THE JOURNAL OF l31om~1c.m. CHEMISTRY Vol. 241, No. 1, Issue of January 10, 1966

Printed in U.S.A.

Studies on Relationships between Structure and Function

of Hemoglobin MIwate *

(Received for publication, June 25, 1965)

NORIO HAYASHI, YUTARO MOTOKAWA, AND GORO KIKUCHI

From the Department of Biochemistry, Tohoku Unive&y School of Medicine, Xendai, Japan

SUMMARY

Functional properties of hemoglobin Mrwate (01~~7 Tyr&A) were studied with particular reference to their relations with the structure of this abnormal hemoglobin.

1. The oxygen affinity of hemoglobin Mrwate (in reality the /3 subunits) was far lower than that of hemoglobin A, and the interaction constant n in oxygen equilibrium was 1.0 at any pH. Apparently no Bohr effect was observed in the pH range 6.3 to 7.5, while a very small Bohr effect (Alog px/ApH = 0.19) could be observed at higher pH values. A lowered Bohr effect was confirmed by the differential titration experi- ments.

2. Hemoglobin Mrwate, like hemoglobin A, dissociates into o$ dimers at higher concentrations of sodium chloride, but the features of the oxygen equilibrium of the half-molecules of hemoglobin Mrwate were not appreciably different from those of the original molecule.

3. Hemoglobin M1wate has two reactive -SH groups per molecule. However, “-SH blocking” by p-chloromercuri- benzoate resulted in no particular change in the value of n and in oxygen affinity, except that the Bohr effect at higher pH values appeared to be lost.

4. Characteristics of the carbon monoxide equilibrium of hemoglobin M1wate were similar to those observed in oxygen equilibrium. However, when the CY subunits were reduced by sodium dithionite, and hence enabled to bind carbon monoxide, the carbon monoxide affinity and the Bohr effect were increased to nearly the same levels as observed with hemoglobin A. Nevertheless, the value of n remained nearly at 1.0.

5. Rates of autoxidation of hemoglobin Mlwate were about 6 times higher than those of hemoglobin A under comparable conditions.

-6. The OL subunits of hemoglobin M1wate were more re- sistant to denaturation by sodium benzoate than the normal hemoglobin.

Thkse data strongly suggest that the substitution of only a single amino acid in one type of subunits profoundly affects the functional properties not only of the abnormal subunits, but also of structurally normal subunits in the same molecule, and possibly of the molecule as a whole. Also it was sug- gested, because of these and some other findings, that the

* This investigation was aided in part by Grant AM-08016-01 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.

conformational changes of the hemoglobin molecule responsi- ble for the Bohr effect, the affinity for ligands, and the shape of the oxygen or carbon monoxide dissociation curve, respec- tively, may be different, even if closely related.

The functional integrity of the hemoglobin molecule has been the subject of many investigations. Recent studies on the oxy- gen equilibrium of some abnormal hemoglobins have provided information valuable for understanding the characteristic fea- tures of the oxygenation of hemoglobin. Benesch et al. (1) reported that the oxygen affinity of hemoglobin H, which is composed of p chains only, was about 12 times higher than that of hemoglobin A, and that the heme-heme interaction, as well as the Bohr effect of hemoglobin H, was greatly reduced. Sin- ilar results were obtained by Horton et al. (2) with hemoglobin Barts, which is composed of four y chains. These findings have been taken as indicating that an interaction between CY and /? or OL and y chains is essential for the conformational changes of the hemoglobin molecule responsible for the heme-heme inter- action and the Bohr effect (1, 2). However, Reissmann, Ruth, and Nomura (3) reported a familial human hemoglobin variant which showed an abnormally low oxygen affinity and apparently no heme-heme interaction, although the Bohr effect of this hemo- globin was of normal magnitude.

