The properties and interactions of the isolated α- and β-chains of human haemoglobin

J. Mol. Biol. (1966) 17, 29-46

The Properties and Interactions of the Isolated 1%- andp-Chains of Human Haemoglobin

V. The Reaction of 1%- and p-Chains

ERALDO ANTONINI, ENRICO BUCCI, CLARA FRONTICELLI, EMILIA CHIANCONE,

JEFFRIES WYMAN AND ALESSANDRO ROSSI-FANELLI

The Institute of Biological Chemistry, University of Romeand the Regina Elena Institute for Cancer Research, Rome, Italy

(Received 6 December 1965)

The mixing of the isolated 0(- and ,8-chainsof human haemoglobin is accompaniedby spectroscopic changes in the Soret region and by changes in the kinetics ofthe reaction with ligands and in sedimentation behaviour. These changes showthat when the chains are combined with p-mercuribenzoate, the system is in a.state of labile association-dissociation equilibrium which is strongly dependentOll pH between pH 7 and 8, and is oxygen linked. The kinetics ofthe spectroscopicchanges reveal that, both in the presence and absence of p-mercuribenzoate in thechains, the recombination is a complex process with an over-all half-time of theorder of one to five seconds at 30°C, when the chains are at micromolar concentration.

1. IntroductionPrevious papers of this series (Bucci et al., 1965; Reichlin et al., 1965; Antonini, Bucci,Fronticelli, Wyman & Rossi-Fanelli, 1965; Reichlin et al., 1966) describe variousproperties of the isolated a- and ,B-chains of human haemoglobin prepared by themethod of Bucci & Fronticelli (Bucci & Fronticelli, 1965; Bucci et al., 1965). Thismethod, it will be recalled, involves treatment of the haemoglobin with an excess ofp-mercuribenzoate at neutral pH, followed by column chromatography. Whenseparated by this method, the chains are in the so-called PMBt form (aPMB or ,BPMB)and contain no free SH groups. However, by treatment with certain reagents such ascysteine or thioglycolate, the PMB may be largely removed to give the SH forms,«SH or ,BSH, in which free SH groups are regenerated. In both forms, SH or PMB, thechains may be regarded as "native" in the sense that they combine reversibly withoxygen; moreover, the aSH and ,BSH chains, when mixed, associate to form a substancewhich behaves essentially like haemoglobin A..

The isolated chains, both in the PMB and SH form, differ widely in many respectsfrom their recombination products, (aPMB+ ,BPMB) and (aSH + ,BSH). This is apparentin immunochemical behaviour (Reichlin et al., 1965), in sedimentation in the ultracentrifuge (Bucci et al., 1965), in the equilibria and kinetics of reactions with oxygenand carbon monoxide (Antonini et al., 1965), and in the absorption spectra of thedeoxygenated forms in the Soret region (Antonini et al., 1965). In the present workadvantage has been taken of these differences, especially of the spectral ones, to

t Abbreviation used: PMB, p-mercuribenzoa.te.

29

30 E . ANTONI N I ET AL .

study the equilibria and kinetics of the reaction between the oc- and fJ-chain s in boththe PMB and SH forms. The equilibrium work was largely confined to the PMBsystem, in which the affinit y bet ween the chains is not as great as in the SH system.It may be emphasized that these t wo systems offer unique opportunities for studyinginteractions of protein subunits generally, and t heir effects on ph ysico-chemicalproperties and fun ctional behaviour.

2. Materials and Methods(a) Haemoglobin chains

The O(PMB. and ,BP!>lB.chains were prepared in t he oxygenat.ed form fro m human oxyhaemogl obin (Bu cci & Frontieell i, 1965; Bucci et al. , 1965 ). In order to obtain t he SHcha ins, t he PMB was removed by dialysis of t he chains against a cysteine solution .Concent rat ions were det ermin ed spectrophotometr ically (Antonini et al ., 1965) and aregiven in terms of haem equiva lents.

(b) S pectrophotometric measurements

These were made with a Beckman DKI or DK2 recording spectroph ot omet er at roomtemperature.

(c) K in etic measurements

These were performed in a Gibson st opped-flow apparatus (Gibso n & Milnes, 1964)equipped with a 2-cm observation t ube, a t 20°C and 30°C.

(d) Ultracentrifuge experiments

These were carried out with a Spinco m odel E cent ri fuge equipped wi t-h aut omatictemperature control a t 17°C. Sediment ation ve loc it ies were ca lculated from the m ovementof t he maximum ordinat e of the schlieren pattern and were corrected to t he den sity andviscosity of water at 20°C.

