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Sedimentation behavior of bovine plasma albumin as a function of urea concentration and pH
Sedimentation Behavior of Bovine Plasma Albumin Function of Urea Concentration and pH Frederick
J. Gutter,
From the Laboratory of Health, TTnited
Elbert
A. Peterson
and Herbert
as a
A. Sober
of Biochemistry, Sational Cancer Institute, Sational Znsiilufrs St&es Public Health Serke, Lkpartment o,f Health, Education, und H.elfare, Bethesda, Mnr~~lnrtd Rewivctl
iIInrc*h
21, l!k57
ISTKOI)l-(‘TIOS
Putnam has reported in a recent review (1, p. S-11)) primarily on t)he txtsis of diffusion and viscosity data (a), that, t,he molecular weight of hovine plasma albumin (BPAA) remains unchanged in the presettw of 8 111 urea. A search of the literature relwled very few sedimentation studies of BPA in cottoentwt’ed urea. I’utnam (I, p. 8’77) referred to unpublished work done in c~ollalwr:~tiott with E. ,Jenxn, in which sedimentation studies of HI’A in 8 M uren re\~c:tled a ~ittale-seditnetttitt~ t)oundary. Charln-ood ($) madt~ srdimcttt:tt,iott and viscosity measuwmettts on BP11 at pH 9.9 in 8 A/ urw in t,hc prcwttc~c of sodium p-chlorortirt.c.uribettzoat,c, and I1I(~Iiettzie ct al. (4) reported on the scdimctttatiott :md diffusion c*oefficients of ISPA in 7 A1I urea at pTI 6.4. The prewttt report,, while supporting 1’utn:tm’s finding of the lack of aggreg:.:ttiott of BI’A at, wry high urea concettttxtiotrs (2)) provides e\&wcr frottl wdimentation studiw t,h:tt tw~crsible :tggreg:rtion of HP.-1 wwr~ in lo\~~:r cortcctttrations of urea, and is dependent, upon pII :IB well as utw ntol:u1)~ the ititrittsic viwosit.y, ity. The swelling of NI’X in urea, :is rrvwled wxs also examined.
pwt,ion of this work. The prot.ein was mndc up to 1.0% by weight, and dialyzed against 100 vol. of this solvent, for 19-24 hr. :rt 5°C. on :I rocking di:tlyxer before iwe. Sedimentation studies were performed at 59,780 r.p.m. in the Spinro synthetic I~oundary cell (6) and model 1;: ult,racentrifuye. l’hotoyraphic conditions and method of measuring sedimentation constant H were as described previously (5), and nil values were corrected for the adiabntic cooling of t,he centrifuge rotor (7, X) Both prot,ein solrltion and solvent viscosities were determined at 25°C. in an OstwaldFenske capillary visrometer with :L water flow time of 168 sec. Protein solut.ion viscosities were measured after overnight dialysis against. ImfYered solw tionr of urea. I)ensities were obtained with :LII A. H. Thomas Co. spec4fic gravity pip1 (1 ml. capacity). Sedimentation const:tnls were t.hen cwrrcctcd to 20” and *olution in w-at,er (Sye, ,). wcording to the equat~ions of Svedherg ant1 Pcdrrsen (9). The vnlidit,y of the correction of srdimentatjiou rates of three-component systems hy this method was discussed in :i previous paper (10). .I11 ultracent riftgcx 1‘1111s w(‘re made at room temperat uro. 11 l!xliLTS
