing 15 to 50 pg of protein were layered on 6-cm gels of 5% acrylamide according to the system of Shapiro, Vifiuela, and Maize1 (9). Electrophoresis was carried out for 2 hours at 5 volts per cm. Gels were stained with Coomassie blue, 0.257,, and destained in 7.5% acetic acid and 5% methanol (10).

Oxygen Equilibria
Hemoglobin solutions of 3 to 57; were deoxygenated in a tonometer modified by Simon (11) from a design of Benesch, MacDuff, and Benesch (12). The concentrations of deosy-, oxy-, and methemoglobins were calculated with the equations of Benesch et al. (12) using the optical densities at 540, 560, and 576 mp.

Sedimentation
velocity ultracentrifugation was carried out in a Beckman model E ultracentrifuge equipped with absorption optics and an automatic photoelectric scanning system (13-l 5). The solutions contained 5 X 1OP moles per liter of hemoglobin which gave an optical density of about 0.5 at 280 mp.
All runs were made at 20" in double sector cells against solvent at 60,000 rpm.
Oxy-and deoxy-AM-hemoglobins were compared to The data were collected with the use of interference optics, recorded on Spectroscopic II-G plates, and read with the aid of a Nikon comparator. An Yphantis sixchamber centerpiece was used.
Each chamber was filled with 10 ~1 of hydrocarbon FC43 and 100 ~1 of solvent or solution. The concentration of protein was kept between 0.03 to 0.05%, and runs were made at 25" and 36,000 rpm.
The partial specific volume of the protein in 6 M guanidine hydrochloride was taken as about 1% lower than that found in water as determined by Tanford and others (20-25 (1 ml) was incubated with 0.5 ml of a 1% hemoglobin solution at 31". Aliquots were removed and the protein precipitated with an equal volume of 5% trichloracetic acid and centrifuged.
The supernatant fractions from each tube were diluted to 2% trichloracetic acid with water and placed directly on an amino acid analyzer in order to determine the amount of histidine released. Pseudo-first order rate constants for the release of histidine were calculated from the slope of a least squares plot of the log of the change in histidine concentration versus time.

Rate of Alkaline Denaturation of N-ar-(Bromoacetoxymethyl)maleimide Hemoglobin
The rates of alkali denaturation were estimated by the method of Singer, Chernoff, and Singer (26).

Ion Exchange Chromatography of Horse Hemoglobin and Its Derivatives
The "fast" and "slow" components of horse hemoglobin (27) were separated as described previously (1). The chromatography and identification of derivatives from the reaction of AM with horse hemoglobin has been described (1). The reaction mixture of AM and human hemoglobin was chromatographed on IRC-50 resin with sodium phosphate buffer, pH 6.4, 0.14 M in sodium ion.
The reaction mixture of AM with cyanomethemoglobin (CNmet-hemoglobin) and carbonmonoxyhemoglobin (CO-hemoglobin) was chromatographed on a column (2.5 x 35 cm) of carboxymethylcellulose (CM52) with a a-liter linear gradient of sodium phosphate buffer, pH 7.15, from 0.05 M to 0.2 M in sodium ions. The column was run at 4" with a flow rate of 40 ml per hour and the elution profile determined by measuring the absorbance at 540 mp.
The specific activity of all derivatives was determined as described previously (1).

AND DISCUSSION
Chemical Properties of N-ar-(Bromoacetoxymethyl)maleimide The reaction of AM with N-acetylcysteine and N-ar-acetylhistidine was studied to find conditions that would allow maximum discrimination between alkylation by the maleimide ring and alkylation by the bromoacetyl portion of the reagent. We also examined the stability of the ester bond in the resulting derivatives where both alkylating groups of the reagent had reacted.
We hoped that studies of the behavior of the substituted reagent in model compounds would assist us in predicting and understanding the behavior found when AM was incorporated into proteins. A simple model compound was prepared by reacting AM with an excess of N-acetylcysteine.
The product released equimolar amounts of succinylcysteine and X-carboxymethylcysteine after acid hydrolysis.
Since these two cysteine derivatives were separable on the amino acid analyzer, it was possible, in subsequent experiments, to quantitatively estimate the relative rates of alkylation by each portion of the AM reagent. At pH 7.2, the relative rate of X-alkylation by the maleimide ring was thirty times faster than X-alkylation by the bromoacetyl portion of the reagent.