(e) Deoxygenation

F or t he spectrophotomet r ic work , the deoxygenat ed derivatives were prepared byadding t o the solut ion s sm all amount s of dithionite, care being taken t o ex clude air, asdescribe d previously (An t on ini et al., 1965). F or the ul tracentrifuge runs two m ethods wereu sed. One in volved t he use of d ithioni t e, as in t he spectro photometric exper iment s; t heother cons isted in deoxygen ating the solution in a tono me ter , which was connected wit hthe ultracentrifuge cell by a short nyl on tube fit ted by a screw device to t he filling h oleof the cell. The t on ometer was detached and t he cell close d wit h a screw pl ug after immer sing the cell in a so lution containing di thionite; t his ens ured exclus ion of air during theprocedure.

3. Results(a) Effect of pH on the spectrum of the mixture of deoxygenated OCPMB and fJPMB

As reported in a previous paper of this series (Antonini et al., 1965), the isolatedoc- and fJ- chains , in both the PMB and SH form, when deoxygenated, show, at pH 7,a small difference of ab sorption in the Soret region from their equimolar mixture(oc + (J) , although they are themselves indistinguishable. The position of the maximumis at a slightly lower wavelength in the case of t he isolated chains , and the corresponding extinction coefficient is less (Ta ble 1).

Since it was hoped that these differences might provide a basis for studyi ng thekineti cs and equil ibria of the interaction between t he cha ins, it was decided toinvesti gate them in grea te r detail over the pH range 6 to 9. The results were asfollows.

31REACTION OF HAEMOGLOBIN a- AND ,B-CHAINS

TABLE 1

Wavelengths of maximum absorption and corresponding molar extinction coefficients ofdeoxygenated chains of human haemoglobin in the Soret region. From measurements at

pH 6·5 in 0·1 to 0·2 M-phosphate buffer

Compound

428

III

428

112

430

126

429

III

428

110

430

124

430

127

(a) The isolated PMB or SH chains maintain their spectrum unchanged throughoutthe range.

(b) The (IXSH + f3SH) system, which may be regarded as the same as haemoglobin A,likewise maintains unchanged its characteristic spectrum, different from that of theisolated chains, over the whole range.

(c) In contrast, the spectral properties of the (IXPMB + f3PMB) system change in astriking way with pH. At acid pH they are those of haemoglobin A; at alkaline pH(> 8), those of the isolated chains; in between, there is a rather sharp transitionwith a midpoint near pH 7·2. This is shown in Fig. 1, in which the optical density at430 miL is plotted against pH. It is important to note that these changes of spectralproperties with pH were reversible and independent of the way in which the finalconditions were achieved; in other words, it made no difference whether the deoxygenated chains were first brought to the desired pH and then mixed, or were first

o

0'9

0'8

FIG. 1. Optical density at 430 mfJ- of the (aPMB + ,BPMB) system as a function of pH in 0·2 M

:phosphate buffer at 20°C. Total haem concentration 7 X 10- 6 M. Dashed line shows optical densityof BOlutions of isolated aPMB_ or ,BPMB-chains at the same concentration and under the sameoonditions.

32 E. ANTONINI ET AL.

mixed and then brought to the desired pH; nor did it make any difference whetherthe reduction (with dithionite) was carried out before or after mixing.

(b) Effect of pH on the kinetics of the reaction of carbon monoxide with (ocPMB + f3PMB)

It had already been shown that the velocity constant for the combination withcarbon monoxide of the isolated chains, both in the PMB and SH forms, is very highand that there is a dramatic decrease when the chains are mixed at pH 7 or less(Antonini et al., 1965). Moreover, the velocity constant for the reaction with carbonmonoxide is essentially independent of pH for both the isolated chains and (ocS H +f3SH),the behaviour of the latter being the same as that of normal haemoglobin.

In view of this and of the spectroscopic results presented in the last section, whichsuggest a progressive dissociation of the (OCP M8 + f3PM8) system into free chains atalkaline pH, experiments were carried out in which mixtures of equal amounts ofdeoxy OCP M8_ and f3PM8-chains were allowed to react with carbon monoxide at differentpH values between 6 and 9. A representative set of results is reported in Fig. 2. Itwill be seen that the kinetics of the reaction change progressively with pH. At pH 6·4all the material reacts at a slow rate; at higher values of pH the reaction becomesmarkedly diphasic, a fast component contributing more and more with increasing pH;

Ti-ne (rnsec)

FIG. 2. Kinetics of the combination of CO with the (",PMB + fJPMB) system as a function of pHin 0·1 M.phosphate buffers at 20°C. Total protein concentration in haem 1·6 X 10- 6 M. CO concentration 12·5 X 10- 6 M. Time 0 corresponds to 3 msec after mixing. Based on optical densitiesmeasured at 421 mn,

REACTION OF HAEMOGLOBIN ex- AND p.CHAINS 33

finally, at pH 8,5, the whole system reacts at a high velocity similar to that of theisolated chains. This behaviour is entirely reversible.