\Vhett BI’A was cw~Muged at pII 4.5 in the :~hset~c~eof urea, 95 % of t hc protxin seditncnled with art &, ,, of 1.1 Svctlbcrg units, 3 % moving faster, with an &,, ,, of about (i. How~vw, itt t*hc: presetwr of 4 ;1[ utwt, at pII 4.5, three wtnpottcxtts wcr(~ swtt. ‘l’hc Anvest moving ~~~npottvttt (LX % of the total) had att S,(,, ,, of 4.1, 26 7~ moved :tt XII S,(,, ,,, of (i.3, :tttd 3X % had :ttt S,,), ,> o f !).!I. I’igttrc IO S~O\\S the rw~lrtt.iott of it fast -moving cwtnpotwtt ft~~tn thv tnaitr pvali 20 mitt. after 1tic> ult r:twt~trifugc rw&~d top spred. l’igtw II), t:tlwtr :rfter 82 mitt, l)\- \\-hiclt titnc the f:t.*itwt seditnettt,ittg peak had movc~l out of thv tirld of visicttt, slto\vs that the slowc~r tttaitt pt~tli h:ttl het~t~ furthw resol\wl ittt o t LVO i~otttpoitcttts. So ptwipita&t of protciti oc~urrc~l. Similar twults \wrt’ ol~i:tittc~l wit,h :I differcttt s:itiiplt~ of cqst:tllittc~ Armout~l~l’,~ (lot, (&3Y2i aiicl \vith l~utn:ut scrutii :tll~utititt (I*‘r:tctiott \‘, I<4 (‘rws; 1 :tlt,h0ugtt iit wch ww tjhcw \va:: lws of the hw\+~st cwttipottc~ttt t11a11itr t 11th13P.2 11wt1 t’or t.hv c~~tnpl~~t~~study. 111‘I‘:rl)le 1 arc prcwtttc4 the twult~s itt 4 111ttrw :tt pH 4.5 ;tttd :I SI~III-. tttaty of ~hc rrsults ol)t:titted when revc~t~11 of the clffwts of -1 alf UW:I :uld lo\\- pH K:IS ;ttttwtpted. lkdysis at pH 4.5, to r~ttto~~~ t,hc urw, ti~r~sc~l thc~ atiiouttt of f:Aest st~ditnetttitig ~~tnpotrr~ttt (:tlthough its Lo, ,, \\-:td IIO\Y higher), whcrws dialysis at pH 8.0 clitniti:itc~d Otis c*otiipottettt~. llaisittg the pH to X.(i, \vhilr ritltc~r tltaittt;tittittg the urc;t c+ollcclltr:ttiolr 01 aftw its t~~w~ov:~l at pIT 1.3, rc~lucwl th(h proportiott of hrwviest c*otnpottetltS t 0 al)out 10 YZ of 1hr tot~ttl protciti. X0 protein Itrt’c’il)if;tt~iott oc~~ttrrcd tluritrg any of thcw pt~wdurc~.
a
FIG. 1. Sedimentation di:qyxns of I3L’A after reaching full speed; (b) 82 min. dt,er proceeds from left t,o right.)
a1 pH rexhing
b 4.5 in 1 .I1 urea. (a) 28 min. full speed. iSedimentation
It \\-a~ found that when BI’A was exposed to 1 X urea overnight in :L 0.1 p \~cronal buffer at pH 8.8, with 110 previous exposure at pH 4.5, no sggregatiou occurred. This fitlding led to a study of the effect of 3 N urea up011 BPA OXTCI the pH riltlgc 3.G8.8. The result,s are sumnmrized in Table II. At pH values below the isoelectric point, NaCl colwxt,ration was increased to minimize charge effcck III the pH range of 3.4-5.3, in 4 N urea,, three compoueut~s were observed in the ultracent,rifuge. At higher pH wlucs, only a single component with a rlormal S,,, ,,. (about 4.0) n-as found. At, pH 4 and below, the &I, ,,. of the slowest-moving component decreased with decreasing pfl. Harrington cd al. (II), wheu investigating BPA in 0.1 p buffer in t,he absenceof urea, observed a similar decreasein ~$0, 9 below pH 4, as well as a decreasein diffusion coefficient end increase in the frictional ratio. Table III shows the effect of urea concentration at pH 4.5. Prom 0 t,o
I 9;
198
GCTTER,
PETKRSOS
TAHL15 pH Dependence
of Sedimentation
AXI)
SOBER
II
of BPA
in 4 Jl
Solvent”
PH
Sro,,,. X 10’3
3.9
3.s
0.25
JI SaCI-0.02
df NaOA-0.28
.lI HOrIc
4.1
0.25
Jf SaCI-0.02
Jf NaOAr--0.15
.lf HOAc
4.3
0.25
df SaCI-0.02
dl NttOAc-O.OY
M H0.4~
4.5
0.15
X SaCI-0.02
JI NaOA-0.06
311 H0.4~
0.06
-11 SaCI-0.04
31 ?;aOAr-0.013
4.9
r .I3 0.
