Biochem. J. (1978) 169, 625-632Printed in Great Britain

The Oxygen-Linked Zinc-Binding Site of Human Haemoglobin

By JOHN G. GILMAN* and GEORGE J. BREWERtCattedra di Enzimologia, Universita diMilano, Via G. Celoria 2, 20133 Milano, Italy

(Received 14 July 1977)

Zn2+ is known to increase the 02 affinity of human haemoglobin. Previous data suggestedthat Zn2+ exerts its effect by directly binding to haemoglobin, rather than by competingwith or binding to 2,3-bisphosphoglycerate. It was also shown that there are two 02-linkedzinc-binding sites in haemoglobin, and that Zn2+ does not significantly alter haemoglobinco-operativity or the alkaline Bohr effect. The effect of Zn2+ on 02 affinity of haemo-globin can also be observed for other haemoglobins as diverse as those ofcow and chicken.This paper presents new data on the haemoglobin-zinc interaction for normal haemo-globin, des-His46fl-haemoglobin and N-ethylsuccinimide-haemoglobin of humans.For normal haemoglobin (0.05mm in tetramers), at 20°C in buffer containing 0.1 M-C1-,02-dissociation-curve experiments showed that the addition of 0.4-0.5 mM-ZnSO4 did notchange the Bohr effect between pH6.71 and 7.29. Similar experiments, with 'zinc-ionbuffers', showed that the value of the Hill coefficient, h, decreased only slightly if theconcentration of free Zn2+ was held constant. For N-ethylsuccinimide-haemoglobin,Zn2+ caused less increase in 02 affinity than for normal haemoglobin. These studies,together with data on the equilibrium binding of Zn2+ to oxy-, deoxy- and des-His1460-haemoglobins, suggest that zinc is chelated in oxyhaemoglobin by at least three aminoacids, two of which are histidine-146fl and cysteine-93fl.

Whilestudying zinc deficiency in sickle-cell anaemia,Oelshlegel et al. (1973) found that Zn2+ increases the02 affinity of human haemoglobin. In the presenceof 2,3-bisphosphoglycerate at 37°C, this 'left-shifting'of the 02-dissociation curve by Zn2+ did not alter thealkaline Bohr effect. Zn2+ appeared to bind stronglyto the haemoglobin and not to exert its effect bycompeting with 2,3-bisphosphoglycerate binding(Oelschlegel et al., 1973, 1974).Gilman et al. (1975) demonstrated the presence of

two strong zinc-binding sites in both human and cow.haemoglobin. They showed that Zn2+ increases the02 affinity ofcow haemoglobin at 37°C (in the absenceof 2,3-bisphosphoglycerate) to about the same extentas for human haemoglobin (in the presence of 2,3-bisphosphoglycerate). Zn2+ did not appear to cause

any significant decrease in haemoglobin co-opera-tivity. Their data also showed that Zn2+ increasesthe 02 affinity of chicken haemoglobin, whichsuggested the relative lack of evolutionary variabilityat the 02-linked zinc-binding site.

Abbreviations used: Bistris, 2-[bis-(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1l,3-diol; zincon, o-{2-[a - (2 - hydroxy - 5 - sulphophenylazo)benzylidene]hydra-zino}benzoic acid, sodium salt.

* To whom reprint requests should be sent, at theDepartment of Chemistry, University of Indiana,Bloomington, IN 47401, U.S.A.

t Permanent address: Department of Human Genetics,University of Michigan, Ann Arbor, MI 48104, U.S.A.

Vol. 169

We here provide data concerning the effect ofZn2+ on the O2-binding behaviour of human normaland N-ethylsuccinimide-haemoglobin, in the absenceof 2,3-bisphosphoglycerate at 20°C. A study of theequilibrium binding of Zn2+ to normal human oxy-and deoxy-haemoglobins, and to human carbon-monoxy-des-Hisl46"-haemoglobin, is also pre-sented. Our results are consistent with the hypothesisthat Zn2+ is chelated in normal human oxyhaemo-globin by at least three amino acids, two of which arehistidine-146fl and cysteine-93/J.

Experimental

Haemoglobin

Haemoglobin was prepared as described byPerrella et al. (1972), with the addition of an equili-bration step in 1 mM-KOH followed by deionizationon a mixed-bed ion-exchange resin column. Thehaemoglobin (1.5-2mM-tetramers, in 0.1 M-KCI)was stored under N2 in Pyrex bottles in the cold(under these conditions, the methaemoglobin contentremained virtually zero for more than 2 months).The des-His146l-haemoglobin (Kilmartin et

al., 1975) was generously provided by Dr. J. V.Kilmartin, Medical Research Council Laboratoryof Molecular Biology, Cambridge CB2 2QH, U.K.

