~-HE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 241, No. 1, Issue of January 10, 1966

P&ted in U.S.A.

Studies on Human Hemoglobin Treated with Various Sulfhydryl Reagents*

(Received for publication, July 12, 1965)

JOHN FULLER TAYLOB, ERALDO ANTONINI, MAURIZIO BRUNORI, AND JEFFRIES WY~~AN From the Department of Biochemistry, University of Louisville, School of Medicine, Louisville, Kentucky, and the Institute of Biochemistry, University of Rome, and “Regina Elena” Institute for Cancer Research, Rome, Italy

SUMMARY

The “reactive” sulfhydryl groups of human oxyhemoglobin (at position 93 of the fi chains) form compounds with cystine, cystamine, dimercaptoethanol, and iodoacetamide. No reac- tion occurs with dithioglycolic acid, dithiobutyric acid, or di- formylcystine. Some reaction occurs with iodoacetic acid but only under special conditions.

The general properties (stability, spectra, molecular weight) of the modified hemoglobins described in this paper (except perhaps for the hemoglobin treated with iodoacetic acid) are very similar to those of unreacted hemoglobin.

The oxygen equilibrium of these compounds has been studied. In all cases, the value of R is similar to that of normal hemoglobin (namely about 2.9) and is approximately invariant with pH. The oxygen Bohr effect is substantially reduced, and the pattern of all the results is similar in spite of the different chemical structures of the blocking reagents.

The hemoglobin treated with cystine shows differential binding of the HPOa ion as between the oxy and deoxy forms at concentrations of HP04’ in the neighborhood of 0.3 M.

This explains the discrepancy between the directly measured Bohr effect for this compound and for hemoglobin treated with cystamine and that calculated from differential titrations. On the other hand, in the case of hemoglobin treated with iodoacetamide, where the newly introduced group carries no charge, there is good agreement between the Bohr effect measured by the two methods.

The kinetics of combination of deoxyhemoglobins with car- bon monoxide and of the dissociation of 02 from oxyhemo- globins has been studied. The kinetic behavior of all the compounds described in the present work is qualitatively similar to that of normal hemoglobin, but different from that of hemoglobin treated with p-mercuribenzoate.

Study of the effects of chemical modification of a protein molecule can yield valuable information on the relation between

* This work was partly supported by research grants from the National Science Foundation (GB-1278 to J.F.T. and to J.W.) and from the United States Public Health Service (AM-02831 to J.F.T.).

structure and function if the site of the modification is known. The sulfhydryl groups of hemoglobin seem to offer an opportunity for such an approach.

As far as is now known, all mammalian hemoglobins contain at least two -SH groups (generally described as “reactive”) that combine readily with various reagents such as organic mercurial compounds. In addition, many, but not all, contain other -SH groups (“unreactive groups”) that do not react unless the mole- cule is opened up in some way, as by acid, urea, or detergents (1). In human hemoglobin Al, the freely reacting -SH groups are known to be located at position 93 of the two p chains (2, 3), and are adjacent to the histidines at position 92, which are in close contact with the iron atoms of the hemes (i.e. the proximal his- tidines) (4). Blocking these two -SH groups affects the oxygen equilibrium of the hemoglobin (2, 5, 6), but since the nature and extent of the effects produced differ with the blocking reagent, it has been concluded that the reactive -SH groups as such do not play an essential role in ligand equilibria (7).

p-Mercuribenzoate and other heavy metal reagents produce alterations in the shape of the 02 equilibrium curve, more so at some pH values than at others, as well as in the affinity of Hb for different ligands (8) ; they likewise affect its kinetic behavior (9). Treatment with N-ethylmaleimide affects the O2 equilib- rium of hemoglobin similarly (2,6).

In a previous investigation, the reactive -SH groups of human hemoglobin were blocked by treatment with an excess of cystine, leading to the formation of a mixed disulfide (5) as reported by Dolbeare (10). The only major way in which this modified hemoglobin differed from normal hemoglobin in its reaction with oxygen was that the normal (or alkaline) Bohr effect was some- what diminished whereas the reverse (or acid) Bohr effect ap- peared to be somewhat increased; the heme-heme interaction, as indicated by a value of the Hill coefficient, n E 3, remained un- changed. In contrast, treatment with glutathione has been said to produce a decreased value of n (11).