- r a. n 5.7 7 3 s.x
I’rea
.I1 HO.Sc
3.0 5.9 10.1 3.4 5.1 9.5 3.8 6.3 Y Y 4.0 6.3 10.0 4.1 6.3 !) Y 4.1 6.2 10.6 3.0 5.7 9.5 4.2 ‘4.2 3.Y 3 .7
Rel.
‘;
44 22 34 47 41 12 42 29 2Y 40 10 50 36 26 3x 44 “0 :(6 5X “8 14 100 100 100 I 00
merit with those of Keurath and &turn (12) and of Neur:tth cutal. (13) for horse serum albumin. In I;ig. I redwed viscaity at, 1 % protein wncentrntion is plotted agninat, urfx molnrity in the present study, and this is compared with results obtained for HI’A by Frensdorff ct a/. (It) and for horse serum albumin by iYeurst.h and &urn (1%). There was :m increase in the slope of the curve in t,he 4-5 X urea region, with I gradual leveling off at, urea concentrations nbovc 5 X. The implication of these findings with regard t.o protein swelling, and their rcla,tion to the drop in A%, ,,, of the slowest component when urea concentration was raised from 4 to 5 M (Table III), will be discussed later.
SEDIMESTATION
BEHAVIOR
OF
BPA
.\ pnrtjial fractionation of the three components oht:ained from HPA iI1 1 JI urea at pH 1.5 \v;L:: made ty heat precGpit:rtioll, following :L prowdurc similar t>o that employed t)y L’utllnm \vith Benc~e-Jones prottk (I, p. 844). After the urea had been removed hy dialysis against 0.01 JI S:lC’l at pII 5.2, about 40 ‘3Gof the RI’A xas prwipitated ty heutilg the> solution for 10 min. at 5R”C. There WLS no prwipit,ation when BPAI, mlexposed to urea was similarly heated. ITltrac.elltrifil~~ll uualysis of t hc superuatnut from the heat. tre:itme:llt sho\vrd that the f:wt,cst-moving cv)mponent had txw1 removed, leaving only the t \vo more ~lo\vly eedimc>litiug peaks. 1>1scuss10s
AIany conflicting report’s have appeared concerning t,he effect of COIIwlltrat cd urea upon RP.1. L)oty and Katz (15) interpreted t,heir light-
a
b
Frc:. 2. Sediment:ttion dLgr:tms of ljP.4: (n) In 7.5 t.tzken 155 min. nfter centrifuge had reached top speed. uren 11y dialysis, keeping pH at 4.5. Picture taken 75 reached top speed. (Sedimentation proceeds from left