N-Ethylsuccinimide-haemoglobin was preparedby reaction of N-ethylmaleimide with cysteine-93f,

625

J. G. GILMAN AND G. J. BREWER

by a method similar to that of Riggs (1961): N-ethyl-maleimide was added to the haemoglobin (in Bistrisbuffer, pH7.02) to a concentration of 1.OmM, andthe reaction was allowed to proceed for 1 h at roomtemperature (23°C).Throughout this paper, haemoglobin concen-

trations are given for tetramers (and generally areeither 0.05mm or 0.1 mM).

Buffers

The pH 7.02 Bistris buffer (0.05M-Bistris/0.1 M-Cl-)for measuring 02-dissociation curves was made witha 1:5 dilution of stock concentrated buffer (0.25M-Bistris/0.081M-HCI containing 0.019M-NaCl); afteraddition ofconcentrated haemoglobin (in 0.1 M-KCI),final volume was reached by adding 0.1 M-NaClsolution. For the pH 7.29 buffer (0.05M-Bistris/0.1M-C-), dilutions were made as for the pH7.02buffer, but with the addition of 6Opl of 1 M-NaOHper lOml of buffer. The pH6.71 buffer (0.05M-Bistris/0.1 M-CI-) was made by mixing equalquantities of the pH 7.02 buffer with a pH 6.38 buffer(0.05 M-Bistris/0.034M-HCI/0.068 M-NaCI). All pHvalues were checked at 20°C, and were always within0.015pH unit of the expected value.

All solutions containing Zn2+ were made fromstock 0.1 M-ZnSO4 (Merck Titrisol).For 02-dissociation curves made with buffers

containing 5mM-NN-bis(carboxymethyl)glycine, thebuffers were made as for pH7.02 above, but withthe addition of a 1:10 dilution of NN-bis(carboxy-methyl)glycine stock solution. Two NN-bis(carboxy-methyl)glycine stock solutions at pH7 were used,one with zinc (0.05 M-NN-bis(carboxymethylglycine/0.045M-ZnSO4/0.1 M-NaCl/0.15M-NaOH) and theother without zinc (0.05M-NN-bis(carboxymethyl)-glycine/O.1M-NaCl/O.1M-NaOH). The final pH ofthe NN-bis(carboxymethyl)glycine-containing solu-tions was 7.06.

02-dissociation curves

The method of Riggs (1951) was used on 0.05mM-haemoglobin, in buffers containing 0.1 M-Cl-. Detailsare as described by Kilmartin & Rossi-Bernardi(1971). Equilibration was for 20min at 20°C, and theA558 was read with a Cary 118 spectrophotometer.For experiments carried out in the presence of

Zn2+, small quantities (up to lOO,ul) of 20mM-ZnS04solution (containing 0.1 M-NaCI) were injected intothe 4ml of deoxyhaemoglobin in the glass tonometer.For experiments in NN-bis(carboxymethyl)glycine-

containing buffers, small quantities of 20mM-ZnSO4were also injected. Since NN-bis(carboxymethyl)-glycine is an effective zinc chelator, the free concen-tration, A, of Zn2+ is given approximately byA = [ZnT]KE, /([NN-bis(carboxymethyl)glycine] -

[ZnT]), where [ZnT] is the total concentration ofZn2+ present, and K1, is the apparent dissociationconstant for the binding of Zn2+ to NN-bis(carboxy-methyl)glycine, as discussed by Cohen & Wilson(1966). These authors quote the pK for the dissocia-tion ofthe third hydrogen ofNN-bis(carboxymethyl)-glycine as 9.73, and the pK for the dissociation of thechelate of Zn2+ with unprotonated NN-bis(carboxy-methyl)glycine as 10.67 (both values measured at20°C in 0.1M-KNO3); they also present a formulathat allows calculation of K., as 10-pH (10-1067/10-9-73). Then, K,, may be calculated at 1O-8M at thepH ofour experiments (pH 7.06). However, this valuemay not be precise for the conditions of our experi-ments, because they were conducted in the presenceof0.095 M-Na+ and 0.003M-K+ [for buffers made withthe zinc-containing NN-bis(carboxymethyl)glycinestock solutions], rather than in 0.1 M-K+.

Zinc-binding studies

Two different methods were used for measuring theequilibrium binding of Zn2+ to haemoglobin, namelyequilibrium dialysis and a ApH method that is not inprinciple very different from the AH+ method ofBenesch et al. (1976).For equilibrium dialysis experiments, ZnSO4

was added to the external buffer, in which wasimmersed a dialysis sac containing 4ml of haemo-globin (in the same buffer); equilibration proceededfor 48 h at 20°C. For 0.05mM-carbonmonoxy normalhuman haemoglobin, 30ml of CO-saturated externalbuffer (0.01 M-Bistris/0.1 M-NaCl) was either at pH6.6or pH7.0 (pH adjusted with HCl). For 0.05mnM-carbonmonoxy-des-Hisl46l-haemoglobin, 30ml ofCO-saturated external buffer (0.01 M-Bistris/0.25M-NaCl) was at pH 7.0. For experiments on 0.1mM-deoxy normal haemoglobin, the haemoglobin wasfirst reduced under N2 in an IL237 tonometer(Instrumentation Laboratory, Lexington, MA,U.S.A.). It was quickly placed in a dialysis sac, whichwas then immersed in 250ml of N2-bubbled externalbuffer (0.01 M-Bistris/0.25M-NaCl, pH7.3). Afterwaiting several hours to ensure thorough deoxy-genation, ifthe haemoglobin wasjudged deoxy (on thebasis of its colour), ZnSO4 was then added to theexternal buffer.

