Structural studies of haptoglobins

ARCHIVP:S

OF

BIOCHEMISTRY

AND

Structural II. Reversible

123, 133-144

BIOPHYSICS

Studies

Dissociation

de Mddecine,

Laboraloire

of Haptoglobins

into Subunits

AI. WAI
of Hp l-l

and

Hp 2-2

A. AI,FSEN

24XD

de Hiochimie, Paris, Received

(1968)

(Prof. France July

M. F. .Ja!lle)

45, rue des Sainls-Pkres,

11, 1967

Chromatographic, sedimentation velocity, and equilibrium experimen1.s have been carried out to investigate the subunit strrlctures of haptoglobins (Hp l-l and Hp 2-2) in relation to their hemoglobin binding and activating properties. DEAE-cellrdose chromatography showed anomalous elution profiles. Bio-Gel P 150 Chromatography revealed, at pH 5.50, near t,he isoionic point, that haptoglobins dissociate reversibly in the concentration range 0.25-2.0 mg/ml, brat, they remain stable at higher protein concentration. Sedimentation and equilibrirlm ultracent,rifugation indicated that, at pH 11.50, Hp l-l and Hp 2-2 are homogenoas and dissociated into subunits of molecular weight 40,000 f 2500. This dissociation is reversible. It is srlggested that Hp l-l possesses an oligomeric structure with a monomer-dimer equilibrium and that Hp 2-2 is made of a subunit of identical molecular weight. in equilibrium with a series of higher order polymeric species. The association of hemoglobin with haptoglobins and t’he consecut’ive increase of the peroxidase activity (1) is closely related to the structure of the interacting molecules (2). Two genetic types of haptoglobin from human serum which show inherited differences by gel electrophoresis (3) have been examined in this study: Hp l-l and Hp 2-2. In previous research (4) the secondary and tertiary structure of these two genetic types was investigated by hydrogen ion titration, by thiol, and by disulfide amperometric titrat,ions, and by optical rotatory dispersion. It was shown that the main difference between Hp 1-l and Hp 2-2 is in the number of disulfide bonds and a more compact structure for Hp 2-2. All other propert,ies were found to be very similar. The present report’ describes t’he reversible dissociation of Hp l-l and Hp 5-Z as studied by chromatography and analytical ultracentrifugation. The data indicate that haptoglobins dissociate reversibly into subunits of identical molecular weight, as previously sugg&ed (4). 1 Charg6

de Rcchcrches

& I’I.N.S.E.R..RI. 133

The results obtained in this study do not fit the model proposed for the molecular structure of Hp l-l and Hp 2-2 by several authors (5, 6) ; however, they are consistent with the finding that the hemoglobin binding properties of the two genetic types are similar when expressed relative t’o a common molecular weight. (7). MATERIALS

AND

METHODS

Pure haptoglobin was obtained from pleural and ascitic fluids by the methods described in a previous paper (4). The protein was concentrated by pressure dialysis in the cold; 8/100 l?skirrg tubing was rlsed (Union Carbide Corp. Chicago, Illinois). Ovalbumin, cryst,allised five t,imes, was obtained from the California Corporation for Biochemical Research (lot No. 503103). Horse carbon monoxyhemoglobitl was prepared according to Cann (8). Haptoglobin was chromatographed OII diet hylaminoethyl cellulose (I)EAE), Whatman l)E 23, and on Bio-Gel P 150 (Bio-Rad Lahorat,ories, Richmond, California). Analytical polyacrylamide gels were prepared with Cyanog\lm 41, dimethylamitro-propiollit,rile and ammonium persulfate pltrchased from E.C. Apparat~l~s (Philadelphia, Pennsylvania): acryl-

13-t

WAKS

,4ND

amide, .V,S’-met.hylene bis-acrylamide and s v 1” T’ tetramethylethylenediamide were pr&:ied’hy Eastmall Organic Chemicals (Rochester, New 1.ork). All other reagents were the same as those mentioned in ollr previoru paper or were the best available commercial prodrict,s llsed witho[lt frut)her purification.