In view of the somewhat confusing results, it seemed worth- while to explore more fully and systematically the effect of other -SH reagents. In this paper, we describe the preparation of derivatives of human hemoglobin in which a basic group (the amino group of cysteamine), or a neutral group (an alcoholic group from mercaptoethanol or a carbamide methyl from iodo- acetamide) is introduced at position 093 (and there only, so far as has been determined). We have examined the ligand reac- tions of these derivatives, more particularly with respect to

241

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242 Hemoglobin Treated with Suljhydryl Reagents Vol. 241, No. 1

equilibrium, but also with brief excursions into kinetic behavior. So far, attempts to prepare any acidic derivative of hemoglobin with dithioglycolic or dithiobutyric acids, or with diformyl- cystine, have failed, although some reaction has been observed with iodoacetic acid under special conditions.

EXPERIMENTAL PROCEDURE

Hemoglobin-Human hemoglobin was prepared from freshly drawn red blood cells according to the standard procedure in use in this laboratory (12). The hemoglobin concentration was determined spectrophotometrically on the basis of the values of the extinction coefficients already given (13). Globin was

TABLE I

Experiments on preparation of human hemoglobin with modi$ed sulfhydryl groups

The reactions were always performed at 4”, except for iodoacet- amide, which was used at 20”.

Reagent and No. of experiments performed

Cystine 1

4 1

Cystamine

2 1

1

3 Diformylcystine

1 Dimercaptoetha-

no1 1 1 1

Glutathione 1

Dithiobutyrate

Dithioglycolate 1 1 1

1

1

Iodoacetamide 3

Iodoacetate

PH Solvent*

10 9.6 P 50 9.6 P

500 9.8 P

10

50 50 50 50

16.5 P

50 50 50

P B B

25

9.5 9.5 9.0 8.0 7.0

8.8

7.0 9.0 9.0

7.0

7.0 9.0

7.0 9.0 7.0 9.0 7.0 9.0

7.0

9.5 8.5 9.1 9.9

P

50 50

P P

50 50 50 50 50 50

P P

P f 2 M NaCl P + 2 M NaCl P f 5 M NaCl P + 5 M NaCl

10

10 22.5 25 22.5

P P P P

Time of ex- msure

hrs

20 20 20

0 0 0

20 20 20 20 20

20 2

20 1.6 20 0.4 44 0.1t

20

16 16

16 16 40 40 40 40

1

20 20 20 20

1.5

2 2

2 2 2 2 2 2

0

0.9 0.75 0.5 0

* P = 0.1 M potassium phosphate buffers; B = 2% borate. t After second treatment. This experiment was performed by

adding fresh reagent to the dialyzed product of the preceding ex- periment and allowing the reaction to proceed for an additional 24 hours.

prepared by the acid-acetone procedure of Rossi-Fanelli, An- tonini, and Caputo (14).

D&u&ides-Cystine was obtained from Merck. Chromato- graphically pure cystamine and diformylcystine were a generous gift from Professor D. Cavallini and coworkers. Dithioglycolic acid, dithiobutyric acid, and P-mercaptoethanol were obtained from Fluka and used without further purification.

Dimercaptoethanol was prepared from the corresponding thiol by cautious oxidation with hydrogen peroxide at pH 9. Dilute H202 (about 5%) was added slowly, keeping the solution cold, until the nitroprusside reaction became negative. This occurred when only a slight excess over the calculated amount had been added. At this point, addition of cyanide, which regenerates free -SH groups, restored the nitroprusside reaction. Excess HzOz was removed by addition of catalase and the solution was used without further purification.

Other Sulfhydryl Reagents-p-Mercuribenzoate and iodoacetam- ide were obtained from Sigma; iodoacetic acid was a commercial product recrystallized by Dr. C. Turano.

Determination of -SH Groups-This was carried out by spec- trophotometric titration with p-mercuribenzoate, according to Boyer (15). Determination of the total number of sulfhydryl groups was made in 0.1 y0 sodium dodecyl sulfate.

Oxygen Dissociation Curves-These were determined by the spectrophotometric method of Rossi-Fanelli and Antonini (16). The hemoglobin concentration was always about 5 mg per ml.