(c) Effect of pH on the sedimentation behaviour of the chains and their mixtures

The sediment at ion behaviour of the isolated chains in both the SH and PMB formsand of their mixtures had already been studied at pH 7, using both the oxy andcarboxy derivatives (Bucci et al., 1965). In view of the results just reported, however,it seemed important to extend these measurements to cover the full pH range, 6 to 9,and to study the deoxy as well as the oxy and carboxy derivatives.

(i) SH systems

The experiments on the SH systems showed that the sedimentation coefficients ofthe isolated chains in dilute phosphate buffer do not change with pH over the range6 t o 9 and are the same for the oxy and deoxy derivatives. The value of 8 20• w for the()(sH-chains was everywhere that characteristic of the monomer; for the pBH-chains itwas that characteristic of a tetramer ({J~H), in agreement with the results of the earlierbut more limited observations. The mixture (a.SH +{JSH}, which may be regarded asre-assembled haemoglobin, behaved just like native haemoglobin A, giving a value of82 0, w of about 4 at all pH values from 6 to 9 both in the oxy and deoxy forms (seeTable 2).

TABLE 2

8edimentation behaviour of oxygenated and deoxygenated derivatives of aSH, pH and the(a.SH + (JSH) system

Compound Solvent pH Concn8 20••(g/100ml.)

Oxygenated (",SH + PSH) 0·05 M-potassium phosphate 7·85 0·18 3·92Oxygenated (",SH + PSH) 0·05 M-potassium phosphate 6·74 0·19 4·15Deoxygenated (",SH + PSH)t 0·05 M-potassium phosphate 7·35 0·18 4·04Deoxygenated (exSH + PSH)t 0·05 M-potassium phosphate 6·56 0·19 4·28

Oxygenated exSH 0·05 M-potassium phosphate 8·30 0·19 1-90Oxygenated ",SH 0·05 M-potassium phosphate 6·60 0·19 1-89Deoxygenated exSHt 0-05 M-potassium phosphate 7-80 0·19 1·91Deoxygenated ",SHt 0·05 M-potassium phosphate 6·48 0·19 1·78

Oxygenated pSR 0-05 M-potassium phosphate 8·00 0·14 3·95Oxygenated pSR 0·05 M-potassium phosphate 6·50 · 0·14 4·47Deoxygenated pSRt 0·05 M-potassium phosphate 7·91 0·14 3-57Deoxygenated pSRt 0·05 M-potassium phosphate 6'35 0·14 4·29

t Deoxygenation was obtained by adding dithionite, as described in the text.

(ii) PMB systems

The PItffi systems behaved quite differently, as will be seen from the results presentedin Table 3 and Figs 3 and 4, which were obtained in 0·05 M-phosphate buffers. In allcases the schlieren diagrams showed only a single symmetrical peak. The sedimentationcoefficient of the isolated chains is essentially independent of pH and the same for the

3


TABLE 3

Sedimentation behaviour of oxygenated and deoxygenated derivatives of rxP M B • and fJPM B •

chains as a function of pH

Compound pH Solvent ConcnS20.W(g/100 ml.]

Oxygenated fJPMB 6·05 0·05 M-potassium phosphate 0·13 1·99Oxygenated fJPMB 6·11 0·05 M-potassium phosphate 0·35 2·30Oxygenated fJPMB 6·11 0·05 M-potassium phosphate 0·51 2·50Oxygenated fJPMB 8·70 0·05 M-sodium borate 0·36 2·70Oxygenated fJPMB 8·70 0·05 M-sodium borate 0,52 2·66Oxygenated fJPMB 9·08 0·05 M-sodium borate 0·13 2·18Deoxygenated fJPMBt 6'30 0·05 sr-potaasium phosphate 0·19 2·29Deoxygenated fJPMBt 8·10 0·05 M-sodium borate 0·19 2·46

Oxygenated IXP M B 6·50 0·05 M.potassium phosphate 0·13 1·61Oxygenated IXPM B 7·24 0·05 M-potassium phosphate 0·25 1·85Oxygenated IXPM B 7·81 0·05 M-potassium phosphate 0·14 1·68Deoxygenated IXPM Bt 6·30 0·05 M-potassium phosphate 0·13 1·85Deoxygenated IXP MBt 8·10 0·05 M·sodium borate 0·15 1·50


4

3

o

2-5

pH

FIG. 3. Sedimentation coefficient of the (IXP M B + fJPMB) system as a function of pH in 0·05 M·phosphate and borate buffers. Total protein concentration, 0·15 to 0·25%. Oxygenated(O); cornbined with CO (0); deoxygenated and then re.oxygenated (il); deoxygenated by evacuation (e);deoxygenated by dithionite (.).