DEANE-Cellulose. Haptoglobin was cromatographed 011 columns 15 cm in height and 2 cm in diameter. The two fractions (A and B) collected in the first step were rechromatographed on colrlmns 15 cm in height and 1 cm in diameter. The fractions obtained after t)he DEAE-cellulose re-run were combined and applied to the top of a column of Bio-Gel 1’ 150, 50 cm in height,, 2.5 cm in diamet.er. Bio-(iel, a porous polyacrylamide gel-filtrat,ion material, was Ilsed in place of Sephadex t,o avoid the interactions hetweell glycoproteins and dextrails that have been described by varioru allthors (9, 10). Gel ,/ilt~!ion. The Bio-(;el P 150 was hydrated for 72 hotus and decanted to remove fines before packing. The brdfer ill 0.1 M KCI, pH 5.50, was degassed. The colrunn ILsed in frolrtal analysis experiments was 55 cm high alld 0.9 cm in diameter (Phoellix precision Itrstrklments Co. Philadelphia, Petrlrsylvania). It was jacketed alld thermostated at 7" + 0.02” by Haake Kt 62 thermostat (Haake, Berlill). The effluent was continllously monia Hellma 0.5.cm flowtored at 278 mp, with ctlvette :tlld a Sargent S.R. recorder attached t,o a Bausch atld Lomb Hpectronic 505 recording spectrophotometer. The colrlrm~ flow rate was set at, 12 ml/hollr by means of au LKB “ReCyChrom” peristaltic pump. The sample, 15 ml of protein soltltiotr, was diluted to the desired concentration and applied to the colrlmu followed by bnfTer. The resldtillg elrltion curve was recorded and showed a plateall, the proteitr concentration of which was eyrutl to that initially applied. P:fflnent volumes were measured by passing the olltput from the peristaltic pllmp ilrto a gradrlated cylinder wit,h which volllmes were measllred at any chart, positiotl. The flow rate alld the chart speed were checked periodically. It) was possible to reproduce ellitioll volumes to f0.1 ml. Ellltioll volrrmes were taken from the initial additiotl of the proteilr sample t,o the middle of the platean rrgioll. The colrum~ was standardized with ovalbllmill, alld horse carboll molroxy-hemoglobin. The void volrlme was measured with a 20s macroglobrllill kindly provided by Dr. R. (iot.

mrl

Elertrophoresis was carried orlt at 500 V and 30 ill a 25 X G.5 X 0.3 cm slab of polyacrylamide

ALFSEN gel, in t,he horizontal apparatus,, described by Raymond and Wang (11). The gels were(i6x Cyanogum al. Whell a continuolls bluffer system was used, a stock sollltion of 0.3 .\I boric acid, 1 N KOH, and 0.02 M KCl, pH 8.6, was diltlted 8 t,imes before use in the gels and the elect>rode chambers. Some electrophoreses were carried orit iit a discontinrtorLs briffer system; for the gels, 18.15 gm Tris and 24 ml of 1 N HCl for 100 ml at pH 8.9 were used; for the electrode chambers a bluffer containing 6.32 gm Tris and 3.9-1 gm ammonia-free glycine for 1000 ml, pH 8.9, was rised.

DETEHMIXTION OF Li~~~4~~~~ SPECIFIC:VOLUME The apparent specific vol[une of haptoglohins was obtained from density determinations of 0.5’)‘) prot,ein solutions in 0.3 M KU. Densities were meastlred ill a 25-ml Brandt pycnometer eqtlilibrated in a constant temperatilre bath at 20” + 0.02”. Weighings accrlrate to 0.01 mg were made with a Sartorills 2601 halauce. Three separate determinations were made on each protein so1~1tion aild v:,,,,, was calculated from t,he following relationship (12) : v-a,,,, =

l/PO-l/c

(P/Purl),

where puand p are the densities of the solvent am1 the solution, respectively, and c is the concerltration in gm/ml.

ANALYTICAL ULTHACEXTRIFUGATION coefficients and molecldar Sediment at ioil weights were determined with a Spinco model E analytical Idtracentrifnge eqltipped with a rotor temperatllre indicator and control unit, a phase plate schlieren diaphragm, and a R.ayleigh interference opt ical system. Sedimentation coclFicients were determined at 20” at a rotor speed of 59,780 rpm; Kel-F center pieces were employed because of the high alkalisity of several hrlfl’ers [Ised. Sedimentation coefficients were calcldated by the Iuual method and expressed as Sk,,,,,. The S vnlrtes were not corrected to the hypothetical values of .s~~, Tu; it sholdd he noted that ill systems consisting of polymeric species iu cy~~ilibrium, corrected ~2~. Lu values will not describe the weight average properties at 20” lmless the cyllilibrium position is temperatllre independent (i.e., AH is zero) (13). Sedimentation cqllilibrirlm experiments were performed essentially as described by Yphantis (la) at low protein concetltration (0.03”,‘,) and a relatively high rotor speed, which assures that the concentratiotl of sedimenting material at the rIpper meniscrls of the solrltion collunn is essentially zero. (iraphs of 111 c verslls .r2 were obtained. The linearity of the plots indicated homogeneity,

STRUCTURAL

STUDIES

OF

HAPTO(;I,OBIN~.