After dialysis, zinc was analysed by the zinconmethod (McCall et al., 1958), with absorption meas-ured at 620nm (haemoglobin was precipitated withtrichloroacetic acid). Haemoglobin concentrationswere determined spectrophotometrically, at 540nm,on cyanmethaemoglobin, by the method of Drabkin(1950), by using the absorption coefficient reported byvan Kampen & Zijlstra (1965). Methaemoglobin wasmeasured by the method of Kilmartin & Rossi-Bernardi (1971), and was always less than 10% oftotal haemoglobin, at the end of the experiment.

1978

626

HAEMOGLOBIN-ZINC INTERACTION

For the ApH method of measuring Zn2+ binding tohaemoglobin, small amounts of ZnSO4 were addedto unbuffered haemoglobin solutions (containing0.25M-NaCl). The binding of Zn2+ to haemoglobindisplaces protons, and also causes a charge change forthe haemoglobin molecule, so that the pH of thesolution changes. If the pH change associated withstoicheiometric binding is known, the concentrationof zinc that has bound can be calculated. This issimply the ratio of the pH change observed to thatexpected for stoicheiometric binding, multiplied bythe concentration ofZn2+ added. The Zn2+ that is notbound is free, so that, by making successive additionsof ZnSO4 and recording the pH change, one canconstruct binding curves.To determine the change inpH with Zn2+ stoicheio-

metrically bound (ApH/Av), it is necessary to addsmall amounts of ZnSO4 to haemoglobin solutionsthat are as concentrated as possible. The reason isseen by considering the equation v = 2KA/(I +KA),for the binding of zinc to two identical non-inter-acting sites on the haemoglobin tetramer (seeEdsall & Wyman, 1958); v is the ratio of mol ofzinc bound per mol of haemoglobin, A is theconcentration of unbound zinc, and K is the associ-ation constant for the binding. For oxy and deoxynormal haemoglobins at a concentration of 0.75mM,0.5 zinc atom per haemoglobin tetramer was added,and the pH change was recorded. Under these con-ditions, if one assumes that K= 1.5 x 105M-1 (thelowest value ofKdetermined in this paper), then morethan 99% of the added zinc would have been bound.Thus the ApH observed should have been that forstoicheiometric binding. For des-His'460-haemo-globin, however, 0.1 mM-haemoglobin was used,and 0.35 zinc atom per haemoglobin tetramer wasadded; in this case, one expects 4% of the added zincto be free, so that the determination of ApH/Av wasnot as precise, andKmay have been somewhat over-estimated.For the experiment on 0.75mM-deoxy and oxy

normal haemoglobins, they were first equilibratedwith N2 or 02 respectively in the IL237 tonometer.0.1 mM-Carbonmonoxy-des-His146 l-haemoglobinwasequilibrated with 02. Then 2.2ml ofthis haemoglobinwas placed in a glass cell, maintained at 20QC, intowhich the appropriate gas (N2 or 02) was flowing.A pH electrode (GK2321C; Radiometer, Copen-hagen, Denmark) was immersed in the gentlystirred solution, and the pH was monitored with aVibron model 33B-2 electrometer. Initially, thepH slowly increased, but it stabilized after about30min.Determinations of ApH/Av were as described

above for 0.75mM-oxy and deoxy normal haemo-globin and 0.1 mM-carbonmonoxy-des-His146fi-haemoglobin. For oxyhaemoglobin, ApH/Av wasapproximately constant between pH 7.5 and pH 7.25,

Vol. 169

at -0.125, and decreased as pH decreased, to -0.133at pH6.96 and -0.157 at pH6.33. For deoxyhaemo-globin, ApH/Av was practically constant at -0.152between pH7.42 and pH7.07, and fell to -0.162at pH 6.82. For carbonmonoxy-des-Hisl460-haemo-globin, ApH/Av was -0.114 at pH7.40, -0.116 atpH7.17 and -0.124 at pH6.99.