Diflerential Titrations-These were carried out as described in an earlier paper (17). The difference in prot.on bound between Hb and HbOt was measured directly by titrating back to the original pH the unbuffered hemoglobin solution after oxygena- tion.

Kinetic Measurements-The kinetics of the reaction of hemo- globin with 02 or CO were measured with a Gibson stopped flow apparatus (18).

RESULTS

Preparation and Some General Properties of Modified Hemo- globins-The attempted reactions were always performed on oxyhemoglobin in aqueous solution at a protein concentration between 2 and 4%. The conditions (pH, salt concentration, temperature, time of exposure, and concentration of the reagent) are specified in Table I, which summarizes all the results.

After the attempted reaction, the hemoglobin solutions were dialyzed in the cold against several changes of distilled water with continuous mechanical stirring for about 48 hours. In the case of cystine, most of the unreacted disulfide was removed prior to dialysis by precipitation at neutral pH, at which cystine is insoluble. In all cases, at the end of the dialysis the hemo- globin solutions were filtered to remove the small amounts of insoluble products, and the number of reactive -SH groups was determined. The results of these determinations are reported in Table I. The modified hemoglobins prepared in this way showed no change of properties (spectra, number of reactive -SH groups, oxygen equilibrium) over a period of at least 10 days when stored at 2-4”.

Table I shows that treatment with cystine, cystamine, and dimercaptoethanol under a wide range of conditions results in the complete abolition of the two reactive -SH groups of the protein. On the other hand, the SH content of hemoglobin remained essentially unchanged after prolonged exposure to diformylcystine, dithioglycolic acid, and dithiobutyric acid even

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Issue of January 10, 1966 .I. F. Taylor, E. Antonini, M. Rrunori, and J. Wynzan

when these reagents wrre present in a large excess, and, in t,he case of dithioglycolate, in wncentrat,ed sodium c~hloride (2 to 5 Xl).

As reported in the literature (19)) iodoacetamidr rracted easily with t’he reactive --SH groups of oxyhemoglobin at neutral pH; in cont,rast,, the reactivity of iodoacetic acid was much kiss and it gave a substantial abolition of the --SI-I groups only at high pIT and then only \vit,h high concentrations or the reagent.

In a few tikations wit.h p-mercuribcnzoat,e in 0.1 y0 sodium dodrcpl sulfate, unkeated human Hb showed -5 reactive --SH groul)s and c:ystamine-trrated hemoplobin showed -3; t’his ~oultl indicate that, t,he rragent did not react est.cnsively, if at all, with any of the “ma&cd” --SH groups at positions PI 12 and a!1 04.

Xo significant amounts of i’crrihemoglobin were dct,ected after csposuw to any of ihc reagents or during storage in the cold for srveral days. This is at \:ariance wit,h what was found by Huisman and I)ozy (11) in the case 01’ the reaction 01 human hemoglobin w&h oxidized glutat’hione, but thr experimental conditions were different. in t’hc t\To cases. Vndrr our cond- t,ions, this reaction was incomplete (as report’ed in Table I).

All modified hemoglobins described here showed just, the same speckal properties, in the visible range, as untreated hemo- globin. They also shoTTed t,he same stabilit,\- during storage or in the course of lhe manipulat’ions.

It, was found that’ when the heme is removed from untreat’ed hemoglobin the resultin, h (I &bin shows one reactive -SH group per q3 unit. This would seem to be the same as Ihe one reactive --SH group per GYP unit’ in the normal protein. It. was found that, t,he free -SII groups oi globin react wit,h cysl~amine in t,he same way as the free --SH groups of hemoglobin. Both facts are of interest, in view of the prosimity of t’he heme t’o the -SH group in lmskion 93 of the /3 chains.