11

1%

dependent on the initial concentration and t,hc total amouttt~of haptoglobin (more than 100 mg) applied to the coIumn. Sharpness varied with the rate of elution; however, a complete separation was never achieved. Each peak (-2 and B of Hp l-l or Hp 2-2) was cottcetttraked bagpressure dialysis to a concentration of 20 mg’ml. Immunoelectrophoresis, polyacrylnmide gel-electrophoresis (Fig. 3) and hemoglobin binding demonstrated :t complete absence of anycomponent other than Hp l-l or Hp 2-2. DEAE-cellulose chromatography of each peak UYLYcarried out. The results of these experiments are summarized in Fig. 4. I’wks ;II and B split, into t,wo more peaks: :t and a’, b and b’. Xo differeuce was observed i F between the t\vo gettet)ic t’ypcs of hnptoglobin. These components gave clectrophoret,ic puttcrns similar to t,hoseshowtt in lcig. I 3. F’innlly, fractions a, a’, h, and b’ were pooled and applied to a Rio-Gel 1’ 130 column. 50 85 120 155 Gel filtration failed to give a.tt effective volume of effluent (ml) fractionation of thcso components; Hp l-l FIG. 1. Elrltion profile obtained from chromaas Hp 2-2 \vere elutcd as citte :qmmctric tography of IIp 1-l 011 a 20 X 2-cm colrml11 of peal; (Pig. 7). I )lX.4E-wlll~lose. Haptoglobin was elllted with Gel jli~~afion and frontal analysis c.rperi0.01 21 wetate huf!fer. 0.08 3%in KCI, pIl1.70, at 20”. ~wcnts.Elution volumes, I’, , pertaining lo gel filtration of Hp l-l and Hp 2-2, arc plotted against concentration (15) in Figs. 5 and 6. With Bio-(kl I’ 130, at, 7”, iu 0.1 .w KC1 at pH 5.50, it, is apparent that the clution volume remains constant, for protein 111 i he ahove equation 111 c is the logarithm of concentrations bet\veen 3 and 2 mg/ml. For prtjtpill collccntration, x2 is the sqllare of the disHp l-l, I’, increases from 25.1 to ‘26.4 ml t:uwc from the center of rotation, I< is the gas n-hen the protein concentration falls from c~~~~st:tlli, 7’ is I he absolute temperature, 1’; is the 2.30 to 0.50 mgjml. Hp 2-2 is excluded from ~wrtial specific volllme, p is the density of the Bio-Gel I’ 150 up to a concentration of 2 sc~llltiot~, and w is the angular velocity. Several - and 0.25 mg/ml t#heelumg/ml; between ‘) rows u-CLW performed at pH 11.50 in 0.3 M KCItion volume increasesfrom 21.G to X.3 ml. KOH or ill 0.3 31 lysine-KC1 bot’fcr. Experiments W:he~~ dilutctd protein solutions (< 2 mg,/ml) wv(‘re dolle at 4”, i”, alld 20”. were concentrated and the elutiou volunw RESULTS ~vas then measured! the points fell on the same curve as in J’igs. .? and fi. CHROAIATOGRrZPHY A typical clutiott profile for Hp l-l is l)E~l EJ-wll~~~losc. The preparation of hap- shown in l;ig. 7, the lower curve being the first derivative, Ad /Al’, ohtnined by using toglobitts (3) is achieved by stepwise elution: 0.5 ml increments of volume. The recorded Hp 1-l with :I 0.01 M ncetat’e buffer, pH elutiott curve (:tbsorption at, 2% trip against 4.70, 0.0s M iu IiCY; Hp 2-2 with the same volume of effluent) consists of an advancing buffer, but 0.1 11in KCl, at pH 5.5. Typical edge, :L plateau regic.)nin \vhich the protein eltttiott profiles :at 20”, show1 in E’igs. 1 and concentration is cc~ual to t.hat initialI)- ;\I)2, cottsisted of two incompletely separated plied, and :I trailing edge. The curve is peaks. The optical density of each peal; \I:W decidedly aq~ntmc~tric, the adwttcittg edge I