There was little variation in the relative rates of alkylation when the temperature was varied between 4" and 30".
At pH 5.5 where both alkylation reactions proceed more slowly, a product can be isolated in which S-alkylation has occurred only at the maleimide ring. These results provide a basis for separating the reaction of the bifunctional AM reagent with hemoglobin and other proteins into two steps. Further evidence that the alkylation can be made to occur in two discrete steps was obtained in the synthesis of another model compound.
To prepare this material, AM was reacted with Nacetylcysteine at pH 5.5, the product purified by chromatography and subjected to acid hydrolysis.
Only succinylcysteine was found.
When N-ar-acetylhistidine was added to the pH 5.5 adduct of N-acetylcysteine and AM and the pH raised to 7.2, equimolar ratios of X-succinylcysteine and l-carboxymethylhistidine were found after product isolation followed by acid hydrolysis and amino acid analysis. Not more than 5% of 3carboxymethylhistidine would have gone undetected in this analysis.
The AM-N-acetylcysteine adduct was stable to hydrolysis between pH 1.5 and 9.5 (less than 5% of the compound amino acid or peptide derivatives could be found by paper electrophoresis after 3 hours of incubation at 25"). Ester hydrolysis, however, was appreciable, amounting to 30% at pH 10.5 and greater than 50% when nucleophilic reagents such as 0.2 M cysteine or 0.5 M NHzOH were incubated with these compounds for 3 hours at 25". These results indicated that the ester bond in AM should be relatively stable at neutral pH even after reaction of the maleimide portion with sulfhydryl groups, but that nucleophilic reagents might be used to sever any covalent bridge introduced into the protein.
Reaction of N-a-(Bromoacetoxymethyl)maleimide with I-luman Hemoglobin Reaction of human oxy-or carbonmonoxyhemoglobin with an equimolar ratio of AM to cyp dimer at pH 7.15 resulted in a derivative where the reactive sulfhydryl group (cys F9(93)@) was alkylated by the maleimide portion of the reagent. This was established by finding 1 mole of succinylcysteine per mole of +3 dimer after acid hydrolysis.
When the reaction was studied with the bromide ion-specific electrode, no bromide release could be detected, indicating that alkylation by the bromoacetyl portion of the reagent had not occurred.
In addition, when 14C-AM labeled in the methyl01 carbon atom, was reacted with human hemoglobin less than 0.2 mole of the methyl01 carbon remained per mole of cr/3 subunit. We interpret these results as indicating that alkylation by the p93 sulfhydryl group went to completion but that hydrolysis of the ester bond occurred before any secondary alkylation by the bromoacetyl portion of the reagent took place.
Loss of the labeled methyl01 carbon atom can be explained on the basis of the known instability of N-hydroxymethyl succinimides (28,29). Thus, if the ester bond of the half-reacted reagent were hydrolyzed, it might be expected that the resulting hydroxymethyl succinimide would decompose releasing formaldehyde.
When 14C-AM, labeled in the methyl01 carbon, was reacted with human oxyhemoglobin, 0.8 mole of 14C per mole of reacted a$ subunit was trapped as the methone derivative of formaldehyde, thus supporting the proposed scheme of decomposition.
The remaining 20% of the label found in the hemoglobin was probably due to Schiff base formation when the released formaldehyde reacted with exposed amino groups on the protein.
The reaction of AM with human hemoglobin failed to produce a cross-linked derivative, but indicated that the reagent (after reaction of the maleimide ring with cys F9(93)@) was suitably oriented with respect to a nucleophilic group on the hemoglobin so that hydrolysis of the ester bond occurred before any alkylation by the bromoacetyl moiety could take place. One of the advantages of an unsymmetrical reagent is the possibility of separating the alkylation reaction into two steps. We therefore studied the relative rates of alkylation by the maleimide and bromoacetyl portions of the reagent with horse oxyhemoglobin.
Alkylation by the maleimide ring could be measured by the incorporation of methylol-labeled %-AM and by the production of succinylcysteine after acid hydrolysis of the reacted protein.