It has already been shown that cynline-treat’ed hemoglobin has t,he same sedimentat,ion behavior as normal hemoglobin (5). In this study, it was found that cysta~iiine-t’reated hemoglobin also has the same sedimentation properties. Thus, s40,2c has a value of about, 4.4 at, a hemoglobin concentration ol’ 5 mg per ml in 0.005 M pot,assium phosphat,e at’ pH 7 in bot’h cases. It was found t,hat cgst’ine-treated hemoglobin in concentrated NaCl solut,ions shows the same drop in the sediment’at’ion coefficient as untreated human hemoglobin (12) (~20,~~ = 3.3, Hb concent,ra- t,ion = 6 mg per ml; 2 M NaCl + 0.1 potassium phosphate; pH 7). We therefore conclude that, at’ least-’ in t,he case of cystJim? and cystamine-treated hemoglobins, there is no considerable modification of the assoc~iat,ioa-dissociation properties of the prokin.

Ox!/gen Equilibrium of JIod$;fLed Hemoglobins-All the experi- ments described here were performed with preparations that show~l compkte absence of react’ive -SH groups. It, was shown t,hat,, for any of Ihr reagent’s used, provided the --SH I?;roups had been complrtely covered, the O2 equilibrium of different preparations was the same irrespectire of the conditions used during the rcact,ion between hemoglobin and the -SH reagent. Moreovel~, several control ctxperirnents on human hemoglobin subjected to t’he same t,realment involved in t’he prcparat~ion of the modified hemoglobin, except’ for the addit’ion of t,he -SH reagent,, showed that’ the oxygen equilibrium was unchanged; this was txue also in the case ol hernoplobin treated Lvit,h acidic disulfides, which failed to abolish the reactive --SH grou~l".

FIG. 1. Oxygen Bohr effect :tlld valws ot 7~ (inset) for cysline- treated hrmoglobill (5 mg per 1~11) at 20”. 0, resulis ill 0.2 IV phosphate; A, results in 0.4 ;M wetale; Cl, rcsull in 0.05 w horntr; 0, results in 0.1 M phosphate bufer to reproduce (~ollditionb previous1.v used (- - -) (5). A., rrsult in 0.4 \I phosph:~lr. - -, unmodified hemoglobin.

0 5 6 7

PH 8 9

FIG. 2. Oxygen Bohr eflect and values of n (inset) lor cys- tamine-treated hemoglobin (5 rrlg per ml) al 20”. 0, results in 0.2 M phosphate; A, results in 0.1 M acelale; 0, results in 0.05 M borate; -- - -, ulrmodified hemoglolArr.

6 7 PH '

Y 10

FIG. 3. Oxygen Bohr effect a11d values of n (inset) for dimer- captoethanol-treated hemoglobin (5 mg per ml) at 20”. 0, re- sults in 0.2 M phosphak; A, resull in 0.4 M acet,ate; U, result in 0.05 M borate; - - -, unmodified hemoglobin.

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244 Hemoglobin Treated with Suljhydryl Reagents Vol. 241, No. 1

1.E

1.c

2.

QOF Q .- 2

C

0.E 6 7

P” 6 9 10

Fro. 4. Oxygen Bohr effect and values of n (inset) for hemo- globin (5 mg per ml) treated with iodoacetamide (open symbols) and iodoacetate (closed sl/mboZs) at 20”. 0 and 0, results in 0.2 M phosphate; a and A, results in 0.4 M acetate; q and n , results in 0.05 M borate; ---, unmodified hemoglobin.

04 r 03 -__-- N 2 B 0.2 -~

:

O1 -o-----

/‘ - 0.5

log (HPO,=) (M)

FIG. 5. Effect of HPO; on log p) at pH ~8.6 and 20” for cystine- treated hemoglobin. Concentration of borate buffer in all cases was between 0.5and 1%. Value of d log pi/d log (HPOd”) given by - - is 1 f 0.1.

The results on the oxygen equilibrium of the modified hemo- vlobins in acetate, phosphate, and borate buffers at an ionic a strength from 0.3 to 0.6 and pH from about 5 to 9 are reported in Figs. 1 to 4. The oxygen equilibria are described in terms of the two parameters, log p+ and n (20). For all the hemoglobins examined at any pH, the individual 02 dissociation curves gave a linear plot of log [y/(1 - I’)] versus log p for values of 7 between 0.1 and 0.9; 7 is the fractional saturation with oxygen. Each inset in Figs. 1 to 4 shows that for all the modified hemoglobins the value of n (a measure of the heme-heme interactions) is near 3, as it is for untreated human hemoglobin under the same condi- tions. On the other hand, the curves of log p+ versus pH (the Bohr effect curves) show large differences in comparison with those of normal hemoglobin.