‘I 1

/

I

136

WAKS

0.D 280

AN I) ALFSEN

mp

4r

volume FIG. 2. Elution of DEAE-cellulose. at 20”.

of effluent

(ml)

profile obtained from chromatography of Hp 2-2 on a 20 X 2.cm column Haptoglobin was eluted with 0.01 M acetate buffer, 0.1 M in KCL, pH 5.50,

FIG. 3. Polyacrylamide gel electrophoretic patt,erns (borate buffer, and B (respectively from left to right) obtained by chromatography Upper pattern : Hp 2-2; lower pattern: Hp 1-l; concentration, 20 mg/ml. with Amidoschwarta 10 B.

being sharper than its trailing counterpart. The elution profile of Hp 2-2 is fundament,ally identical and contains some slight inflection points on the trailing edge. According t.o Winzor and Scheraga, such a pattern

pH 8.6) of peaks ,4 on DEAF,-celltdose. The gel was st.ained

is characteristic of a reversible association equilibrium (16). By conkast, ovalbumin, a nondissociating protein, yields a gel f&ration pattern in which the trailing edge is sharper than the advancing one (Fig. S).

STIIUC’TIJIL4I,

STUI)TW

OF

D.E.A.E. Cehbse iflg

1 and

TThPTO(;I,ORTNS.

137

II

s---HP--I

21

B (HP)

A (HP)

D.E.A.E. cellulose rerun b (HP)

a’ (Hp)

a (Hp)

b’ (HP)

peaks a 1 a‘ -2 b c b’ Bio - Gel P - 150

HP FIG. 1. Scheme of chromatographic experiments carried out terization of the peaks A and B obtained from rechromntography perimental conditiolrs are described in the text.

haptoglobills. on I)FAE-cellulose.

CharacEx-

I

I

Ve

with

ml.

0

I

I

I

1.0

2.0

3.0

mg/ml

C. FIG. 5. The elution voltune, V,, of Hp 1-l on a, 50 X 0.9-cm Bio-Gel P 150 coIumn a function of protein concentration. The protein was in 0.1 M KCl, pTT 5.50.

at 7’ as

ve

ml.

27

26

25

0

0

1.0

2.0

volume V,, of IIp as in Fig. 5.

2-2 as a function

3.0

w/ml

C. FIG. (j. The elut,ion experimental conditions

of protein

cottcetrtratjiott.

Same

A 280

30

20

10

0 J-&l

AV 02

0.1

15

18

21

volume FIG. 7. Elutiotr profile mg/ml) ou a 50 X 0.9-cm pH 5.50, at 7”. The lower (right) edges.

(upper colttmtt curves

24

of effluent

27

30

(ml)

curve) obtaitted in t.he chromatography of Bio-Gel 1’ 150 previottsly equilibrated are the first derivative of the advancing 138

33

of Hp 1-l (3.4 with 0.1 M KCI, (left,) and trailing

36

STRUCTURAL

A280

STUI)IES

OF

HAPTOGLOBINS.

139

II

2-

l-

o1.5 -

1.0 -

1.5 -

68

volume

78

of effluent

98

(ml)

FIO. 8. Elllt,ion profile (dapper clu-ve) obtained in the chromatography of ovalbumin mg/ml) on a 50 X 2.5.cm colmnn of Sephadex G-100 previously equilibrated with phosphate, pH 7.90, at 20”. The lower cilrves have the same sigrlificajtee an in Fig. 7. I'A~~AL

SPECIFIC:

(4.4 0.01 M

VOLUME

obtained for Hp l-l and Hp Z-2 The 8,,,, in 0.3 al KC1 \vas 0.660 f 0.005 ml/gm at pH 5.50 and 0.667 ml/gm at pH 11.50, in disagreement, with the value of 0.766 ml/gm reported by Hermnnn-Boussier (17). The partial specific volume calculated (Cohn and Ed&l, Ref. IS) from bhe amino acid and sugar composit~ion given by Cloarec et al. (19) was found to be 0.710 ml/gm for Hp 1-l a.nd Hp Z-2. This value is lower than that given by Hermann-Boussier and higher than our experimental data. The low P,,,, of hsptoglobins is most probably due to t,heir sugar cont.ent, (lS.5 54). Gibbons has shoivn (20) that t,he low partial specific volume of glycoproteins is direct)ly related t,o their sugar content. (r: of sugars is in the 0.5500.660 ml/gm range). However, it is possible that the discrepanqy between the calculated and our experimental I-. of haptoglobins value may be due to some uncertainty in the extinction coefficient, of Hp 1-l and Hp 2-2 (17 ) “1).