In the present paper we report some unusual functional prop- erties of hemoglobin MIwate observed in oxygen and carbon monoxide equilibrium experiments. Hemoglobin MJIIwate has an abnormal cr chain (4, 5) ; the histidine in position 87 of this chain is substituted by tyrosine (5). Hemoglobin MIwats is identical with hemoglobin MKankakee (6). The a subunits of hemoglobin MIwatp usually stay in the ferric state, but do not react with cyanide, azide, fluoride, and hydroxyl ions and are reduced by dithionite only sluggishly (7). Only the /I subunits of the hemo- globin MIwate molecule are able to bind oxygen reversibly (7). A part of this work has been reported in a preliminary form CS).

EXPERIMENTAL PROCEDURE

Preparation of Hemoglobin So&ions-Hemolysates from the blood sample of a patient with hemoglobin MIwate disease (the nigremia of Tamura and Takahashi (9)) were freed from stroma and were dialyzed against 0.0125 M ammonium phosphate buffer, pH 7.1, and subjected to chromatography on Amberlite XE-64

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80 Functional Properties of Hemoglobin Mlwute Vol. 241, No. 1

y 0.5 -

FIG. 1. Oxygen dissociation curves of hemoglobin MI,,.,,, and hemoglobin A. Hemoglobin, 8 X low5 M as heme, in 0.08 M phos- phate buffer, 20”. O-O, pH 7.0; O-O, pH 7.5; X-X, pH 8.5. Curves in the figure represent the theoretical curves for 12 = 2.8 (hemoglobin A) or 12 = 1.0 (hemoglobin MI,~,~,).

equilib&ed with the same buffer. The normal hemoglobin fraction was completely eluted by washing the column with the equilibration buffer. The hemoglobin MIwate fraction remaining on the column was eluted with 0.5 M sodium chloride, pH 7.0. Hemoglobin A solution used as a control was prepared from nor- mal adult blood in similar manner, but without the column step. The hemoglobin fractions thus obtained were dialyzed against distilled water, and centrifuged again before being used. All procedures were performed at about 4”. Methemoglobins A and MIwate were prepared by ferricyanide oxidation of the cor- responding oxyhemoglobins, followed by dialysis against dis- tilled water. Hemoglobin concentrations are expressed in terms of heme determined as the alkali-denatured globin hemochrome, assuming the millimolar ext,inct#ion coefficient at 559 rnp to be 30.6 (10). A Hitachi recording spectrophotometer was used for all photometric experiments.

Determination of Oxygen and Carbon Monoxide Dissociation Curves-Oxygen dissociation curves were determined spectro- photometrically by the method of Allen, Guthe, and Wyman (11) at 20”, but with a tonometer attached to the optical cell of l-cm light path, and were calculated from the absorbance changes at 560 rnp. The pH of the reaction mixture was determined with a glass electrode pH meter at the end of each experiment. Air was used in experiments with hemoglobin A, whereas pure oxy- gen gas was employed in experiments with hemoglobin Mrwate, because the latter had a lower oxygen affinity. The carbon monoxide equilibrium was determined in a similar manner, with carbon monoxide-nitrogen gas mixt.ures, and was calculated from the absorbance changes at 540 rnp and 570 mp. Nitrogen and oxygen gases were obtained commercially. Carbon monoxide was prepared from formic and sulfuric acids, and purified by passing the gas through sodium hydro‘xide solution, alkaline dithionite solution, and, finally, distilled water.

Differential Titration of Oxygen-linked ilcid Groups-Oxyhemo- globin solution (3.0 ml) in 0.1 M sodium chloride was introduced into a titration chamber mainmined at 20”, and the pH of the solution was adjusted to 7.3. ,4 glass electrode pH meter, attached to a recorder, was used in the titration experiment. The solut.ion was completely deoxygenated by passing oxygen- free nitrogen gas through the chamber; then 0.01 N HCl was added with the aid of a syringe microburette until the original pH value of 7.3 was restored. The same solution was subse- quent,ly reoxygenated, and the pH was brought back to 7.3 by titrating with 0.01 N NaOH. Values of oxygen-linked acid

groups were calculated from the amounts of HCl and NaOH consumed.