After values of ApH/Av had been determined, theassociation constant K could be measured by usingrelatively unconcentrated haemoglobin (0.1mM orless). For this, 2.2ml of 0.05mM-oxyhaemoglobin or0.1 mM-deoxy normal or carbonmonoxy-des-His146l-haemoglobin was handled as for the determinationof ApH/Av. Haemoglobins were then titrated with1 or 2,u1l increments of 20mM-ZnSO4, up to a ratio ofzinc atoms added per haemoglobin tetramer of about2.5. By comparing the pH change observed for eachincrement with that expected from the values ofApH/Av, the Scatchard (1949) plot could beconstructed.Low values of A are impossible to measure

accurately with the ApH method. This is because Ais proportional to the difference between two ApHvalues, and the relative error is largest when the twovalues differ by very little. This limits the values of vfor which data can be obtained. Thus, for the deoxy-haemoglobin data plotted in Fig. 3(b) below, haemo-globin was initially at pH7.42, and was titrated topH 7.10 with ZnSO4. However, the eight pointsshown in Fig. 3(b) are only for the pH range 7.25 to7.12, for values of v from 1.10 to 1.96.

Results and DiscussionEffects of zinc on the 02-dissociation curve ofhaemoglobin

Fig. 1 shows the effect of zinc on the 02 affinity(logP50) and co-operativity (h) of normal humanhaemoglobin. Data were obtained for 0.05 mM-haemoglobin in the presence of various concen-trations of ZnSO4 at three pH values, 7.29, 7.02 and6.71 (buffers were 0.05 M-Bistris/0. 1 M-Cl-).The data confirm the claim of Oelshlegel et al.

(1974) that zinc does not alter the alkaline Bohreffect of haemoglobin. The difference between logP50for pH7.29 and 6.71 is 0.324 in the absence of Zn2+,and 0.316 for the maximum concentrations of Zn2+used.The data of Fig. 1 may be used to estimate the ratio

of the association constants of Zn2+ for oxy- anddeoxy-haemoglobin (K. and Kd respectively). Fora given free-Zn2+ concentration A, the decrease inlogP50 (relative to zero zinc) is given by AlogP50=-0.5 log(1 +KoA)/(1 +KdA) (see Baldwin, 1975). IfA is very large, AlogP5o = -0.5 log(Kl/Kd). For themaximum concentrations of Zn2+ that were used,AlogP50 (averaged for the three pH values tested)was -0.678, giving the estimate of K0/Kd as 22.7.

627

628 J. G. GILMAN AND G. J. BREWER3.00 (a)2.75 A

(2

A2.50 A 5 *.2 ~~~~~A

Q

2.25 A

2.00 _ go

1.75

.4

0.9 1.3

(b)0.8 1.2

0.7 O

0.6 1.0

0.5 0.9

0.40 0' ,o 0.4 0.0 0 1.0 2.0 3 .0

0 [hae iZnT]/[haemog1obi

0.3

0.2

0.1

0~~~~~

0 1.0 2.0 30 40 50 60 70 8 90 101.0

Fig. 1. 02-dissociation-curve datafor haemoglobin in thepresence ofZn2+Values of the Hill coefficient h (a) and logP50 (b) are plotted against the total zinc concentration, which is given as themolar ratio of total Zn2+ (represented by [ZnT]) to haemoglobin tetramers (represented by [haemoglobin]); P5o isthe partial pressure of oxygen (in torr) at which the haemoglobin is 50%/ saturated with 02. Human haemoglobinconcentration was 0.05mM in 0.05 M-Bistris buffers containing 0.1 M-Cl-. A, pH 6.71; U, pH 7.02; *, pH7.29. For theinset, data on more concentrated haemoglobin solutions are taken from Gilman et al. (1975). Those experimentswere performed at 37°C and pH7.4 in bicarbonate buffer in the presence of 5.5%. C02: v, cow haemoglobin (0.76mM,no 2,3-bisphosphoglycerate); *, human haemoglobin (0.87mM, approximately equimolar amount of 2,3-bisphospho-glycerate present). Means+s.D. are given when more than two experiments were performed for any experimentalcondition, and solid lines connect the means of the data for the various zinc concentrations at a given pH. For zerozinc concentration, the means are for six, six and five experiments for pH6.71, 7.02 and 7.29 respectively; at maximumzinc concentrations used, the means are for five, six and four experiments respectively. For clarity, standard deviationsare omitted for values of h, but are less than 0.14 for zero and maximum zinc concentrations. The dotted line is atheoretical curve, computed assuming two identical zinc-binding sites, with the association constant K. for oxy-haemoglobin of 1.5 x 106M-1, and the ratio of K. to Kd (for deoxyhaemoglobin) of 22.7 (see the text). Then, for a givenfree Zn2+ concentration A, AlogP50 = -0.5 log(l +K.A)f(l +KdA) (see the text). It was necessary to estimateA from theknown values of total added Zn2+ ([ZnT]). This was done by assuming that the haemoglobin was always in the oxy state,so that the equation v = 2K.A/(1 +K.A) could be used. This procedure gave a minimum estimate for A, since oxy-haemoglobin binds Zn2+ better than does deoxyhaemoglobin. The theoretical curve thus derived corresponds fairly wellto the experimental data, except for low values of [ZnT], where the error, owing to estimatingA for oxyhaemoglobin only,would be the greatest.