The Bohr effect curve for cystine-treated hemoglobin shown

in Fig. 1 is significantly different from that reported in the pre- vious paper (5). It was soon discovered that the difference was caused by the different concentrat.ions of the buffers employed in the two sets of experiments; control experiments in a buffer of the same composition as the one used before confirmed this hypothesis. These results suggest the presence of unusually

O5 6 7 PH

8 9 10

FIG. 6. Oxygen Bohr effect and values of n (inset) for cystine- treated hemoglobin (5 mg per ml) in 2 M NaCl at 20”. -, Bohr effect as obtained from differential titrations under the same condit.ions (see Fig. 8) ; - - -, unmodified hemoglobin in 2 M NaCl (see Antonini et al. (17)).

T-

i5d 0 0.5 1.0 1.5

log p

FIG. 7. Values of log [?/ (1 - Y)] versus log p for cystine-treated hemoglobin (5 mg per ml) in 2 M NaCl, pH 7.1, 20”. Different symbols indicate different experiments. The slope of the line is 3.

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Issue of January lo,1966 J. F. Taylor, E. Anton&i, M. Brunori, and J. Wyman 245

large specific ion effect’s in some of these modified hemoglobins. Several additional sets of experiments were undertaken to explore this possibility.

The first set involved a study of the effect on log p+ of varying the concentration of the HPOg ion, at a constant pH of about 8.6, in the cystine-treated hemoglobin. At this pH, since the buffering power of phosphate is almost negligible, the pH was controlled with borate buffer at a concentration of 0.5 to 1.0%. The results are reported in Fig. 5, which shows log p+ versus log (HP04=). It will be seen that a transition from log p+ = 0.09 to log p: = 0.36, which corresponds to an approximately 2-fold change in p*, is realized within a range of log HP04= of less than

-05’ I I I I

10

I /

10 I

I

I

6 7 PH 8

9 10

FIG. 8. Difference in the number of protons bound (per heme) by deoxy- and oxyhemoglobin (AH+) as a function of pH at 20’. Top panel, results for cystine-treated hemoglobin in 0.3 M NaCl (0) and 2.0 M NaCl (0); middle panel, results for cystamine- treated hemoglobin in 0.3 M N&l (0); Zvlcrer panel, results for iodoacetamide-treated hemoglobin in 0.3 M NaCl (0). Hemo- globin concentration was in all cases between 5 and 15 mg per ml. ---9 results for unmodified hemoglobin obtained under the same

FIG. 9. Comparison of directly observed values of log p$ (C-0 and O---O) with those calculated from differential titrations (- - -) for cystine-treated Hb (fop panel), cystamine- treated hemoglobin (middle panel), and iodoacetamide-treated hemoglobin (lower panel). All data at 20”. For cystine-treated Hb are included points in 0.1 and 0.2 M phosphate.

TABLE II

Dissociation of oxygen from oxyhemoglobins in presence of NuzSz04 at 20”

The values of the first order rate constants given here are the average of observations made at two wave lengths (560 and 577 mp). The two values agree within 5%.

Hemoglobin derivative

Normal

Cystine-treated

Cystamine- treated

Iodoacetamide- treated

p-Mercuriben- zoate-treated (2 moles/mole of heme)

p-Mercuriben- zoate-treated (20 moles/mole of heme)

pH 7.0 pH 9.15

kcsec-‘)

40

55

68

36

47 (initial) 12 (final)

54 (initial) 12 (final)

i-

Kinetic behavior

Homoge- neous

Homoge- neous

Homoge- neous

Homoge- neous

Grossly di- phasic

Grossly di- phasic

kcsec-3

14

18

24 (initial) 15 (final) 14

25 (initial) 14 (final)

24 (initial) 12 (final)

Kinetic behavior

Homoge- neous

Homoge- neous

Diphasic

Homoge- neous

Grossly di- phasic

Grossly di- phasic

1 unit. The value of d log ps/d log (HPOP) at the midpoint of the transition is very nearly 1.

The second set consisted of measurements of the oxygen eauilibrium of the cvstine-treated hemodobin in concentrated Y conditions (21).