REACTION OF HAEMOGLOBIN oc· AND ,B. CHAINS 35

carboxy, oxy and deoxy derivatives. In contrast, the sedimentation coefficient of themixture ((XPMB + flPMB) is strongly dependent on both pH and the presence of ligand(02 or CO) (see Fig. 3). In the case of the liganded material, it drops from a value ofabout 3, which is characteristic of a dimer, at pH 6, to a value of about 2'3, somewhatabove that for a monomer, at pH > 8. In the case of the unliganded material, it dropsfrom a much higher value, namely about 4·4, which is that for a tetramer, at acid pH,to the same value, "" 2·3, at alkaline pH. The transition is very steep indeed, andoccurs over a small range of pH close to 8. It is apparent that the ((XPMB + flPMB)

system is indeed in a state of labile association-dissociation equilibrium, which is notonly strongly proton linked, but is also strongly oxygen linked, something whichcould not have been predicted from any of the previously reported observations.

It may be remarked that the deoxygenated material employed in these experimentswas obtained either by the gasometric procedure described under Materials andMethods or by the addition of dithionite; the two procedures gave essentially identicalresults. In most cases the behaviour of the same solutions after reoxygenation wasstudied; whenever this was done, the results were the same as those obtained withoxygenated solutions which had not been previously deoxygenated. Also, thereversibility of the pH effects was verified.

All the experiments on the PMB systems just described were carried out in 0·05 M·phosphate buffers. A few observations were also made on the mixture ((XPMB + flPMB)

in 0·2 M-phosphate and 0·3 M-NaCI + 0·05 M-phosphate. The results, which aresummarized in Table 4, were significantly different from those in 0·05 M-phosphate

TABLE 4

Effect of sodium chloride and phosphate on the sedimentation behaviour of the oxygenatedand deoxygenated derivatives of the ((XPMB + flPMB) system

Compound Solvent pHConcn

S20,w(g{lOOml.)

Oxygenated (OCP MB + f3PMB) 0·3 M-NaCI + 0·05 M-potassium phosphate 6·27 0·25 2·40

Oxygenated (OCP MB + ,BPMB) 0·3 M-NaCI + 0·05 M-potassium phosphate 7·20 0·20 2·54



Oxygenated (OCP M B + ,BPMB) 0·2 M-potassium phosphate 6·37 0·37 2·80Oxygenated (OCP M B + ,BPMB) 0·2 M-potassium phosphate 7·10 0·20 2·52Oxygenated (ocP MB + ,BPMB) 0·2 M-potassium phosphate 7·63 0·34 2·54Oxygenated (OCP M B + ,BPMB) 0·2 M-potassium phosphate 8·31 0·20 2·38

Deoxygenated (OCP MB + ,BPMB)t 0·3 M-NaCI + 0·05 M-potassium phosphate 6·25 0·20 4·43

Deoxygenated (OCP MB + f3PMBJt 0·3 M-NaCI + 0·05 M-potassium phosphate 7·02 0·20 4·29

Deoxygenated (OCP M B + ,BPMB)t 0·2 M-potassium phosphate 6·44 0·19 4·14Deoxygenated (OCP M B + ,BPMB)t 0·2 M-potassium phosphate 7·39 0·19 3·70


36

4

E. ANTONINI ET AL.

pH 6'1

--e:>-_--(")-- -----'.J-pH 8'1

FIG. 4. Concentration dependence of the sedimentation coefficient of the oxy or carboxyderivative of the (",PMB + fJPMB) system at three different pH values in 0·05 M-potassium phosphatebuffers.

alone. Thus the sedimentation coefficient of the oxygenated derivative was independent of pH and had everywhere a value of about 2·3; in the case of the deoxygenatedderivative, the transition in the value of the sedimentation coefficient appeared tooccur at a somewhat lower pH. This shows that the (OCP MB + f3PMB) system is sensitiveto specific ion effects which are oxygen linked.

(d) Kinetics of the aesociation-dieeociatio» reaction of deoxygenated ex- and f3-chains

From the preceding observations, it seems clear that the optical density changeobserved at 430 mp' when deoxygenated chains are mixed is due to the association ofthe two different kinds of chain, ex and 13. It was studied therefore in a series ofexperiments in which temperature, pH and concentration of the reacting chains wereall varied in an attempt to obtain information about the kinetics of the associationprocess.

(i) exS H _ and f3sH-chains

Figure 5 shows the time course of the optical density changes, at 430 mp., observedwhen equal amounts of exSH• and f3sH- chains are mixed. These changes correspond tothe difference in extinction, reported previously, between the isolated chains andre-assembled or normal haemoglobin. The changes were so slight, in relation to thetotal values of the optical densities, that it was impossible to extend these experimentsover more than a small concentration range, or to vary the observations in suchobvious ways as by keeping the concentration of one kind of chain constant andaltering that of the other.