1978

HAEMOGLOBIN-ZINC INTERACTION

The inset to Fig. 1 presents data on more-con-centrated solutions of cow (0.76mM) and human(0.87mM) haemoglobin, the latter in the presenceof an approximately equimolar amount of 2,3-bis-phosphoglycerate (these data are taken from Gilmanet al., 1975). Zinc decreases logP50 in a similarmanner for human haemoglobin (with 2,3-bisphos-phoglycerate) and cow haemoglobin (without 2,3-bisphosphoglycerate) at 37'C and pH7.4 in thepresence of CO2. The values of logP50 show anapproximately linear decline as Zn2+ concentrationincreases from zero to [ZnT]/[haemoglobin] = 2,followed by a levelling-off; this suggests the presenceof two 02-linked zinc-binding sites per haemoglobintetramer. (The data on 0.05mM-haemoglobin showa less-steep decline in logP50 as ZnSO4 is added,because [ZnT]/[haemoglobin] represents a much lowerZn2+ concentration in those experiments than in theexperiments with more concentrated haemoglobins.)The upper part of Fig. 1 shows that the Hill

coefficient, h, the measure of co-operativity, initiallydeclines as ZnSO4 is added to the haemoglobin,but then recovers to close to the original value asZnSO4 concentration increases. A similar effect wasnoted by Tomita & Riggs (1971) for the interactionof haemoglobin with 2,3-bisphosphoglycerate. Theysuggested that the low value of h observed couldbe due to the fact that the concentration of allostericeffector varied during the course of an experiment.For example, oxyhaemoglobin binds zinc more than20 times as effectively as does deoxyhaemoglobin,as shown above; therefore the free Zn2+ concen-tration is highest for deoxyhaemoglobin, andgradually decreases as. the haemoglobin becomesoxygenated.To test this explanation, 02-dissociation curves

were obtained for haemoglobin in 'zinc ion buffers'.Fig. 2 shows a plot of logP5o and h against A/K.,.A (the free Zn2+ concentration) was held constant asthe result of the interaction between Zn2+ and thechelating agent NN-bis(carboxymethyl)glycine (seethe legend to Fig. 2 and the Experimnental section forthe definition of K., and for a description of theprinciples involved). The upper part of Fig. 2, whichgives the plot of h against A/K.,, shows that h wasalmost constant as zinc concentration increased. Thisdemonstrates that most of the decline in apparent h,for the experiments in Fig. 1, was due to the variationin free Zn2+ concentration that occurred during thecourse of an experiment.

Zinc-binding studies

Equilibrium binding of Zn2+ to haemoglobin wasinvestigated for normal and des-His146fl-haemoglobinafter the effect of Zn2+ on 02 affinity was known,but before we studied the interaction of Zn2+ withN-ethylsuccinimide-haemoglobin (see the next sec-

Vol. 169

3.25

-Z 3-00 -in on ME s

2.75 s') rnA

0.75

0.70

0.650

N 0.600

0.55

0.50

0.45

U

(a)

j (b)

*--..U.|..

*-... ...

0 5 It0 1 5 20 25 30 35

Fig. 2. 02-dissociation-curve data for haemoglobin in zinc-ion buffers atpH7.06

Values of h (a) and logP.o (b) are plotted againstA/K1,, where A is the free Zn2+ concentration. In theseexperiments, A was kept constant by the presence of5 mM-NN-bis(carboxymethyl)glycine, a chelatingagent, and K1, represents the apparent dissociationconstant of Zn2+ for NN-bis(carboxymethyl)glycine.K1, should be of the order of 10-8 M at pH7.06, andvalues of A/K1, were computed by using theapproximation A/K., = [ZnT]/ [NN-bis(carboxy-methyl)glycine]- [ZnT]), as described in the Experi-mental section. The averages±S.D. are given forA/K1, = 0 and A/K., = 9, for five and four experi-ments respectively (other points represent individualexperiments). The dotted line is a theoretical curve,constructed in a manner similar to that for thetheoretical curve of Fig. 1 (see the legend to Fig. 1),except that A/K., is given as described above, and theassumption is made that K.K1, = 6.74x 10-2.

tion). The data in the present paper are thus beinggiven in roughly chronological order, to illustratethe development of the hypothesis concerning thelocation of the 02-linked zinc-binding site.