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246 Hemoglobin Treated with Sul~hydryl Reagents Vol. 241, No. 1

TABLE III

Combination of carbon monoxide with deoxyhemoglobin at pH 7 and 20”

Hemoglobin concentrations, 2.5 ELM; 0.1 M potassium phosphate buffer. Observations at X = 421 and 430 mp.

Hemoglobin derivative

Norrnd. Cystine-treated.. Cystktmine-treated. Iodoacetamidc-treated. p-Mercuribenzoate-treated

(2 moles/mole of heme). p-Mercurihenzoate-treated

(20 moles/mole of heme).

Initial vcond order rate con-

stant from evtrapolatlon at zero time

Y-1 St--’ x 10-j

0.95 0.95 1.50 1.45

1.45

1.50

Kinetic behavior

Autocatalyt’ic Autocatalytic Autocatalytic Autocatalytic

Nonautocatalytic

Nonautocatalytic

(2 $7) N&l solutions. The results of these experiments are shown in Fig. 6. As in the case of normal hemoglobin, high concentration of salt reduces the Bohr effect and decreases the oxygen affinity, especially at alkaline pH. As in the case of normal hemoglobin, the value of n remains unchanged, or even increases slightly, in 2 M NaCl (Fig. 7), although the sedimenta- tion experiments show dissociation of the oxygenated molecule into halves.

The third set of experiments consisted of differential titrations in 0.3 M NaCl of the oxy and deoxy derivatives of the hemo- globins treated with cystine, cystamine, and iodoacetamide. Specific ion effects would be expected to show up as a discrepancy between the Bohr cffeet as directly measured and as calculated from such titrations. The results of these experiments, which are reported in Fig. 8, show that the difference in proton bound be- tween Hb and HbOs is reduced after abolition of the reactive -SH groups. The Bohr effect curve (log p: versus pH) cal-

culated by graphical integration (17) from these data for each of the modified hemoglobins is compared with the directly ob- served curve in Fig. 9. While for iodoacetamide-treated Hb,

as for normal Hb, the Bohr effect is about the same whether measured by O2 equilibrium in the presence of a buffer or by differential titrations, there is a large difference in the case of cystine and cystamine-treated hemoglobins. This finding will be discussed lat’er.

Kinetic Experiments on Modified Ilemoglobins-The results on the O2 equilibrium of the modified hemoglobins described above led us to investigate the kinetics of their reactions with gaseous ligands. In spite of the difficulty of interpreting such kinetic

1

0

0

W- 4

0

:

a

I0

0

.“r

i 5 20 40

.msec

FIG. 11. Dissociation of oxygen from oxyhemoglobin in pre- sence of Na&04 in 0.1 M phosphate bufrer, pH 7.0 and 20”. He- moglobin concentrations, 1 mg per ml in every case. 0, results for unmodified hemoglobin; (>, results for cystine-t.reated hemoglo- biu; l , results for p-mercuribenaoate-treated hemoglobin (20 moles per mole of heme). Observations at X = 5G0 mp.

W” a W0 a 8

t

0 0 50 100 150 200 250 300

--+ msec

FIG. 10. Combination of carbon monoxide wit,h deoxyhemoglobin in 0.1 M phosphate buffer at pH 7.0 and 20”. Hemoglobin concent,ration of 0.1 mg per ml iu every case. 0: results for unmodified hemoglobin; (>, results for iodoacetamide-treated hemoglo- bin; 0, results for p-mercuribensoate-t.reated hemoglobin (20 moles per mole of heme). Observations at X = 421 mp.

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Issue of January 10, 1966 J. F. Taylor, E. Antonini, M. Brunori, and J. Wyman 247

neasurements, these results may be useful for a qualitative :xplanation of the changes of behavior brought about by the various modifications of the protein. Kinetic measurements also seemed interesting in view of the gross changes in the kinetics of lemoglobin treated with p-mercuribenzoate reported by Gibson tnd Roughton (9).

In the present case, the kinetics of the over-all combination of leoxyhemoglobin with carbon monoxide and the over-all dis- sociation of 02 from HbOz in the presence of Na2S204 were in- gestigated. Parallel measurements were carried out on the various modified hemoglobins, on normal hemoglobin, and on lemoglobin treated with p-mercuribenzoate. The results are meported in Tables II and III and Figs. 10 and 11.