Alkylation by the bromoacetyl portion of the reagent could be measured by observing the amount of bromide ion released with the use of a bromide-specific electrode. When AM was reacted with horse oxyhemoglobin at pH 7.15, as shown in Fig. 2, a IO-fold differenbe was observed between the rates of alkylation of cys F9(93)/3 by the maleimide and carboxymethylation of val NAl (1)p by the bromoacetyl portion of AM. Pseudo first order rate constants were calculated from reaction mixtures with a lo-fold molar excess of reagent (5 X 10V3 M) to hemoglobin (5 x lo+ M).
When hemoglobin that had been alkylated first by N-ethylmaleimide at cys F9(93)/3 (EM-oxyhemoglobin) was used in the reaction, no bromide ion release could be detected (Fig. 2), thus showing that it is necessary to anchor the reagent at cys F9(93)@ before the second reaction can occur. As can be seen in Fig. 2, the alkylation at val NA1(1)/3 proceeds to at least 80% completion in this case. In other hemoglobin preparations from the same horse, the extent of carboxymethylation was somewhat lower.
The reasons for these variations are not known.
Ap interesting feature of the reaction is that the labeled methyl01 group was retained when carboxymethylation occurs before the ester bond in AM hydrolyzes.
We have been unable to ascertain whether the methyl01 group remains attached to the succinimide ring or is distributed among various lysine residues in the form of Schiff bases. If esterolysis occurs before carboxymethylation, the label is lost as formaldehyde and no secondary alkylation occurs. To understand more about the course of the reaction, horse carbonmonoxy-AM-hemoglobin, prepared at pH 7.15 and purified by column chromatography (l), was studied by osmometry under conditions that cause dissociation of the liganded tetramer into a$3 dimers (0.4 M MgC12, pH 7.0). The results shown in Fig. 3 demonstrated that the AM derivative of horse CO-hemoglobin dissociates to the same extent as unmodified horse CO-hemoglobin.
Furthermore, when oxy-AM-hemoglobin was subjected to ultracentrifugation in 6 M guanidine hydrochloride, a molecular weight of 18,700 was obtained compared to 18,500 for horse CO-hemoglobin and 17,500 for myoglobin, all determined in the same run. Thus, the isolated AM derivative does not have a /3-p' covalent bridge although it has reacted completely at the cys F9(93)P and val NAl(l)P of both /3 chains. The possibility that the reagent links the cys F9(93)/3 sulfhydryl group to the NHz-terminal valine of the same fi chain is considered most unlikely because the size of the reagent is small compared to the distance between these residues.
Furthermore, if such a cross-link occurred, the p chain would have to be considerably distorted since a major portion of the p chain lies between the 93/3 sulfhydryl group and the NHQ-terminal group of the same chain.
That such distortion does not occur is shown by the x-ray crystallographic results of Moffat on AMhemoglobin (30).  I  I  I  I  I  I  I  I  I  I  I  I  I  I  2  4  6  8  IO  12  If the reagent actually forms a covalent bridge between /3 chains which subsequently undergoes hydrolysis, we thought that it might be possible to demonstrate the presence of a transient covalent bridge with acrylamide gel electrophoresis in sodium dodecyl sulfate.
It is known that under these conditions multimeric proteins are dissociated into monomers and that their rate of migration is linearly related to the logarithm of their molecular weight (10). Aliquots of the reaction mixture of AM and hemoglobin were removed at various times and run on acrylamide gels with buffers containing mercaptoethanol and sodium dodecyl sulfate. The results of this experiment are shown in Fig. 4. It can be seen that unmodified hemoglobin and myoglobin migrate at nearly the same rate. As the reaction with AM proceeds, a new band is formed which migrates at a rate consistent with a molecular weight of 32,000 (Fig. 5). All the radioactivity was associated with this band which was then eluted and hydrolyzed.
The amino acid analysis showed that it was composed only of /3 chains and that the ratio of AM to /3 chains was 1: 1. When the j3-/3 dimer isolated from the gel was subjected to gel electrophoresis again after having been &red in the same electrophoresis buffer for 3 days, only a single These results provide direct evidence for the tra,nsient formation of a covalent linkage between /3 chains in the AM-hemoglobin derivative.
They also demonstrate that the ester bond in the derivative is stable when it is removed from the environment of the native hemoglobin molecule. An examination of the 2.8 A model of horse oxyhemoglobin (31) suggested that the imidazole group of his HC3(146)P might be in a position to catalyze the hydrolysis of the ester bond in the reagent once a reaction had taken place at cys F9(93)/3. This possibility was ruled out, however, by the finding that the pattern after sodium dodecyl sulfate acrylamide gel electrophoresis of the reaction mixture of AM with carboxypeptidase A-treated hemoglobin was identical with that obtained with undigested hemoglobin after reaction with AM.