91 IIIWL nt pH 4.5. I’icture (b) After remowl of 7.5 M min. after centrifuge hnd to right.)
1
0 0
I 05
I 1.0
1.5
I 20
,
CONCENTRATION
FIG. 3. R.educed viscosity of Bl’A as a function of urea expressed in g./lOO ml. Conditions: concentration (ttbscissn) 2nd containing: NaOAc, adjusted t,o pII 4.5 II-ith HOhc, urea; l 4 M nren; 0 no urea. 200
concentration. Protein 112 0.15 112 N&I-0.02 (> 7.5 X urea.; 0 5 112
0
2
3 UREA
4 5 MOLARITY
6
7
8
scsttering results as indicating t,hnt the albumin n~olwulc had undergone approximately isotropic swelling in concentrated urea solutioiis. I&y and Edsall (IG) found no prot’ein aggregation by light-scnt,tering when bovine mercaptalbumin WIS dissolved iu 8 AU urea solutiow, apart from the mercury dirnerizutiou reaction. However, t’herc have been several reports of aggregation of serum albumin iu the preseuce of collceutrated urea (14, 17, 18, 3, 4, 19). &uz~uaru~ (L(3), from optical rotation and viscosity studies of RI’;1, has inferred &at the aggregation which he observed in t,he prescncc of urea iuvolvcd an exchange reaction between :L sulfhydryl group of one molecule aud :I disulfide board of mother, as originally post&ted by Huggius cf al. (‘LO). 111a later paper, IGuzrnauu and L)ouglns (21j discussed t,he various forms in which unfolded serum slburniu might mist’ in cwweut,mted urea, depending upon conditions of pH md protein c~onccutration. They stated th;it, the exchange rwctioll previously cited (20) does uot occur at an appreciable rata below pII 3 aud that’ the disulfidc cross liuks are not disturbed. Kay and Ed&l (l(i) repwtcd t,hat “at pII \-alucs of 8 or below, secondary :Lggreg:Ltionreac-
202
GUTTER,
PETERSON
ANI)
SOBER
tions, due perhaps t,o sulfhydryl-disulfide interchange, are virtually absent.” Our sedimentation studies, however, demonstrate that aggregation does occur at acid pH at certain urea concentrations. The partial reversal of this aggregation by changing pH, or complete reversal by increasing urcn concentration, is in accord with previous reports that at acid pH there is little or no sulfhydryl-disulfide rearrangement. One would not expect the disulfide bonds in protein molecules to be susceptible t,o cleavage by urea, and this supposition is supported by t,he work of Anfinsen et al. (22) wibh ribonuclease, and of Greenstein (23) with insulin. Bot)h proteins remained active in concentrated urea, suggesting strongly t,hnt the disulfidr bonds, necessary for activit,y, were not affevted. S:lroff cd al. (24) have reported other da,t:t which suggest that the aggregnt,ion they observed in serum albumin at low and very high pII values \vns not t,he result of intermolecular covalent, bonding. At, pH 3.5, they found heavy components in human and bovine swum albumin, 1vit.h srdimentatioll weffi(Gnt,s of :lpproximatjely 6 and I9 Svedberg units, at high salt) ~onc~crltr:tt’iorls or after long periods of storage in 0.1 X salt. These effects at Ion- pH, which occurred in the absence of urea, were reversible by dialysis at :I higher pII. We have confirmed their rwults and ha\-<, show1 t,hat) in addition to t’he vhangcs they ohserred at low pH, considel:My mow :tggrcg:lt,ion owurred in the presrwe of 3 ~11 urw. The e\-idence suggests that, t,he aggregation reported here arises from rearr:lllgcment.s in hydrogen-~)onding or rlect,rost,atic linkages. In our study, the challgcs in reduwd viscosity (l;ig. 4) and \,alues for int.rinsic viscosity obtjained from I’ig. 3 could be interpreted as a marked irwrcase in swelling of the El’=2 molecule &en urea concentrat,ion was increased frorn 4 to 5 ,21, followed 1,~ a more gradual s\z-elling as urea caoncentration was further increased. This would explain the drop in the component (Table III) when going from 1 *c ?O,\r of the slowest,-moving to 5 31 urea, :uld indicates t,hat the vornponent, having an S,O, w of 1. I X 10-l” in 4 111 ure;~ corresponds to that, showing an I!$“, qr of 2.9 in 5 JI urea and 2.5 in 7.5 JI urea. This identity is further suggested by the work of l’ut,n:lm ct al. (2) 011the molecular lveight of RPA in 8 &i’ urcn from Although a molecular weight \&osity and diffusion measurements. somewhat, higher than normal was reported in that study, c~ombination of our value of 2.4.5 )( 10-l” for t,hc Sze, ,v of I3P.A in 8 M urea [cxtrapolated from our wlues at 5, 6, and 7.5 111, and ill fairly c~losc agreement &,h that of CJh:irl\\-ood (3)] with Putnam’s value of 1.01 X IO-’ for the
diffusion coefficient in 8 M urea (2) gives a molecular in reasonable agreement with that for native BPA.