Fig. 3 shows Scatchard plots for Zn2+ binding tooxy- and carbonmonoxy-haemoglobin, deoxyhaemo-globin and carboxy-des-Hist46P-haemoglobin. Filledsymbols represent data obtained by equilibriumdialysis, and open symbols are for data obtained by theApH procedure (described in the Experimentalsection). The data were analysed as described in thelegend to Fig. 3. Equilibrium-dialysis data for carbon-monoxy-haemoglobin gave values for the associationconstant of 1.1 x 106M-1 at pH7.0 and 5 x 105M-1 atpH6.6, whereas ApH data for oxyhaemoglobingave the value of 1.9 X 106M-1 for the pH range

629

L.)u'

J. G. GILMAN AND G. J. BREWER

x

0

0 0.5 1.0 1.5

V

Fig. 3. Scatchard plots of equilihaemoglobin

Zinc-binding data were obtaineddialysis (filled symbols) and asymbols) (see the Experimentaldescription of the ApH method)of zinc bound to mol of haemofree Zn2+ concentration. (a) Ox)normal haemoglobins; U, pH7.Cmonoxyhaemoglobin (equilibrpH6.6, 0.1 M-KCl,carbonmonoxlibrium dialysis); El, pH7.11oxyhaemoglobin (ApH). (b) Decand carbonmonoxy-des-His'46'pH7.0, 0.25M-NaCI, carbonhaemoglobin (equilibrium dialys0.25 M-NaCl, oxy-des-His'"'-hA, pH7.3, 0.25M-NaCl, deoxylibrium dialysis); A, pH7.42-7.oxyhaemoglobin (ApH). The stFigures have been drawn tdialysis data points, estimatin3b, the line was drawn forglobin only, because the dat;globin were too scattered to allo'meaningful line). Values of theK were then computed for the strby using those straight lines, acc4of Scatchard et al. (1957) forfor multiple classes of binding s

covered, 7.11-6.83 (Fig. 3a). For deoxyhaemoglobin,(a) the ApH data gave a value for the association

constant of 1.8 x 105M-1 for the pH range 7.42-7.10(Fig. 3b). For carbonmonoxy-des-His'46,l-haemo-globin, equilibrium dialysis gave an associationconstant of 1.5 x 105M-' (pH 7.0), whereas the ApHdata gave a value of 2 x 10Gm- for the pH range7.17-6.97 (Fig. 3b). For all haemoglobins, the data

,*\a indicate two identical principal binding sites forzinc per tetramer.

D \ These binding data thus demonstrate that deoxy-haemoglobin has a considerably smaller associationconstant for Zn2+ than does oxyhaemoglobin, inqualitative agreement with deductions from 02-dissociation-curve data (see the legend to Fig. 1).

(b) Carbonmonoxy-des-His146,-haemoglobin binds Zn2+approximately as weakly as does deoxyhaemoglobin;this suggests that histidine-146f, is crucial to the strongbinding ofZn2+ observed for oxyhaemoglobin.

In deoxyhaemoglobin, histidine-14611, an alkalineA Bohr group, is involved in a salt bridge to aspartic

acid-94f, of the same chain (Perutz, 1970). Thisfact could account for the weaker binding of Zn2+ to

A deoxy- than to oxy-haemoglobin: histidine-146,8would be less free to participate in the chelation of

A^ Zn2+ in deoxy- than in oxy-haemoglobin, since indeoxyhaemoglobin that residue is constrained,whereas in oxyhaemoglobin it is relatively free to

2.0 2.5 3.0 move.

One may ask, at this point, why Zn2+ does not alterthe alkaline Bohr effect of haemoglobin, if zinc

ibrium zinc binding to is binding to an alkaline Bohr group. The hypothesisI just presented suggests the reason, namely that Zn2+[by both equilibrium binding to deoxyhaemoglobin may interfere onlyApH method (open minimally with the salt bridge between histidine-14616section for a detailed and aspartic acid-94fl. In deoxyhaemoglobin, Zn2+globin, and A is the binding may occur as chelation to two other aminoy and carbonmonoxy acids, but not significantly to histidine-14616. [Two),0.1M-KCl, carbon- other amino acids are suggested by the order ofium dialysis); v, magnitude of the zinc-binding constant for deoxy-Lyhaemoglobin (equi- haemoglobin (105M-'); a single histidine side chain-6.82, 0.25M-NaCl, would be expected to bind Zn2+ with an association)xy normal, and oxy- constant of only 102-5M-1 at pH7, from data of^-haemoglobins. *, Gurd & Goodman (1952).]imonoxy-des-His'"'- Thus Zn2+ may be reasonably strongly chelated;is); 0, pH 7.17-6.97,aemoglobin (ApH); to deoxyhaemoglobin despite the minimal partici--haemoglobin (equi- pation of histidine-146,B in the chelate. The very10, 0.25M-NaCI, de- switch to oxyhaemoglobin that liberates the alkalinetraight lines in thesethrough equlibrium-g by eye (for Fig.des-His'"4'-haemo-

a for deoxyhaemo-w the drawing of anyassociation constantrongest binding sites,ording to the methodestimating K valuesites; the assumption

was made that there are two identical strong bindingsites, which appears reasonable, judged from thedata. For the ApH data, a computer program wasdevised to fit the data points for each experiment to astraight line, by the method of least squares; theprogram then calculated values of K by the aboveprocedure. Values ofKthus calculated, for the data ofthis Figure, are given in the text.