The time course of these changes does not correspond to a simple second-orderreaction, the shape of the kinetic curves being in all cases sigmoid. (The usual plot for

REACTION OF HAEMOGLOBIN IX- AND ,B-CHAINS 37

0·05

0·04

ci6 0·03'I

0·02

0·01

FIG. 5. Time course of optical density changes at 430 mp. on mixing deoxygenated IXSH_ and,BsH.chains in 0·2 ::II-potassium phosphate buffer of pH 7·05 at 30°C. Concentration of each type ofchain after mixing: curve I, 1·5 X 10- 6 M; curve 2, 0·75 X 10-6 M; curve 3, 0·375 X 10- 6 M. Totaloptical density changes (between time 0 and 00): curve 1, 0·071; curve 2, 0·036; curve 3, 0·017 .

a second-order reaction, IjLJ O.D. versus time, gives curves everywhere concaveupwards.) On the other hand, over the concentration range examined, the half-timesfor the reaction appear to be proportional to the reciprocal of the initial concentrationof the two chains. The magnitude of the second-order velocity constant correspondingto these half-times is roughly 8xl05 M- 1 sec- 1 •

The effect of changing the pH is shown in Fig. 6. The shape of the kinetic curve isthe same at pH 7 and pH 8'5; the reaction is, however, a little slower at the morealkaline pH. As expected, in view of the results obtained by static spectrophotometry,the total optical density change is the same at the two pH values.

The effect of temperature is shown in Fig. 7. The shape of the kinetic curve isinvariant with temperature, just as with pH. On the other hand, the rate of thereaction at 20°0 is 2·6 times slower than at 30°0; this corresponds to an apparentactivation energy of about 16 kcaI.

0-03

do"""l

FIG_ 6. Effect of pH on the time course of optical density changes at 430 mILon mixing deoxygenated ~SR_ and ,esR-chains: results in 0·2 M-potassium phosphate buffer at pH 7·05 (-l',-l',-);results in 0·18 M-K2HP04 + 0·05 M-tris buffer at pH 8-45 (-0-0-). Concentmtion of eachtype of chain after mixing, 0·75 X 10- 6 M. Total optical density changes: at pH 7-05, 0-036; atpH 8-45, 0-037.

0-03

0'02 20°C

do'<:t

0-01

FIG. 7. Effect of temperature on time course of optical density changes at 430 mIL on mixingdeoxygenated ",SR. and ,esB.chains in 0-2 M.potassium phosphate buffer of pH 7·05. Concentrationof each type of chain after mixing, 0-75 X 10- 6 M. Total optical density changes: at 20°C, 0-040;at 30"C, 0-036.

REACTION OF HAEMOGLOBIN ce- AND p·CHAINS 39

(ii) (J.PMB_ and f3PMB- chains

It was shown above that, over the pH range 6 to 9, the ((J.PMB + f3PMB) systembehaves as an equilibrium mixture in which the free (J.- and f3-chains are in equilibriumwith an association complex. At micromolar concentrations, association approachesan upper limit at pH values well below 7. Most of the kinetic experiments wereperformed near pH 7, where the association is of the order of 80% of its maximum.A set of kinetic curves, at thi s pH, obtained under conditions similar to those employedin the O(SH + f3SH experiments described in Fig. 5, is shown in Fig. 8.

The results are very similar to those obtained with the SH system, except that theabsolute rates, under the same conditions, are uniformly about three times slower.At both 20°0 and 30°0 the kinetic curves are sigmoid. As in the other case, the half.time of the reaction is inversely proportional to the initial concentration of the chains.The effects of temperature and concentration on the half-time of the reaction are

Time(seC>

FlO. 8. Time course of op ti cal den sity changes at 430 mfL on mixing deoxygenated cePMB_ andpPMB.chains in 0·1 sr-potessium phosphate buffer of pH 7·0 at 30°C. Concentration of each type ofcha in after mixing: cur ve 1, 1·75 X 10 - 6 M; curve 2, 0·87 X 10- 6 M; curve 3, 0·43 X 1O- 6 :1lL Totaloptical density changes: curve I, 0·084; curve 2, 0·041; curve 3, O·OIl .


shown in Fig. 9. It will be seen that the effect of temperature is the same as for theSH system.

The reaction of the <xPMB and f3PMB chains was also explored at pH values above 7.The results are shown in Fig. 10. Both the extent and the rate of the reaction decreaseprogressively with rising pH; on the other hand, the shape of the kinetic curvesremains essentially unchanged.

15

1.)( 10-6C

FIG. 9. Half-tdmes of the optical density changes at 430 m,.. on mixing deoxygenated (XPMB. and,BPMB.chains in 0·1 M-potassium phosphate buffer of pH 7·0. Abscissae give reciprocal of concentration of each type of chain after mixing.