the
We are indebted to Dr. viscosity measurcmenl,s
William R. Carroll for and t,heir intjerprctntion. ~'oTE
ADDED
Is
helpful
weight
discussions
of 59,000,
conrcrning
PmoF
Suhsequcntly to submission of this paper for publication we have learned, in a personal communication from I)r. Joseph F. Foster of l’urdlle University, that some aggregation of BPA occurs in the duralumin ultracentrifuge cell because of contamination with trace amounk of aluminum. However, further work showed that ure:i-induced aggregation occurred to fhe same extent, in the Kel-F plastic cell as in t,he duralumin cell.
1. Treatment of BPA with 3-5 111urea at’ pH 4.5 resulted in the appearance of three ultracentrifugal components. Only one component n-as seen below 3 N and above 6 X urea. 2. Three peaks, mit’h &o, w values of approximately 1, 6, and 10 Svedberg units, were observed in 4 Al urea over the pH range 3.G5.S. Only one component, with a normal S x), 1,.of about 4, appeared at pH \;alues of 5.5 and higher. 3. A considerable increase in the intrinsic viwosit’y of BYA occurred at urea concentrations above 4 M, particularly in t,he int’erval between 4 and 5 ,M. The reduction in Sz,,, 1Vat 5 JI from that seen in 4 M urea was interpreted as being due to a marked increase in swelling of t,he albumin molecule. 4. Possihlc mechanisms have been discussed for t,he aggregation of BPX in urea observed by sedimentation techniques, and conditions under which either partial or complete reversal occurred have been presented. REFEIZENCES 1.
E‘. w., in “The Proteins", (13. iYeurat,h and Ii. Bailey, cds.), Vol. I, B. Academic Press, New York, 105:s. 2. PI:TN.IM, F. Iv., !&ICKSON, J. O., VOLKIX, Ii:., AKI) NEIRATH, H., J. &IL. Physiol. 26, 513 (1943). 3. CHARLWOOD, P. A., Cnn. .I. Che~x. 33, 1013 (1!)55). 4. MPKXXZIE. H. A., SMITH, M. B., AKI) WAKE, 11. (;., A'ntrc~ 176, 738 (1955). 5. I~~ELEs,G., AND GYTTER, F.J.,J. Am. Chem.Soc.73,3770 (1051). 1'1-TNAZI,
Part
6. PICKELS,E.G.,HARRINGTON, W.F., ANU SCHACI-IMAN,H.K.,P~OC.N~~~.A~~~. Sci. U. S. 38, 043 (1952). 7. WA~:GH, 1). F., AND YPIIANTIS, 1). A., i&v. Sci. I&r. 23, 609 (1952). 8. BIANcaERI.4, A., AXI) KEOELES, G., J. .lm. Chem. Sot. 76, 3737 (1954). 9. SVEDBERO, T., ANI) PEDERSEN, K. O., “The Ultracentrifuge.” Clarendon Press, Oxford, 1940. 10. GUTTER, F. J., SOBER, H. 4., AND PETERSON, E. A., Arch. Biochem. Biophys. 62, 427 (1956). II. HARRJSGTON, W. F., JOHNSON, l'., AND OTTEWJJ,L, IL H., B&hem. J. 62, 569 (1956). 12. SEURATH, H., AND SAUM, A. %I., J. Biol. C"hew7. 128, 347 (1939). 13. ~EURATIX, H., COOPER, G. It., ANI) ERICKSON, J. O., J. Rio!. Chem. 142, 240 (1942). l-1. FRENSDORYF, H. Ii., WATSON, IM. T., AND KAI~ZI\IANN, W., J. ilrt~. Cke,,r. Sot. 75, 5167 (1953). 15. I)OTY, P., AND KATZ, S., Abstr:~cts 118th I\lecting, americ:m Chemical Society, p, I4C. Chicago, Ill., 1950. 