It is evident that, at least qualitatively, the kinetic behavior of the modified hemoglobins described in the present paper is similar to that of normal hemoglobin and different from that of hemoglobin treated with p-mercuribenzoate.

DISCUSSION

Treatment of hemoglobin with an excess of certain disulfide compounds leads to disappearance of the reactive -SH groups of the protein, presumably due to the formation of a mixed disul- tide (5, 10). Such treatment provides a mild and specific way of blocking these -SH groups and also offers the opportunity of introducing different chemical groups into the protein molecule at a known point, namely position 93 of the p chain. Of the various disulfides tried in the present experiments, only certain ones reacted. These were cystine, cystamine, and dimercapto- ethanol. In each case, the reaction is highly specific, in the sense that only the free or reactive -SH groups of the protein appear to be covered. This is primarily shown by the fact that the masked or unreactive -SH groups (at position ,8112 and ~~164) remain unchanged after the treatment.

It will be seen from Table I that, with one exception (cystine), disulfides containing carboxyl groups fail to react. This would suggest that electrostatic effects involving negative charges in the neighborhood of the reactive -SH groups play an important role in determining the reaction, and in this connection it is significant that residue fl94, the one next to the reactive -SH group, is aspartic acid. The exception, cystine, contains two amino groups which ionize at a pH close to that of the experi- ments. However, the failure of the other compounds to react persisted even in 2 to 5 M NaCl, at which electrostatic interac- tions should be largely eliminated by the shielding effect of the salt.

The reaction behavior of the disulfides seems to be duplicated in the case of the alkylating agents. Thus, the uncharged iodoacetamide molecule reacts readily with free -SH groups to give a homogeneous and stable compound. On the other hand, the negatively charged iodoacetate ion gives slow and incomplete reaction and even that only under special conditions. The rather extreme conditions used in the iodoacetate reaction might give rise to nonspecific modification of the protein or to reaction at other points of the molecule.

The spectra, stability, and behavior in the ultracentrifuge of the modified hemoglobins, except perhaps for the iodoacetic acid-treated hemoglobin, which was not studied further, are very similar to those of untreated hemoglobin and indicate that the treatment employed is a very mild one which leaves most of the general properties of the molecule unchanged.

The central feature of the present work is represented by the

results on the oxygen equilibrium of the various modified hemo- globins. As mentioned in the introduction, it has been suggested that the -SH groups of hemoglobin play a prominent role in the heme-heme interaction and in the Bohr effect. This was based mainly on experiments on the oxygen equilibrium of hemo- globin treated with mercurial -SH reagents (2). However, later results on cystine-treated hemoglobin showed that, at least in this compound, abolition of the reactive -SH groups was not accompanied by any significant change in the heme-heme interaction (5). The much more extensive data presented here on the five modified hemoglobins with no free -SH groups sub- stantiate the conclusion that these groups are not directly in- volved in the heme-heme interactions. These compounds all show a high value of n, about the same as in normal hemoglobin, which is nearly independent of pH.

The value of n for hemoglobin treated with iodoacetamide as reported in the past, is lower than that reported here (2). This might be due to an incomplete reaction of the -SH groups in the previous experiments, since functionally heterogeneous ma- terial will show a value of n lower than that of the individual components of the mixture.

The characteristics of the Bohr effect in the various modified hemoglobins are brought out in Figs. 1 to 4. Although there are significant differences between them, all the compounds show a Bohr effect smaller than that of normal hemoglobin. At the same time, they all have a considerably increased oxygen affin- ity, particularly at low pH. In this respect, they differ from the phenylmercuric hydroxide derivative studied by Snow (22), which has the same Bohr effect as normal hemoglobin.

Benesch and Benesch (6) reported that the reaction of the -SH groups at position 93 in the /3 chains, with iodoacetamide or mercurials, produced no change in the Bohr effect. This conclusion was reached on the basis of measurements of Afi+ (difference in proton-binding capacity on oxygenation) at pH 7.3. The Bohr effect of hemoglobin treated with iodoacetamide has been studied here over a lafge pH range, measuring log p+ as a function of either pH or AH+. The results show that, although the maximum value of Al?+ is not greatly changed, the “tota.1” Bohr effect is smaller than in normal hemoglobin (Fig. 8).