r FIG. 9. Sedimenijatiotl velocity patterns of 0.6% Hp 1-l (left) and Ilp 2-2 (right) ill 0.3 M KCI, pH 5.50, at 20”. I’ictln-es were lake11 50 minlltes after attaiuing a speed of 50,780 rpm. 01 allyIt of the schliereu diaphragm of W.

Hp l-l and Hp 2-2 have been examirled by sedimentation velocity experiments in the pH range from 9 to 12, in \\-hich the hydrogen ion t.itration :tnd spectropllotometric titration curves have been shown to be reversible (4). At 20”, in 0.3 11 IiCl, from pH 5..‘5, lvhich is not far from the isoionic point of the protein, to pH 9.5> Hp 1-l scdimcnts with a

140

WAKS

ANI)

ALFSEX

s alJu 5.0

9.00 10.80 11.30 11.50

4.0

11.80 12.00 3.0

t

9.0

I 9.5

I IO

I 10.5

I 11

I 11.5

12

PH

Fro. 10. ITariat,ioll iti the apparent sedimentation coefficient of Hp 1-l as a flmction of pH. Solid circles represent the irreversible part of the clmve; diamonds, back titration dat,a. Solid f riangles : experiments after alkylation of the proteitl wit,11 iodoacetnmide at pH 8.60.

FIG. 11. Sedimentation velocity patterns of IIp 1-l (left) and Ilp 2-2 (right) at 20” in 0.3 M KCl-KOH, pH 11.50, at a concentration of 5 mg/ ml. Pictllre was taken at a phase plate angle of GO”, ,znd 92 minutes after reaching a rotor speed of 59,780 rpm (Hp l-l) ; at an angle of 50”, 82 minutes after reaching a rotor speed of 58,780 (Hp 2-2).

symmetrical boundary, with a sedimentation coefficient’ of 4.10 (Fig. 9). Hp 2-2, in COIItrast, displays a heterogenous pattern with at least t)wo components. The sedimentation coefficientj of the slow component was calculated to be 3.5, and that of the fast,, 7.5 (Fig. 9). It is clear from Fig. 10, that, in the pH range 9.5-12, t,hc sedimentation coefficient of Hp l-l decreases with increasing pH. The curve obtained by plotting S,,,, against

3 .5

7.1

3.5

6.5

3.5

(i.6

2.0 2.8 2 .9

pH is completely reversible between pH 9.5 and 11.5, as the S,,,, values decrease from 4.10 to 2.90. At higher pH values, an irreversible transformation occurs. This irreversible change is preventred by addition of iodacetamide to the protein at pH $60. Under these condibions the S values do not, decrease further than 2.9. Figure 11 shows the schlieren pattern for a sedimentat,ion velocity experiment on Hp l-1 in 0.3 I\I IiCl-KOH, at pH 11.50. The symmetrical peak observed throughout the course of the experiment suggests a relat,ively homogenous species. The variat’ion of &,,,, for Hp 2-2 I\-ith increasing pH values is illustrated by the data in Table I. In t’he pH range 9-11.3 t’here are only slight variatious of S:,,,, . A fast transitjiou occurs between pH 11.30 and 11.50, and a single 2.9s peak is observed between pH 11.5 aud 12. This transit,ion is completely reversible, i.e., at any pH value between 12 and 11.5 the back titration of Hp 2-2 shows the same t,wo peak schlieren pat,tern observed in Fig. 9, with unchanged sedimentation values of 7.1 and 3.5. Siuce a decrease in sedimentation coefficient cau be attributed to a dissociation of macromolecules into subunits, to an increase of the frictioual coeficicnt, or to a combination of dissociation and disorganization, the molecular weights can be calculated from t,he combination of sedimentation coefficients and intrinsic viscosities. However, such determinations are dependeut on the choice of a hydrodynamic model (22). In order to eliminate such uncertainties, sedimentation equilibrium experiments were performed ou a 0.03% solution of Hp l-l and Hp 2-2 at a rotor speed of 27,690 rpm. Figures 12 and