Sedimenlation Analyses-Sedimentation velocities were meas- ured at 20° with a Spinco model E analytical ultracentrifuge at 59,780 rpm, with a synthetic boundary cell. A Toshiba R2 red, filter was placed in the light path. The partial specific volume of the hemoglobins was assumed to be 0.748 ml per g (12).

Autoxidation 0~” Ozyhemoglobins-Rates of autoxidation were measured by following the absorbance changes at 542 rnp and 578 mp. The oxyhemoglobin A samples used were isolated from the same blood sample from which hemoglobin MIwate was pre- pared. Reactions were performed in a Thunberg-type cuvette filled with pure oxygen gas, because of the low oxygen affinity of hemoglobin MIT,,vate.

RESULTS

Oxygen Equilibrium of Hemoglobin dlrwotc--The oxygen dis- sociation curves of hemoglobin MIwate and hemoglobin A at different pH values are compared in Fig. 1. In the figure, the fractional saturation of the hemoglobin with oxygen, represented by Y in Hill’s empirical equation Y = Kp”/(l + Kp”), is plotted against the logarithm of the partial pressure of oxygen, poZ. Three important differences can be seen between the oxygen equilibria of these two hemoglobins. Firstly, the interaction constant n, calculated from Hill’s equation, was found to be 1.0 for hemoglobin M,,,,, at each pH, while the n value for hemoglobin B was in the neighborhood of 2.8. Secondly, the oxygen affinity of hemoglobin M Ix.ate was much lower than that of hemoglobin A; the oxygen pressure required for half-saturation (p;) of hemoglobin MIwate was about 5 times higher than that of hemoglobin A at pH 7.0. Thirdly, the Bohr effect of hemo- globin MIwate was very small. Results of detailed studies of the effect of pH on the oxygen affinity of hemoglobin MIwate are shown in Fig. 2 Apparently no Bohr effect could be ob- served in the pH range of 6.3 to 7.5. The maximum value of Alog ph/ApH calculated for hemoglobin MIwatr was about 0.19, whereas that for hemoglobin A was about 0.50. The Bohr effect was also determined by the differential titration method (Table I). The results obtained with the titration method were essentially similar to those obtained in oxygen equilibrium ex-

OeV PH

FIG. 2. Effect of pH on the oxygen affinity of hemoglobin Mrwate and hemoglobin A. O-O, hemoglobin components from the patient’s blood; O-O, hemoglobin A from normal human blood. ~024, oxygen pressure in millimeters for 50% saturation. Other condit,ions are similar to Fig. 1.

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Issue of January 10, 1966 N. Hayashi, Y. Motokawa, and G. Kikuchi 81

periments. The oxygen-binding properties of the normal hemo- globin fraction isolated from the patient’s blood were indis- tinguishable from those of hemoglobin A prepared from normal blood.

lZflect of Sodium Chloride on Oxygen Dissociation Curves of Hemoglobin Ml,,t,-Oxygen affinity of hemoglobin MIwate was slightly increased by the increased addition of sodium chloride, as shown in Fig. 3, but the value of ps obtained even in the pres- ence of 2 M sodium chloride remained far lower than that of hemoglobin A obtained in t’he absence of sodium chloride (cf. Fig. 2). Also, n values of the oxygen dissociation curves were not appreciably changed in the range of sodium chloride concen- trations employed.

Values of sedimentation constants of hemoglobins A and M 1wate in the presence and absence of sodium chloride are shown in Table II. The values of s 20,W of both hemoglobins ,4 and M mate were reduced to practically the same extent by the addi- tion of sodium chloride under comparable conditions. These results suggest that hemoglobin MIwate is largely dissociated into identical half-molecules, olfl and c@, in the presence of higher concentrations of sodium chloride, as had been shown to be the case for hemoglobin A (12-15). In all runs, only a single symmetrical boundary was observed in the sedimentation pat- terns. It should be noted that since these experiments were run under an atmosphere of air, approximately 20% of total hemoglobin MIwate would, because of its low osygen affinity, be expected to stay in the deoxygenated form under this condition (cj. Figs. 1 and 3). The dissociation of the hemoglobin MIwatp molecule, however, did not appear to be significantly affected by the concomitant presence of this amount of deoxyhemoglobin, although it has been reported that deoxyhemoglobin from nor- mal human blood is somewhat more resistant than the oxyhemo- globin to dissociation by sodium chloride (14).