Properties oj N-a-(Bromoacetoxymethyl)rruzleimide Hemoglobin
Oxygen Equilibria-Oxygen saturation curves were determined on the reaction mixture of AM and oxyhemoglobin rather than on the chromatographically purified derivatives since AM-oxyhemoglobin is easily oxidized to methemoglobin. The oxygenation curve was hyperbolic, the value of the interaction constant (n) obtained from the Hill equation* was 1.1 to 1.2, and the logarithm of the partial pressure of oxygen at halfsaturation (log ~50) was 0.4 compared with the log ~50 value of 1.02 for normal hemoglobin tested under the same conditions. There was no detectable Bohr effect. Furthermore, we have shown that two products are formed in the reactions of AM with oxyhemoglobin, one having val NAl(l)@ carboxymethylated and the other having val NAl(l)P free (I), but the presence or absence of the carboxymethyl group apparently does not affect the degree of cooperativity that is observed. These Issue of April 25, 1971 D. J. Arndt and W. Konigsberg results show that cooperative interactions were almost completely eliminated when AM reacted with oxyhemoglobin. When AM was reacted with deoxyhemoglobin, the reagent alkylated cys F9(93)P and then the ester bond in the reagent hydrolyzed.
Oxygen saturation experiments carried out with this derivative showed that cooperative interactions were preserved (n = 2.0, log pso = 0.8). 4 Dissociation Behavior-Although we have previously shown by osmometry and acrylamide gel electrophoresis that the intermolecular bridge in AM-hemoglobin is short lived, it was of interest to see if the presence or absence of ligand in AM-hemoglobin affected the equilibrium between tetramers and dimers in the same way as it does with unmodified hemoglobin (32)(33)(34). The results of sedimentation velocity ultracentrifugation of AM-hemoglobin, under a variety of conditions, with and without ligand, are presented in Table I form. Since the degree of dissociation of tetramers into dimers appears to be associated with the sigmoid oxygenation equilibria in native hemoglobin (32)(33)(34), the similarity in the dissociation properties of the oxy and deoxy forms of Ah4 oxyhemoglobin is consistent with results of the oxygen saturation curves which indicate almost total absence of cooperativity. Furthermore, the similarity in the sedimentation velocities of oxy-AM-hemoglobin and normal liganded hemoglobin suggest that horse AMhemoglobin is constrained in an oxy-like conformation.

Rate of Digestion with Carboxypeptidase
A-The rates of digestion of oxy-and deoxyhemoglobins by carboxypeptidases A and B were first studied by Zito, Antonini, and Wyman (35). They were able to show that cbrboxypeptidase A removed tyroaine and histidine from the COOH end of the /3 chain three times as fast with oxyhemoglobin as compared with deoxyhemoglobin.
Since these rates are dependent on the presence or absence of ligand, the susceptibility to carboxypeptidase A can be considered a linked function.
In contrast to the results with carboxypeptidase A, the rates of digestion of oxyhemoglobin by carboxypeptidase B were 50 to 80% greater than those for deoxyhemoglobin.
The rates of digestion of oxy-and deoxy-AM-hemoglobin were examined and compared to the results obtained with unmodified oxy-and deoxyhemoglobin. more closely related to normal oxy-than deoxyhemoglobin. Since the rate of digestion of ANI-oxyhemoglobin is insensitive to the presence or absence of ligand, this provides further evidence that ligand exchange in AM-hemoglobin is not) linked to the same conformational change that occurs in normal hemoglobin.
In the case of AM reacted wit,h deoxyhemoglobin, the rates of digestion in the oxy and deoxy states were similar to those found for normal oxy-and deoxyhemoglobin.
These results are consistent with the other observations on ANI-deoxyhemoglobin, namely that the cooperative interactions and the functionally important conformational changes are preserved." Rate of Alkali Denduration-No difference was found between the rate of denaturation by alkali on AM-and normal horse hemoglobin indicating that no measurable alkali-sensitive instability was introduced by the modifying reagent.