0 42 400

I

I 600

I

I 800

I

I 43 000

I

I .200

I

I 400

I

I 600

I

I 800

13 show plots of In c against x?, at. pH 11.50. For homogenous materials, such plots give straight’ lines. The experimental dam of Hp l-l and Hp 2-2 give a straight, line, and t.he molecular weight calculated from t,he slope at 7” is 42,000 for Hp l-l and 43,000 for Hp 2-2, when in 0.3 M KCI-ROH. At ‘LO”, in a 0.3 IVI lysine-KC1 buffer, a value of 48,7SO was found for both Hp 1-l and Hp 2-2, Ah P = 0.710 ml/gm. With FaDI, = 0.667 ml/gm the molecular weight would be 38,000.

On the basis of the sediment’ation velocity and equilibrium data, it may be concluded that, at pH 11.50, Hp l-l and Hp 2-2 diss0ciat.e reversibly into subunits of ident.ical molecular weight. DISCWISION/ ,

Human haptoglobins of genetic type l-l and 2-2 are made up of two types of polypeptide chains: a-chains designated 10~(F or S) for Hp l-1, 2cufor Hp 2-2; and @chains. Models proposed for the molecular structure of haptoglobins are based on the post,ulation (23) of two allelic genes,Hpl and Hp2; the latter would arise from a partial duplication of Hpr and dict,ates t,he syruhesis of 2a-polypept,ides. The ‘a-chains cont,ain a repeated amino acid sequence, each portion of which is approximately equivalent to one la-chain (24) and would be twice as large as the la-chain (25). The p-chain, on t.he other hand, is common t’o bot’h genetic t’ypes. The molecule of Hp 1-l has been described as a single monomer consisting of a pair of k-chains and a pair of &chains (26). Hp 2-2 would be made up of a series of stable polymers of increasing molecular weight (3, ci), with a number of 2or- and P-chains not well defined, probably in t,he samemolecular proportions. It’ is clear that the data presented in this study do not fit, such models. The anomalous elut.ion profile on DEdEcellulose chromatography may be due either to impurities or to artifacts. However, polyacrylamide gel electrophoresis does not reveal any impurities even at high protein concentration (20 mg/ml) ; the two components, A and B, elut,ed from the column bind t,he same amourlt of hemoglobin (27).

These chromatographic patt’erns have been found in all experimcms performed with different batches of DE=\E-cellulose (Whatman DE 50, DE 23, Kodak and Scrva). The possibility of an artifact is considered to have been eliminated. According to Keller and Giddings (2S), and more recently Bet.hune and Kegeles (29), such a pattern can be interpreted in terms of a reversible association equilibrium. The results of gel filtration chromatography WI Bio-Gel P 150 are in agreement with this irnerpretation; the increase in the elut’ion volume as a function of decreasing concentrat,ions of Hp 1-l and Hp 2-2 indicates a reversible dissociation into subunits of smaller molecular weight. The shape of the elution profiles is also consistent with the presenceof an association equilibrium in Hp l-l and 2-2. At pH 5.50, near the isoionic point of huptoglobins, a complete dissociation is not attained at the protein concentration used (see Note added in proof). The hydrogen ion titration curves published in our first paper (4) display an irreversible zone from pH 2.50 to 9, in which some aggregation was observed by ultracentjrifugation experiments (27), and a reversible zone from pH 9 to 12, in which a reversible decreaseof t,he apparent sedimentation coefficient occurs. From these data, a reversible dissociation in the alkaline pH range becomes evident for both genet,ic types of haptoglobins. The possibility of a change in molecular shape, by unfolding, which could produce a corresponding decrease in sedimentation coefficient, can be eliminated since the molecular weight of t,he subunit, calculated by sedimentation equilibrium, is half the molecular weight of Hp l-l at neutral pH (S.5,OOO). Such reversible dissociation could not occur if covalent linkages were involved in the structure of the 2a-chain. It would be difficult to propose a model of a subunit made up of one a- and one fi-chain which has the same molecular weight for Hp l-l and Hp 2-2, if the ‘Lcr-chainhad a molecular weight approximately twice that of a lachain. The difference in the subunit molecular weight, would be about 5900 (molecular