Effect of p-Chloromercuribenxoate on Oxygen Equilibrium of

TABLE I Titration of oxygen-linked acid groups of hemoglobin A and

hemoglobin Mrwate

Hemoglobin, 3 X lo+ M as heme, in 0.1 M sodium chloride, pH 7.3, 2o”.

Hemoglobin

Hemoglobin A......................... Hemoglobin MI,.~,.

H+ concentration

moles/wle pro2ein

2.2 0.4

.O\ O\o, 00 - - -

FIG. 3. Effect of sodium chloride on the oxygen affinity of hemoglobin Mrwate. Hemoglobin, 8 X 10e5 M as heme, in 0.008 M phosphate buffer, pH 7.0, 20’.

Sedimentation

TABLE II coeficients of hemoglobin A and hemoglobin Mrwale

in presence of sodium chloride

Concentrations of hemoglobins A and Mrwate were 0.8 g/100 ml, and 0.7 g/100 ml, respectively. All experiments were performed in 0.05 M phosphate buffer, pH 7.0, at 59,780 rpm.

520,21; Sodium chloride

Hemoglobin A Hemoglobin Mlwrrte

M s s

0 4.39 4.33 1.0 4.02 3.95

2.0 3.60 3.60

O.lL . ' IO

p 0*~0,tn’l?,0 5 10

FIG. 4. Oxygen dissociation curves of hemoglobin Mr,,.%te in the presence (closed points) and absence (open points) of CMB. He- moglobin, 8 X 10-S M as heme, in 0.08 M phosphate buffer. CMB, 8 X 1o-5 M, 20’. o-o, o--o, pH 7.0; O-0, W---W, pH 8.0. Lines in t,he figure represent the slope for n = 1.0.

Hemoglobin M I,,t,-Hemoglobin MImate was found to have 2.0 reactive -SH groups per molecule as determined by the Boyer method (16, 17). The value of 2.0 is the same as that found with hemoglobin A by other investigators (18, 19). Also the binding of CMB by hemoglobin MIwate was very fast and was completed promptly. These results suggest that the reactive -SH groups in the hemoglobin M iwate molecule are very similar to those of hemoglobin A and possibly are located on position 93 of the /I chains as established for hemoglobin 9 (20).

Oxygen dissociation curves of hemoglobin MIwatc determined in the presence of CM131 are shown in Fig. 2. No apparent change in the n value resulted from the addition of CMB, and the oxygen affinity also remained low. However, the Bohr effect of hemoglobin MIwate, which could be observed at higher pH values in the absence of CMB, appeared to be lost by the addi- tion of CMB.

Carbon Monoxide Equilibrium of Hemoglobin Mrwate-The characteristic features of the carbon monoxide equilibrium of hemoglobin MIwate were essentially similar to those of the oxy- gen equilibrium when the a subunits remained in the ferric state (Figs. 5 and 6). The carbon monoxide affinity of hemo- globin MIwate was approximately one-tenth that of hemoglobin A; the n value was apparently 1.0; and the Bohr effect was very small. As reported in our previous paper (7), however, the

1 The abbreviation used is: CMB, sodium p-chloromercuri- benzoate.

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Functional Properties of Hemoglobin ATlluate Vol. 241, n-o. 1