1978

630

HAEMOGLOBIN-ZINC INTERACTION6

Bohr protons would permit histidine-1461J to swinginto position and provide the third side chain of thechelate, thereby raising the association constant forZn2+ by some 20-fold.

Effects ofzinc on the 02-dissociation curve ofN-ethyl-succinimide-haemoglobin

If Zn2+ does bind to histidine-146fl in oxyhaemo-globin, examination of the three-dimensional oxy-haemoglobin model (Perutz et al., 1968) suggeststhree possible locations for the additional aminoacids of the chelate: in the vicinity of histidine-143flor cysteine-93fl of the same fl-chain, or histidine-2flof the other fl-chain.The last possibility is effectively ruled out by the

data of the insert to Fig. 1. Zinc increases the 02affinity of cow haemoglobin, in the absence of2,3-bisphosphoglycerate, in a manner similar to thatfor human haemoglobin in the presence of 2,3-bisphosphoglycerate, even though cow haemoglobinlacks histidine-2fl. These same data also suggestthat it is unlikely that histidine-143fl is involved inthe binding of Zn2+, since that residue is at the2,3-bisphosphoglycerate-binding site (see Perutz,1970). If histidine-143fl were involved in the bindingof both 2,3-diphosphoglycerate and Zn2+, thenthe presence of 2,3-bisphosphoglycerate might beexpected to enhance the degree to which Zn2+causes a left-shift of the 02-dissociation curve.One concludes that the 02-linked zinc-binding sitemay be in the vicinity of cysteine-93fl. The experi-ments on N-ethylsuccinimide-haemoglobin test thispossibility.Blockage of cysteine-93fl, by reaction with N-

ethylmaleimide, is known to prevent the formation ofthe salt bridge between histidine-146fl and asparticacid-94fl. For N-ethylsuccinimide-haemoglobin,therefore, the alkaline Bohr effect is decreased by50%, 02 affinity increases, and co-operativity dimin-ishes, though not drastically (see Baldwin, 1975).If one accepts the premise that Zn2+ binding involveshistidine-146fl in oxy- but not in deoxy-haemoglobin,one might expect N-ethylsuccinimide-deoxyhaemo-globin to bind Zn2+ about as well as does oxy normalhaemoglobin. This is because much of the constrainton histidine-146fl in deoxyhaemoglobin would beremoved in N-ethylsuccinimide-haemoglobin, andhistidine-146fl should therefore be freer to swinginto position and participate in Zn2+ chelation.So, for N-ethylsuccinimide-haemoglobin, Zn2+ wouldbe expected to bind very strongly (K> 106M-1) toboth the deoxy and oxy states, unless cysteine-93,f(orgroups close by) was directly involved in the chelationof the Zn2+, in which case both the oxy and deoxyforms of N-ethylsuccinimide-haemoglobin wouldbind Zn2+ rather weakly.

Vol. 169

2.752.50

t 2.252.00.75

0.25

0.20

0. 15

0. 100Wo

I I

(a)

(b)

0.05 I

0.

0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

[ZnT]/[haemoglobin]

Fig. 4. 02-dissociation-curve data for N-ethylsuccinimide-haemoglobin at pH7.02 in the presence ofZn2+

Values of h (a) and logP50 (b) are plotted against thetotal Zn2+ concentration, which is given as the molarratio of total Zn2+ ([ZnT]) to haemoglobin tetramers([haemoglobin]). Means+-S.D. are given when morethan two experiments were performed for anyexperimental condition, and otherwise the actualpoints for the data are given. For zero Zn2+, the meanis for eight experiments; for [ZnT]/[haemoglobin]equal to 2.0, 4.0 and 6.0, the means are for threeexperiments each. The broken and dotted lines aretheoretical curves constructed by the procedure usedto compute the dotted line of Fig. 1 (see thelegend to Fig. 1). For the broken line, the associationconstant for oxyhaemoglobin is assumed to be 106 M-1and the ratio of association constants for oxy- todeoxy-haemoglobin is 2.71, whereas for the dottedline the association constant for the oxy state isassumed to be 3 x 104M- and the ratio of theconstant for the oxy state to that of the deoxy stateis 3.0.

Fig. 4 shows the effect of Zn2+ on the 02 affinity(given as logP50) and co-operativity (represented by h)of human N-ethylsuccinimide-haemoglobin atpH 7.02. Comparison with Fig. 1 shows that, in theabsence of Zn2+, the N-ethylmaleimide treatmentdecreased logP5o from 0.70 to 0.22, and h from 2.86to 2.33. Zinc affected the 02 affinity of N-ethyl-succinimide-haemoglobin much less than that ofnormal haemoglobin. The addition of 0.5mM-ZnSO4to the N-ethylsuccinimide-haemoglobin decreasedlogP50 by 0.217, whereas the addition of 0.4-0.5mM-ZnSO4 to normal haemoglQbin dwroased log P5