Since the association-dissociation equilibrium of the (<xPMB + fJPMB) system is proton 'linked, it is clear that if the pH is suddenly changed, the equilibrium will be disturbedand that there will be an approach, or "relaxation", to a new equilibrium. The studyof the rate of this process should provide valuable information about the dissociationand association velocity constants. Experiments involving a sudden change of pH,produced by injection of a buffer of desired pH, were carried out in the stopped flowapparatus.

Figure 11 shows the results of such an experiment in which a solution containingequal concentrations of a.P MB• and f3PMB. chains at an initial pH of 8'3, and hence nearlyall dissociated, was suddenly brought to pH 6·7. Both the shape of the kinetic curvesand the half-times are similar to those observed in the experiment on the directcombination of the chains at pH 6·7.

Figure 12 shows the results of a converse experiment in which a mixture of thechains, initially at pH 6'5, was suddenly brought to pH 8·5. In this case the reactionis an almost complete dissociation of the originally almost completely associatedmaterial. It appears to follow a simple first-order course with a velocity constant ofabout 0·5 to 0·7 sec-I.

Time (sec)

F IG. 11

o

0·02

3Time {se C>

FIG. 10

0'05

0'04

0'01

0·03ci6

'<:l ,-~

,o6"1

0'021- I / I 0· 01

FIG. 10. Effect of pH on the time course of optica l density changes at 430 mp. on mixing deoxygenated ",PMB. and flPMB·chains in 0·05 M.pho sphatebuffers at 30°0 . Concentration of eac h type of chain after mi xing, 1·5 X 10- 6 M . Total optical density chang es : at pH 6,4, 0·078 ; at pH 7·3, 0·048.

F IG. 11. Time course of optical den sity changes at 430 mp' on mixing a solution of (otPMB+ flPMB) at pH 8·3 in 0·2 M-K.HPO. with 0·2 M.K H . PO.; thefinal pH was 6·7. Total protein concent ration in haem bef ore mixing e-> 2 X 1·5 X 10- 6 M. Tot al optical density change, 0·039 .

42

ci6

"<1

0·05

0-04

0-03

0-02

0-01

o

E. ANTONINI ET AL.

3

Time {seC>

FIG. 12. Time course of optical density changes at 430 mp. obtained on mixing a solution of(OCPMB + f3PMB) at pH 6·5 in 0·05 M-phosphate with 0-05 M-sodium borate of pH 9,1, the final pHwas 8·4. Total protein concentration in haem before mixing, 2 X 1·7 X 10- 6 M.

4. DiscussionThe results, both those described earlier and those reported in this paper, demon

strate that the re-assembling of haemoglobin from the isolated IX- and ,a-chains is aspontaneous and rapid process. The fact that re-assembled haemoglobin A showsessentially the same physico-chemical and functional properties as the original proteinis evidence of the great precision of the process and of the high degree of specificityof the subunits for one another. Just as the folding of a polypeptide chain is uniquelydetermined by its amino acid sequence, so the assembly of the subunits of the tetrameris uniquely determined by their identity. Thus the quaternary structure of haemoglobin A or haemoglobin H is the final and exclusive expression of the informationcontained in the primary structure of the component chains, which in the case ofhaemoglobin A are synthetized in genetically distinct loci. It is noteworthy that thereconstitution of haemoglobin occurs equally well whether the chains are mixed inthe oxygenated or deoxygenated form, although the quaternary structures of the

REACTION OF HAEMOGLOBIN IX- AND ,B-CHAINS 43

liganded and unliganded forms of haemoglobin A are different (Cullis, Muirhead,Perutz, Rossmann & North, 1962; Wyman, 1964; Antonini, 1965).t

The ()(.SH. and ,8sH-chains form a very stable complex (haemoglobin A) over thewhole pH range from 5 to 10, both in the oxygenated and deoxygenated form. Evenunder conditions where there is a strong dissociation of the tetramer into dimers, veryfew monomers appear to be present and the dimers are of composition rx,8 (RossiFanelli, Antonini & Caputo, 1964).

As compared with the SH chains, the PMB chains show considerably less tendencyto associate. This is obviously related to the success of PMB as an agent for obtainingthe isolated chains in the method of Bucci & Fronticelli. A mixture of the two kindsof chain (rxPMB+ ,8PMB) dissociates to a large extent at pH > 7, when in the deoxygenated form, and to an even greater extent and at lower pH values when in theoxygenated form. Thus the association-dissociation equilibrium of the rxPMB+ ,8PMBsystem provides an interesting example of an oxygen-linked process (Wyman, 1964).

It is pertinent to ask to what extent this linkage of dissociation with oxygenationmight be expected to affect the character of the oxygen equilibrium-it must clearlyhave some influence. If we assume, somewhat arbitrarily on the basis of Fig. 3 that,at pH 6,5, the chains in the oxy form are 5% associated and in the deoxy form 95%associated, then we calculate, by essentially the same procedure as that developedearlier (Wyman, 1964, section 18), that at 50% oxygenation the associationdissociation transition should make a contribution to the Hill parameter n of approximately 1·4; i.e, if n were 1 in the absence of the transition, it should be 1·4 as a resultof it. The transition should also lead to a skewing of the equilibrium curve.