0.11 9: I 4005 0.01 0.05 0.1 cl5 1.0 50

PC0 t mmHg

FIG. 5. Carbon monoxide dissociation curves of hemoglobin Mrwate and hemoglobin A isolated from the patient’s blood. He- moglobin, 5 X 10e5 M as heme, 20’. O-O, hemoglobin Mnvate in 0.08 M phosphate buffer, pH 7.0; 0-0, hemoglobin Mrwate in 0.08 M phosphate buffer, pH 7.8; O-U, hemoglobin A in 0.08 M phosphate buffer, pH 7.0; n - - - - -m, hemoglobin A in 0.08 M

phosphate buffer, pH 7.9; Q--Q, hemoglobin MI,,~*~~ in borate- phosphate buffer containing 3.6 X 10e2 M sodium dithionite, pH 6.9; @-CD, same as above but at pH 7.6; A- - - - -A, hemo- globin A in borate-phosphate buffer containing 3.6 X 10F~ sodium dithionite, pH 6.8; A- - - - -A, same as above but at pH 8.2. Lines in the figure represent the slope for YL = 2.0 (hemoglobin A) or 12 = 1.0 (hemoglobin MI,“,~,).

C

-1

-IN

8 n

$ -I -2

-3

-@-OS

‘.

\ %? Q,

O\O

0 al \

I 7 8 PH

FIG. 6. Effect of pH on the carbon monoxide affinity of hemo- globin MI,“,,, (circles) and hemoglobin A (squares). Hemoglobin, 5 X 10-e IVI as heme, 20”. e---O, n --~, in 0.08 M phosphate buffer; O-O, U&--U, in borate-phosphate buffer containing 3.6 X 10m2 M sodium dithionite. pCO$, carbon monoxide pressure in millimeters for 50% saturation.

o( subunits of hemoglobin MIwate can be reduced, though very

slowly, by dithionite under an atmosphere of nitrogen. Full

reduction usually requires about 30 hours until full reduction is

accomplished, and the absorption spectrum of hemoglobin MIwate after complete reduction is very close to that of deoxyhemoglobin

A. Also, in completely reduced hemoglobin Mrwate, both Q and 0 chain subunits are able to bind carbon monoxide.

When t.he completely reduced hemoglobin MIwate was em-

ployed, both the carbon monoxide affinity and the Bohr effect were found to be markedly increased, and the observed magni- tudes of carbon monoxide affinity and Bohr effect were almost similar to those obtained with hemoglobin A, as can be seen from Figs. 5 and 6. The values obtained with hemoglobin A were in good agreement with those reported by other workers (21, 22). It is noteworthy, however, that the n value of carbon monoxide dissociation of hemoglobin Mlwilte was still nearly 1.0, even when fully reduced hemoglobin Mr,,t, was employed. Although it is known that the oxygen equilibrium of hemoglobin

B is affected by salts (22-24), the observed changes in carbon monoxide equilibrium of hemoglobin MIwate in the presence of dithionite cannot be due solely to the salt effect of the added dithionite. The carbon monoxide affinity of hemoglobin A was indeed increased by the addition of dithionite, but the ext’ent of the increase observed with hemoglobin A was only about one- tenth of that observed with hemoglobin MIwate. Thus, we may conclude that the observed increases in carbon monoxide affinity

3

FIG. 7. Percentage formation of hemichromes of methemo- globin A and of CY chain subunits of methemoglobin MI,,,, at various concentrations of sodium benzoate. Hemoglobin, 5 X 1O-5 M as heme, in 0.1 M phosphate buffer, pH 7.4. Hemichrome formation was measured after 1 hour in benzoate at 1%‘. Results of two independent experiments are plotted for hemoglobin A (0, a,) and CY subunits of hemoglobin Mrwzte ( l , @) . Curves in the figure represent the theoretical curves for n = 6.0 (hemoglobin A) or n = 9.0 (CY subunits of hemoglobin MI,,~,) calculated from the empirical equation y/l00 = K [benzoate]“/(l + K [benzo- ate]“), where y represents percentage of hemichrome formation.

FIG. 8. Rates of autoxidation of hemoglobin Mr,xistc ar 1C 1 hemo. globin A. Hemoglobin, 5 X lo+ M as heme, 37’. Open poznts are for hemoglobin A, and closed points are for hemoglobin M rwate. O---O, B---m, in 0.08 M phosphate buffer, pH 7.0; ApA, A--A, in 0.08 M phosphate buffer, pH 7.0, containing 2.0 M sodium chloride; O-O, O--O, in 0.16 M sodium acetate buffer, pH 5.0. Reactions were conducted under an atmosphere of oxygen.