PTRUCTURAI,

STUI~TES

jveight of one la-chain, Ref. 25) and should be evident from the sedimentation cquilibrium experiments (14). The thiol groups and disulfide bonds seem to bc involved in the structure of Hp 1-l and Hp 2-2. IMferent eicctrophoretic pat,terns have been obtained by Smithies (30) for Hp 2-2 by disulfide-bond cleavage in the presence of 2-mercaptoethanol and diet’hanol disulfide at pH S.7. Hoxvcvcr, the major component obtained in these experiments had :t sediment:tt.ion coefficient. close to t.hnt, of Hp l-l. It, 11:~s been sho\vn in the present study that the reversible dissociation of Hp l-l is not, modified by nlkylation \vith iodoacet,amide of the SH groups exposed at pH 8.6. Han-ever, further dissociat.ion, which is irreversible, is prevented. The diasociat.ion of Hp Z-2 is ent,irely reversible in the pH range {j-l’-,; the higher number of disulfide bonds, 1 II-ice as many as in Hp 1-l (a), probably prevents an irreversible dissociation. In fact, it appears t,hat t.he la-chain contains one internal disulfide bond and that the Z-chain contains t,\vo such bonds under the expcrimental condit)ions described by Smit’hics et. nl. hlorcover, under some conditions, the single L)cr component sho\vcd three zones by gel elcctrophvresis that, differed only in the presence or absence of internal disulfide bonds (31). The discrepancies betbveen the data present,ed in this study and those published by Connell et al. (25) rnfy. be due t)o differences in experimental condltlons. The behavior of the isolated %-chains can be different ivhen hound to p-chains in the intact, molecule. Connell et al. have noted a greater tendency to aggregation n-ith %-chains than u-ith la-chains; thus, it is possible t’hat the sedimentat,ion coefficients obtained (1.4 for 2,) still correspond to a,ggregates. At alkaline pH, in the highly charged molecule, this aggregate dissociates and leads to a protomer (33) of identical molecular jveight for Hp l-l and Hp 2-2. The nature of the intermolecular forces involved, jvhich maintain the subunits t,ogether, is ccrt,ainly of a different nature than of t.hose involved in the molecule of hemoglobin (32). High salt concerlt~r:ttions (0.3 11

OF IIAI”l’O(

1%

:T,OBIP;S. I I

MgCl?) do not promote any dissociation; solvcnt,s like formamide arc also ineffective (27). It, seems that the ionization of some: charged groups is the determining feature in the disso&tion into protomers. The parallelism bet\\-ecltt the spc,ctroE)hotornetric tit.rntion curve of the tyrosines in Hp 1-l (4) and t’hc slope of the plot of S,,,,, against. pH (Fig. 10) suggests a possible role of those t\-rosine groups \vhich have an :anomnlous high pl< (-1). If the tvrosines arc not involved by t hemsclvcs ’ itI maintaining the subunits together, they are at least modified at the same time as dissociation occurs. The number of protons involved in this dissociation corresponds to the number of tyrosinc: groups calculated from the potentiometric and spect.roI)hot.omctric titrat,ion curves. In conclusion it, is clear from this set of expcrimcnts that bot,h Hp 1-l and Hp 2-Q undergo reversible association-dissoci:~tiorl equilibria with protomers of identical molecular Jveight. HI, l-l is a dimer, and Hp 2-2 is probably a polymer of a higher order. However, t,hc rate of equilibrat,ion should be different in the tv\-o genetic species. The role of these equilibria in t’he interaction v,Tith hemoglobin is still to be defined. .Yole adtletl in prooj. N. Cittanova, from this laboralory, has recerjtly denlonstrat.ed 1,~ sedinientaliorl cyltilibrirun experiments at. pII 3.50, that IIp l-1 tlissociatcs at low protein concentrations. This work was carried o\lt while visiting Dr. H. Edelhoch’s laboratory. (C.E.B., N.I.A.1\I.D., N.I.FT., Bethesda.) Sirrrere thanks go to Dr. Edelhoch for his aasistallc‘e and hospitality. .4CKNOWLEIX ;MENTS We wonld like to thank I’rol’essor J. Tonrlelnt for his constant. illtcrcst ill o,n- work, and Atldrfie BarnColld for her help ill the ultracentrifrlge esperirnellts (Laboratoire de Biologic PhysicoChiIniyue, Fac*lllt6 des Sciellrcs, Orsay). We are rnrlnh indel)ted to Drs. (;. A. Gilttert., Ii. L~mry, nlld J. Wylnati for stirnldatitrg discI1ssions dllritlg I he course of this investigation. This work was slIpported in part. by a research grant from the I)616gntion (:6tl1!rale ?LIn I~ec*herche %nrtifiqlle et Techlliqrle.