The general parallelism between the sedimentation behaviour, the kinetics ofreaction with carbon monoxide and the absorption spectrum in the Soret region ofthedeoxygenated PMB system, suggests that all these properties are correlated andreflect a proton-linked association-dissociation equilibrium between forms containingrx,8 units and the free, monomeric a- and ,8-chains.

It is tempting to try to account for the spectroscopic behaviour shown in Fig. 1quantitatively. Let us assume that, under the conditions of these experiments, thechange in f (fraction of total chains present as monomers) results exclusively from achange in the equilibrium constant of a dimer-monomer equilibrium. Then:

I-f-- =2LC=L'f2

(1)

where C is the total concentration of the chains. The variation of L with pH whichgives rise to the observed pH dependency off must be due to a difference between theionization of certain dissociation-linked acid groups in the dimers and monomers.

t It was observed by Huehns, Beaven & Stevens (1964), using the technique of starch gelelectrophoresis, that the reaction of IX-chains of human haemoglobin prepared by the method ofHuehns, Dance, Shooter & Beaven (1962) with native haemoglobin H to form haemoglobin Awas a diphasic process. A small fraction of the material reacted "instantaneously", but the restreacted much more slowly over a period of 24 hours. The difference between this result and oursmight be ascribed either to a difference between our ,BsH-chains and native haemoglobin H, to adifference between our IXsH-chains and those prepared at acid pH by the method of Shooter, or tothe fact that our observations were made on the deoxy derivatives, theirs on the cyan-metderivatives. However, as reported in previous papers of this series, when the chains prepared bythe Bucci procedure were mixed, either in the oxy or carboxy form, the changes in sedimentationand electrophoretic properties and in the kinetics and equilibria of reactions with ligands werecomplete within the time required for the measurements.


(2)

1·0.-----==--------------------------,

..... 0·5

pH

FIG. 13. Fraction J of chains present as monomers in relation to pH for the (ctP MB + flP MB)

system (see text). Values calcula ted from optical dens it y changes consequent on mixing theisola ted chains, assuming that at acid pHJ -+ 1 (0). The curve is cal culated on the hypothesisthat asso ciation consists exclus ively of the reaction ctP MB + flP MB -+ ctP MB fl P MB, and t ha t the reare three association- linked acid groups wh ich are masked in t he dimer (pK ;;._ 9) and exposedin the monomers (pK ~- 6), the asso ciation constant (1 - f)11 2 being 10- 2 at pH 8·09 underthe conditions of the experiment.

The governing equation is:

dlog L' = dlog L =_ L1E.dpH dpH

Here, if L is defined as the association constant for the equilibrium, JB is the differencebetween proton bound by the dimer and that bound by the two component chainsfrom which it is formed. The smooth curve in Fig. 13 is calculated by settingL1B = - 3 at every value of pH less than 8'09, where L' is assumed to be 10- 2 • Thisis equivalent to postulating that there are three dissociation-linked acid groups,presumably two for one kind of chain, one for the other, each of the pK values ofwhich lies somewhere above about 9 in the dimer and somewhere below pK 5·5 inthe monomer. These groups (presum ably carboxyl) could be regarded as being maskedin the dimer and being unmasked when the dimer splits into the two componentchains . This, of course, applies to the system in the deoxygenated st ate. The agreementbetween points and the curve is reasonably satisfactory, although the model is, ofcourse, highly arbitrary and ignores the effect of tetramers resulting from polymerization of the ocfI units.

Perhaps the most significant feature of the pre sent study is the light it sheds on thekinetics of the combination of the chains. All that we observe is, of course, the spectralchanges which accompany the mixing of the chains, and even in thi s observation weare limited to the deoxygenated chains. The experimental evidence indicates that the

REACTION OF HAEMOGLOBIN ex- AND .a-CHAINS 45

spectroscopic change is due to the association of unlike chains in pairs and to thearrangement of the new molecule in a specific conformation. The mere polymerizationof chains does not produce a spectral change, since fiSH (or haemoglobin H) (Benesch,Gibson & Benesch, 1964), which is tetrameric, has the same spectrum as fiPMB, whichis predominantly monomeric. Moreover, haemoglobin A does not change spectrumwhen dissociated into dimers (rxfi dimers) in concentrated salt solutions. On the otherhand, haemoglobin CPA and the haemoglobin-haptoglobin complex, both of whichcontain IX- and fi·chains but are conformationally different from deoxygenated haemoglobin A, have a spectrum similar to that of the isolated chains (Chiancone, Wittenberg, Wittenberg, Antonini & Wyman, 1966).