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Issue of January 10, 1966 N. Hayashi, Y. Moiolcawa, and G. Kilcuchi 83

and in the Bohr effect of hemoglobin MIwate were primarily the consequence of the reduction of the a subunits.

Conformation Change of Methemoglobins A and MIWate Effected by Sodium Benzoate-Methemoglobin A is converted to a hemi- chrome-like compound when it is allowed to react with sodium benzoate at room temperature (25). The hemichrome shows an absorption peak at 536 ml*, and the yield of hemichrome is a function of the concentration of benzoate added. At pH 7.4, the absorption spectra of methemoglobin A and its hemichrome are isosbestic at 596 rnp. Methemoglobin MIwate is also con- vert.ed to its hemichrome by the addition of benzoate. With methemoglobin Miwate, however, the spectral change, along with the increased addition of benzoate, was irregular and there occurred large absorption changes at 596 rnp according to the concentrations of benzoate added; finally, when the benzoate concentration was increased over 1.5 M, the spectrum of hemo- globin MIwnte became apparently indistinguishable from that of the hemichrome of hemoglobin A. It is conceivable that the observed absorption change at 596 ml* with methemoglobin MIwate is due solely to its abnormal (Y subunit, the absorption spectrum of which is considerably different from that of methe- moglobin A (7), since the spectral changes of the ,f3 subunits of methemoglobin MIwate are expected to be identical with those of methemoglobin A under comparable benzoate concentrations (7). On these assumptions the extent of hemichrome formation from the abnormal a! subunits of hemoglobin MIwate at different benzoate concentrations was calculated on the basis of absorp- tion change at 596 rnp and plotted against the logarithm of the concentrations of benzoate added (Fig. 7). Hemichrome forma- tion from hemoglobin A calculated from absorption changes at 536 mp is also shown in Fig. 7. The n value of the sigmoid curve for hemoglobin A was 6.0, and 50% of the formation hemi- chrome of hemoglobin A was obtained at a benzoate concen- tration of 0.56 M. In contrast, considerably higher concentra- tions of benzoate were required to effect hemichrome formation from the abnormal o( subunits of hemoglobin MIwate, and 50% hemichrome formation was reached at 0.82 M benzoate. Also, the n value of the sigmoid curve of hemoglobin MImate was found to be 9.0. These results indicate that the OL subunits of hemoglobin Mlwate are more resistant to conformational changes by benzoate than those of the normal hemoglobin.

Autoxidation of Hemoglobin M1,ate-The rate of autoxidation of hemoglobin MIwate was about 6 times higher than that of hemoglobin A at either pH 5.0 or 7.0 (Fig. 8). Also, the rate of autoxidation of both hemoglobins was increased equally by about 4 times when 2.0 M sodium chloride was added.

DISCUSSION

The observed abnormality in oxygen as well as carbon mon- oxide equilibria of hemoglobin MIwrtte can be taken as a functional reflection of the structural abnormality of the hemoglobin MIwate molecule. It is particularly noteworthy that the oxygen affinity of hemoglobin MIwate was significantly lower than that of hemo- globin A. Hemoglobin Mrwat, showed no heme-heme inter- actions (as judged by the n value of the oxygen dissociation curve), and also showed no Bohr effect, or only a very small one. The observed low oxygen afhnity of hemoglobin MIwate contrasts with the cases of hemoglobin H (1) and hemoglobin Barts (2). Although the (Y chain subunits in hemoglobin MIwate are func- tionally inactive, they interact strongly with the functionally act.ive 0 subunits in the same molecule, thus limiting seriously

the oxygen affinity of the p subunits. The decrease in the inter- action constant n therefore does not necessarily mean a decreased interaction between the different types of chains. The char- acteristic features of the oxygen equilibrium of hemoglobin M rwate remained essentially unchanged in 2 M sodium chloride, which has been shown to cleave the hemoglobin MIwate molecule into symmetrical half-molecules. Therefore, the interaction which brings about the abnormally low oxygen affinity of hemo- globin MIwate seems to work primarily between ar and /I chain subunits, but not between identical @ dimers.