The fact that a substantial part of the Bohr effect remains even when the -SH groups are covered by an uncharged substituent (mercaptoethanol or iodoacetamide) is strong evidence against the idea that the -SH groups are themselves a direct source of Bohr protons. The similarity of the Bohr effects in the deriva- tives that had been treated with cystine, cystamine, and dimer- captoethanol shows further that the newly introduced ionizable groups are not oxygen-linked even though they are located in a region of the molecule which is greatly affected by the conforma- tional change accompanying oxygenation (23).

Perhaps the most interesting feature of the results is that brought out in Fig. 5, namely the great sensitivity of the oxygen affinity of the cystine-treated hemoglobin to small concentrations of (HP04-) ion. This would suggest a very specific binding of that ion by the protein over a small and critical concentration range, a matter which will be analyzed in detail later on in another connection. It would seem to correspond to t,he lack of agreement between the Bohr effect as directly observed in phosphate buffers of concentration -0.2 M and as calculated from differential titrations in the absence of buffer in sodium chloride solutions (see Fig. 9). This lack of agreement is es- pecially evident in hemoglobin treated with either cystine or

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248 Hemoglobin Treated with Sulfhydryl Reagents Vol. 241, No. 1

cystamine, in both of which the newly introduced group carries 6. BENESCH, R., AND BENESCH, R. E., J. Biol. Chem., 236, 405

a charge, a single positive charge in the case of cystamine and (1961).

one positive and one negative charge representing a dipolar ion 7. ROSSI-FANELLI, A., ANTONINI, E., AND C~PUTO, A., Advances

in the case of cysteine; in contrast, in the compound treated with in Protein Chem., 19, 74 (1964).

iodoacetamide, this discrepancy is absent or extremely small, as 8. RIGGS, A., AND WOLBSCH, R. A., J. Gen. Physiol., 39, 585

(1956). it is in normal hemoglobin. 9. GIBSON, Q. H., .~ND ROUGHTON, F. J. W., Proc. Roy. Sot. Lon-

The kinetic data are in qualitative agreement with the equilib- don, Ser. B, 143, 310 (1955).

rium results. The shapes of the kinetic curves for both the dis- lo* DoLBEAREt F. A.~ Doctoral dissertation, University of

sociation and combination reactions of the disulfides and iodo- Louisville, 1961.

acetamide-treated hemoglobins are the same as those of normal 11. HUISMAN, T. H. J., AND DOZY, A. M., J. Lab. Clin. Med., 60,

302 (1962). hemoglobin, although there may be some alteration in the abso- 12. ROSSI-FANELLI, A., ANTONINI, E., AND CAPUTO, A., J. Biol. lute values of the constants. In this respect, the compounds Chem., 236, 391 (1961). studied in this paper are different from hemoglobin in which the 13. ANTONINI, E., BRUNORI, M., CAPUTO, A., CHIANCONE: E.,

-SH groups have reacted with p-mercuribenzoate. In the RO~SI-FANELLI, A., AND WYMLN, J., Biochim. et Biophys.

latter case, the kinetics of the hemoglobin reactions are char- Acta, 79, 284 (1964).

acterized by a marked heterogeneity which, like the decrease of n 14. ROSSI-FANELLI, A., ANTONINI, E., AND CAPUTO, A., Biochim.

et Biophys. Acta, 30, 608 (1958). observed in the oxygen equilibrium (8), may result from the pres- 15. BOYER, P. D., J. Am. Chem. Sot., ‘76, 4331 (1954). ence of the reagent in the framework of the polypeptide chains 16. ROSSI-FANELLI, A., AND ANTONINI, E., Arch. Biochem. Bio-

rather than from the abolition of the reactive -SH groups. phys., 77, 478 (1958). 17. ANTONINI, E., WYMAN, J., BRUNORI, M., BUCCI, E., FRONTI-

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John Fuller Taylor, Eraldo Antonini, Maurizio Brunori and Jeffries WymanStudies on Human Hemoglobin Treated with Various Sulfhydryl Reagents

1966, 241:241-248.J. Biol. Chem. 

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