Although the spectral changes may not give the full picture of the kinetics ofcombination of the chains, at least they give a minimum picture-the true kineticscan hardly be simpler than the spectroscopic kinetics. In the SH system the totalprocess which takes place when the chains are mixed may be written as:

(A)

A possible scheme, among others, which is consistent with the main features ofthe rather limited kinetic data presented here is the following (see Jen-Yuan Chien(1948)):

fi, -+ 4fi (a)

rx + fi -+ rxfi* (b)

IXfi* -+ IXfi (c)

2rxfi -+ rx2 fi2 (d)

This involves one conformation change, namely step (c), during which the speotroscopic change is supposed to occur. It is clear that if the reaction goes to completion,then the observed spectroscopic changes cannot be more complex than correspondsto the totality of the reactions up to that point.

As we have seen, the over-all speed of the process, calculated as a second-orderreaction, is of the order of 106 M- 1 seo" '. This is surprisingly high in view of itscomplexity; however, it is much less than that of recombination of haem with globin(over-all second-order velocity constant v--> 108 M- 1 sec- 1 (Gibson & Antonini, 1960).Here, however, the process directly observed is a different one, namely, the combination of haem with the chains. Unfortunately, no other similar data on there-assembly of a protein from its constituent chains are available with which tocompare these results.

Changing the pH from 7 to 8·5 has little effect on the kinetics of the reaction ofSH chains, although both chains almost certainly carry a high net charge at pH 8·5.Unspecific electrostatic interactions between the chains as a whole would seemtherefore to playa relatively small role. In contrast to pH, temperature has a largeeffect, but it is difficult to interpret this in terms of activation energies in view of thecomplexity of the situation.

The kinetics of the reaction of deoxygenated o,:PMB_ and fiPMB.chains is remarkablysimilar to that of o,:SH and fiSH, both in respect to the shape of the curves and to theconcentration dependence of the half-times of the reaction; also the effect of ternperature is alike in the two reactions. It is evident that, since the isolated fisH.chainsexist as tetramers, the fiPMB.chains as monomers, the fi~H -+ 4fisH dissociation cannot

46 E . ANTONINI ET AL.

be a rate-limiting step in reaction (a). But apart from details, it is remarkable that socomplex a pro cess as the assembly of a large molecule like haemoglobin from itsvarious component parts should be as fast as it is.

This work was partly supp orted by a grant fro m t he National Science Foundation t oone of us (J . W .). We t hank Mr Mario Sanchion i for ski lfu l technical assistance.

REFERENCE S

Antonini, E . (1965). Physiol. Rev. 45, 128.Antonini, E., Bucci, E., Fronticelli , C., W yman , J. & R ossi.Fanelli , A. (1965) . J. Mol.

R iolo 12, 375.Benesch, R., Gibs on , Q. H . & Benesch , R. E . (1964) . J. Biol. Chem. 239, 1668.Bucci, E . & Fronticelli, C. (1965). J. Biol. Chem. 240, P C551.Bucci, E., Fronticelli, C., Chianc one cEi, Wyman, J., Ant onini, E. & R ossi-Fanelli, A.

(1965). J. Mol. Biol. 12, 183.Chiancone, E., Wittenberg, J. B ., Wittenberg, B., Anton in i, E. & Wyman, J. (1966).

Biochim. biophys. A cta, in the press.Chien, J ..Y. (1948). J. Amer. Chem. Soc. 70, 2256.Cullis, A. F., Muirhead, H., Perutz, M. F., Rossmann, M. G. & North, A. C. T. (1962).

Proc, Roy. Soc. A, 263, 161.Gibson , Q. H. & Antonini, E . (1960) . Bio chem, J. 77, 328.Gibson , Q. H. & Milnes, L. (1964 ). Biochem. J. 91, 161.Hueh ns, E. R., Beaven, G. M. & Stevens, B. L. (1964). Biochem , J. 92, 444.Huehns , E. R., Dance, N ., Shooter, E. M. & Beaven , G. H. (1962) . J. Mol . Biol. 5, 511.R eichlin , M., Bucci, E ., Fronticelli , C., "Wyman, J., Ant onini, E ., I oppolo, C. & Rossi-

F anelli , A. (1966) . J. M ol. Biol. 17, 18.R eichlin, l\1., Bucci, E., Fron ticelli , C., Wyman, J., Ant on ini , E . & R ossi-Fanelli, A. (1965).

J. Mol. Biol. 12, 774.Rossi-Fanelli, A ., Ant onini, E . & Cap uto, A. (1964) . A dvanc. Protein Chem. 19, 73.Wyman, J. (1964). Advanc. Protein Chem. 19, 223 .

The properties and interactions of the isolated α- and β-chains of human haemoglobin

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Transcript of The properties and interactions of the isolated α- and β-chains of human haemoglobin