The carbon monoxide affinity and the Bohr effect in the carbon monoxide equilibrium of hemoglobin Mlwate were found to in- crease to almost the same levels as those of hemoglobin A when all four subunits of hemoglobin MIwate were reduced to the fer- rous state. These results indicate that the carbon monoxide reactivity of p subunits was released from a restriction by the abnormal LY subunits (or from a strong interaction between a! and /3 subunits) when the CY subunits were enabled to bind carbon monoxide. Nevertheless, the n values of the carbon monoxide dissociation curves of completely reduced hemo- globin MIwrtte were again found to be nearly 1.0. These findings strongly suggest that conformational changes of the hemoglobin molecule responsible for the Bohr effect, the affinity for ligands, and the shape of this dissociation curve, respectively, may be different. The results of studies by Antonini et al. (26, 27) on the unusual functional properties of carboxypeptidase digests of normal human hemoglobin and on the effect of brom- thymol blue upon the oxygen equilibrium of human hemoglobin also appear to support this view. In hemoglobin MIwatc, the histidine in position 87 of the CY chains is substituted by tyrosine. This histidine in position 87 has been supposed to be the site of linkage between the protein moiety and the heme iron in the o( chain of hemoglobin A (28). It would be particularly interest- ing to compare the carbon monoxide dissociation curve of totally ferrous hemoglobin MIwate with the carbon monoxide equilibria of hemoglobin MBoston (4) or Mosaka (29), in which the substitu- tion of histidine by tyrosine in the a! chain occurs not in position 87, but in position 58.

Hemoglobin MIwate has t.wo reactive -SH groups per mole- cule, as does hemoglobin A. Blocking of the reactive -SH groups of hemoglobin MIwatc by CMB did not change the oxy- gen affinity near neutral pH, although it eliminated the Bohr effect which was otherwise observable at a higher pH. This is further evidence that the Bohr effect and the oxygen affinity are controlled by different types of interactions. It is suggestive that Taylor, Antonini, and Wyman (30) showed that the n value of hemoglobin A was not affected at all by the substitution of -SH groups by a half-cystine residue, and that Benesch and Benesch (18, 31) showed that the -SH hydrogen could be dis- placed by a number of substituents without influencing the Bohr effect of hemoglobin A.

The rates of autoxidation of hemoglobin MIwate were about 6 times higher than those of hemoglobin A under comparable pH conditions, even in 2.0 M sodium chloride. These observations again support the view that reactivities of one type of subunits in hemoglobin are affected or controlled by the other type of subunits in the same molecule. Although it is well known that the rate of autoxidation of hemoglobin is a function of oxygen pressure (32), the observed difference in rate may not be an ex- pression merely of the difference of oxygen affinity between hemoglobins MIwate and A, since the experiments were performed

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84 Functional Properties of Hemoglobin MIwat, Vol. 241, No. 1

under pure oxygen gas to ensure the full saturation of any re- active hemoglobin subunits. Somewhat higher autoxidation rates have also been reported for hemoglobin H (33)) hemoglobin Zurich (34), and a variant of hemoglobin M reported by Kiese, Kurz, and Schneider (35).

Acknowledgments-We are indebted to Dr. A. Tamura (de- ceased in 1964), Iwate Labour Casualty Hospital, Hanamaki, for his kindness in supplying the nigremia blood sample. We also wish to thank Dr. J. Wittenberg, Albert Einstein College of Medicine, for his help in preparing the manuscript.

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Norio Hayashi, Yutaro Motokawa and Goro Kikuchiiwate

Studies on Relationships between Structure and Function of Hemoglobin M

1966, 241:79-84.J. Biol. Chem. 

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