631

632 J. G. GILMAN AND G. J. BREWER

by 0.678 (the average for the threepH values ofFig. 1).This decrease in logP50 for normal haemoglobinwas interpreted as showing that the ratio of theZn2+ association constants for oxy- and deoxy-haemoglobin was 22.7. The same analysis applied tothe N-ethylsuccinimide-haemoglobin data of Fig. 4gave a value for this ratio of only 2.71.The dotted and broken lines in Fig. 4 are theoretical

curves that were computed by the procedure used forthe dotted curve of Fig. 1 (see the legend to Fig. 1).For Fig. 1, the theoretical curve was computed byassuming that the association constant of Zn2+ foroxyhaemoglobin was 1.5 x 106M-1 and reasonablygood agreement with the data was obtained. ForFig. 4, however, the assumption of a high value of theassociation constant for N-ethylsuccinimide-oxy-haemoglobin (106M-1), was in poor agreement withthe data (broken line). Only by assuming a low valuefor the association constant of N-ethylsuccinimide-oxyhaemoglobin was it possible to obtain reasonablygood agreement with the experimental data, as shownby the dotted line (for which the value was taken as3x104M-').

It is concluded that the data of Fig. 4 imply thatgroups in the vicinity of cysteine-93f are involvedat the 02-linked zinc-binding site. The fact thatcysteine is one of the strongest zinc-binding aminoacids (Albert, 1961) suggests that cysteine-93,B mayitself be involved in the chelation of Zn2+, alongwith histidine-146fl, and at least one other (unknown)residue located nearby.

We thank Dr. L. Rossi-Bernardi for his hospitality toJ. G. G. during his stay in Italy, for discussions and forsuggesting the experiment on des-His46,1-haemoglobin.We thank Dr. J. V. Kilmartin for providing the des-His'46,l-haemoglobin, Dr. P. Righetti for technical help,Dr. M. Luzzana for computer assistance and Dr. F. R. N.Gurd for discussions. J. G. G. was supported in part bythe Consiglio Nazionale delle Richerche (Italy) (Decretono. 197213) and the National Institutes of Health (U.S.A.)(1 FO 2 HL55564-02).

ReferencesAlbert, A. (1961) in Biochemists' Handbook (Long, C.,

ed.), pp. 95-96, E. and F. N. Spon, LondonBaldwin, J. M. (1975) Prog. Biophys. Mol. Biol. 29,

225-320Benesch, R., Edaiji, R. & Benesch, R. E. (1976)

Biochemistry 15, 3396-3398Cohen, S. R. & Wilson, I. B. (1966) Biochemistry 5,

904-909Drabkin, D. L. (1950) in MedicalPhysics (Glaser, O., ed.),

vol. 2, pp. 1039-1088, Year Book Medical Publishers,Chicago

Edsall, J. T. & Wyman, J. (1958) Biophysical Chemistry,vol. 1, chapter 11, Academic Press, New York

Gilman, J. G., Oelshlegel, F. J., Jr. & Brewer, G. J. (1975)in Erythrocyte Structure and Function (Brewer, G. J.,ed.), pp. 85-101, Alan Liss, New York

Gurd, F. R. N. & Goodman, D. S. (1952) J. Am. Chem.Soc. 74, 670-675

Kilmartin, J. V. & Rossi-Bemardi, L. (1971) Biochem. J.124, 31-45

Kilmartin, J. V., Hewitt, J. A. & Wootton, J. F. (1975)J. Mol. Biol. 93, 203-218

McCall, T. T., Davis, G. K. & Stearns, T. W. (1958) Anal.Chem. 30, 1345-1347

Oelshlegel, F. J., Jr., Brewer, G. J., Knutsen, C., Prasad,A. S. & Shoomaker, E. B. (1973) Biochem. Biophys. Res.Commun. 53, 560-566

Oelshlegel, F. J., Jr., Brewer, G. J., Knutsen, C., Prasad,A. S. & Schoomaker, E. B. (1974) Arch. Biochem.Biophys. 163, 742-748

Perrella, M., Rossi-Bemardi, L. & Roughton, F. J. W.(1972) in A. Benzon Symp. 4: Oxygen Affinity ofHemoglobin and Red Cell Acid-Base Status (Roth, M.& Astrup, P., eds.), pp. 177-203, Munksgaard,Copenhagen

Perutz, M. F. (1970) Nature (London) 228, 735-739Perutz, M. F., Muirhead, H., Cox, J. M. & Goaman,

L. C. G. (1968) Nature (London) 219, 131-139Riggs, A. (1951) J. Gen. Physiol. 34, 23-40Riggs, A. (1961) J. Biol. Chem. 236, 1948-1954Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672Scatchard, G., Coleman, J. S. & Shen, A. L. (1957) J. Am.

Chem. Soc. 79, 12-20Tomita, S. & Riggs, A. (1971)J. Biol. Chem. 246, 547-554van Kampen, E. J. & Zijlstra, W. G. (1965) Adv. Clin.

Chem. 8, 141-186

1978