Two Stage Reactions of N-or-(Bromoacetoxymethyl)maleimide with Horse Hemoglobin
The difference in reaction rates of the maleimide and bromoacetyl portions of the reagent was used to demonstrate the presence of mixed species of /3 chains in a tetramer and to prepare hemoglobin derivatives with carboxymethyl groups on val NAl(l)P having free sulfhydryl groups at residue cys F9(93)P. In this experiment, CNmet-hemoglobin was reacted at pH 5.7 with a 2-fold excess of AM labeled with l4C in the acetyl group.
After the excess reagent had been removed by gel filtration, it could be shown that 1 mole of reagent had been incorporated into 1 mole of the hemoglobin (based on radioactivity) and therefore no esterolysis had occurred. A lo-fold excess of unreacted CO-hemoglobin was then added to the -SHreacted CNmet-hemoglobin and the pH adjusted to 7.2. No bromide ions were released at pH 5.7 but at the higher pH nearly 80% of the theoretical amount of bromide ions in the reagent were liberated. The reaction products were then chromatographed on Cm-cellulose to give a pattern shown in Fig. 6 The results of this hybridization experiment suggested that it might be extended to test the degree of fit (with respect to the distance between the 93/l sulfhydryl group on one /3 chain and the NHrterminal group of the other /l chain) in the formation of mixed tetramers (c@alfll) where each ap subunit is derived from either a different species or is in a different liganded form of the subunit of the same species.
To test this, the following experiment was performed. CNmet-hemoglobin was reacted with a 2-fold excess of AM at pH 5.7. After removal of excess reagent the solution was flushed with Nt, adjusted to pH 7.2, and a IO-fold excess of deoxyhemoglobin (relative to the amount of CNmet-hemoglobin) was immediately added. Bromide ion release amounted to 0.6 mole per mole of AM. Excess cysteine was then added to trap any unreacted reagents and bromoacetate which might have been released by esterolysis.
Carbon monoxide was bubbled through the solution which was then filtered through Sephadex G-25.
The amount of radioactivity in the most retarded zone amounted to 0.2 residue based on the original amount of (YP subunits of CNmet-hemoglobin used. The hemoglobin in the void volume was rechromatographed on Cm-cellulose to give a pattern shown in Fig. 7. All the radioactivity was associated with the CNmet-hemoglobin (Peak 1) which was free of CO-hemoglobin.
No radioactivity was found in the CO-hemoglobin (Peak 2). As we have shown (l), the reaction of AM with either liganded or unliganded hemoglobin results first in alkylation of the cys F9(93)@-SH group by the maleimide ring, whereas with liganded hemoglobin the second step results in alkylation of the NH2 terminus of the other p chain in the tetramer; with deoxyhemoglobin the second step results in complete hydrolysis of the ester bond in AM. Our results can be interpreted in two ways, either the (c@)~~?'-(4 CNmet tetramer did not form, in which case the introduction of deoxyhemoglobin after raising the pH would have had no effect on the -SH-reacted AM-CNmet hemoglobin, i.e. the secondary alkylation should occur solely within the CNmethemoglobin tetramer. If the mixed (&?)deoxy-(&?)CNmet tetramer does form, its quaternary structure must be different from that of oxy-or deoxyhemoglobin tetramers at least with respect to the distance between cys F9(93)/3 and ral NAl(l)P' since the second alkylation occurs exclusively with the CNmethemoglobin tetramer.
The reason for considering the latter interpretation is based on the results obtained by Guidotti (36) on the behavior of mixtures of deoxy-and CNmet-hemoglobin in solution.
Since he was able to infer the existence of mixed CNmet-deoxy tetramers by osmotic pressure measurements we thought that (c$~)~~~~Y-(c@) A"-CNmet tetramers could also form under the conditions of our experiment. The use of reagents that would covalently link fl chains and where the reagent bridge could not be severed under mild conditions might be used to indicate the degree of fit between sets of unlike CYP dimers.
We are now attempting to prepare a reagent which would be similar to AM but would have an amide instead of an ester linkage between the maleimide and the bromoacetyl portion of the molecule.
Despite ester hydrolysis, which severs the covalent bridge, reagents similar to AM may prove useful for two-step labeling of proteins other than hemoglobin such as 3-phosphoglyceraldehyde dehydrogenase where a reactive sulfhydryl group is situated near an E amino group of lysine and a catalytically important histidine residue (37).