2.

NAGEL,

lt.

L.,

I:OTHMAN,

R,I.

C.,

BRADLEY,

144

WAKS T.

B.,

AND

11. &I.,

R.4n-NEY,

J. Jjiol.

ANI) Chem.

240, PC 4513 (1965). 3. SMITHIES, 4. WAKS,

o., M.,

Biophgs. 6. SMITHIES, (1959). 6. 1\[oILETTI, L.,

Rull. 7. WAKS, 11..

~~ioche,,~.

ALFSEN, 113, 301 (1%X). .Ldom.

J.,

.J. 61, 629 (1955). il.. -1rch. Hioch,em. Prdein

CHEF’I’EL,

Sot. AKD

Chem. ALESEX,

Chem.

11. I., Hiol. A.,

ASD

I'.,

Riochem.

J. 96,

WaRD, 1). N., AND AILNO~, them. 12, 296 (1965). 11. RA~MCINII, S., .~ND WANG, lo.

12.

them. 1, 391 (1960). SCHA(-HJIAN, H. K.,

Biochemist New York 13. NICHOL, I,. W., 386 (1965)

r>-,” p. (1959). AND

CLOAltEV, Biochem.

(1965). .‘tna/.

Bio-

,I?Ld.

Hio-

J.,

“IiltracelltrifIlgatiotl 259. Academic

ROY,

65

(1964). 595

fix. s., Y.

14,

(1966)

48, X-13 fjiophgs.

Res. Corrr,nun. 23, 62 (1966). 8. Cask, J. I<., Ijiochemisfry 3, il 9. ANDRETVS,

19.

CLOAMW,

Compt.

AND

0..

SLFSEY

11. B., Biochemistry

itt Press, 4,

13. YPH~NTIS, 1). A., Biochenaislr,t/ 3, 2% (1964). 15. GILBERT, G. A., ,\‘crhre 210, 299 (1966). 16. WINZIJR, D. J., ANI) QCHERAGA, H. A., Biochemisfr!/ 2, 1263 (1963). 17. HER~~BN-BOI.SSIEI(, (;., These Doctorat scienres, p. 99. Paris, Foulon (1960). 13. &us. K. .J., AND l+:usa~r,, J. T., “Proteitts, Amino Acids, x~ttl Peptides,” p. 370 Rein hold, New York (1933).

I,., -\IoIwwr, Rend. Acarl.

J. ANI) Sci.

257,

RAFELSON, 983 (1963).

M.,

20. GIDBOSH, I<. A., “Clycoproteins” (A. Gottschalk, ed.), p. 61. Elsevier, Amsterdam. (1966). 21. (:I,OAILE(‘ I,., These I)octorat Sciences, p. 6. Paris, ‘;Fottlon (196l). 22. S('HERAGA, H. A., ASI) MENDELKEHN, I,., .J. Atnl. Chern. sot. 75, 179 (1953). 23. SMITHIES, O., AND WALKER, 7X. F., Nalwe 176, 1265 (1955). 21. SMITHIES O., Cold Spring Harbor Symp. Qraanl: kio(. 29, 309 (1964). 25. (IONXELI,, (:. IS., SMITHIES, O., AND DIXON, (i. II., J. MO/. Mol. 21, 225 (1966). 26. SHIM, B. s., hxn BEARN, -4. G., J. Exptl. Med. 119, 611 (1964). 27. WAKS,M., aND ALFSE%, A., Unpublisheddata. 28. KELLER, R. A., AND (:IDDINGS, J. C., J. Ch,roma/og. 3, 205 (1960). 29. BETHUNE, J. I,., ANI) KEGELES, G., .J. Ph+n. Chem. 65, 133 (1961). 30. SMITHIES, O., Science 150, 1595 (1965). 31. RMITIIIE~, O., Cossmr,, (:. E., AND I)ISON, G. II., .I. Mol. Bid. 21, 220 (1966). 32. KIRSHXER, A. G., Ph.D. Dissertat,ion, Duke Ultiversity (1963). 33. MONOD, J., WYMAX, J., INI) CHAXGEU~, .J. P.; J. Mol. Bid. 12, 88 (1965).