16. KAY, C. &I., AN) I':IMAI,L, J. T., birch. Biochem. Biophys. 65, 354 (1956). 17. KAVZMANN, W., in “The Mechanism of Enzyme Action" (W. I>. McElroy and B. Glnss, eds.), p. 105. Johns Hopkins Press, Baltimore, 1954. 18. HOSPELNORN, V. L)., CROSS, B., .~ND JENSEN, E. V., J. Arrl. Chem. Sot. 76, 2827 (1954). 19. KAUZMASX, W., J. Celllrlar Con~p. Physiol. 47, Suppl. 1, 113 (May, 1956). 20. HCCXXNS, C., TAPLEY, I>. F., AND JENSEX, E. V., Natwe 167, 592 (1951). 21. KAUZMANN, W., AXD I~OCGLAS, R. G., JR., 4rch. B&hem. Biophys. 65, 106 (1956). 22. AXFINSEX, C. B., HARRJNCZTON, W. F., Hvrwr, Aa., LINDERSTR~M-LANG, K., OTTESEN, M., AND SCHELLMIIN, J., Biochim. et Biophys. Acta 17, 141 (1955). 23. Quoted as Ref. 167 of NEURAT~I, H., GREENSTEIN, J. P., PI-TNAM, F. W., AND ERICKSON, J. O., Chwn. k?os. 34, 15’7 (1944). 24. SAROFF, H. ,4., LOER, G. I., AND SCHERAGA, H. A., J. Am. ChevL. Sot. 77, 2908 (1955).
J. Gutter,
From the Laboratory of Health, TTnited
Elbert
A. Peterson
and Herbert
as a
A. Sober
of Biochemistry, Sational Cancer Institute, Sational Znsiilufrs St&es Public Health Serke, Lkpartment o,f Health, Education, und H.elfare, Bethesda, Mnr~~lnrtd Rewivctl
iIInrc*h
21, l!k57
ISTKOI)l-(‘TIOS
Putnam has reported in a recent review (1, p. S-11)) primarily on t)he txtsis of diffusion and viscosity data (a), that, t,he molecular weight of hovine plasma albumin (BPAA) remains unchanged in the presettw of 8 111 urea. A search of the literature relwled very few sedimentation studies of BPA in cottoentwt’ed urea. I’utnam (I, p. 8’77) referred to unpublished work done in c~ollalwr:~tiott with E. ,Jenxn, in which sedimentation studies of HI’A in 8 M uren re\~c:tled a ~ittale-seditnetttitt~ t)oundary. Charln-ood ($) madt~ srdimcttt:tt,iott and viscosity measuwmettts on BP11 at pH 9.9 in 8 A/ urw in t,hc prcwttc~c of sodium p-chlorortirt.c.uribettzoat,c, and I1I(~Iiettzie ct al. (4) reported on the scdimctttatiott :md diffusion c*oefficients of ISPA in 7 A1I urea at pTI 6.4. The prewttt report,, while supporting 1’utn:tm’s finding of the lack of aggreg:.:ttiott of BI’A at, wry high urea concettttxtiotrs (2)) provides e\&wcr frottl wdimentation studiw t,h:tt tw~crsible :tggreg:rtion of HP.-1 wwr~ in lo\~~:r cortcctttrations of urea, and is dependent, upon pII :IB well as utw ntol:u1)~ the ititrittsic viwosit.y, ity. The swelling of NI’X in urea, :is rrvwled wxs also examined.
pwt,ion of this work. The prot.ein was mndc up to 1.0% by weight, and dialyzed against 100 vol. of this solvent, for 19-24 hr. :rt 5°C. on :I rocking di:tlyxer before iwe. Sedimentation studies were performed at 59,780 r.p.m. in the Spinro synthetic I~oundary cell (6) and model 1;: ult,racentrifuye. l’hotoyraphic conditions and method of measuring sedimentation constant H were as described previously (5), and nil values were corrected for the adiabntic cooling of t,he centrifuge rotor (7, X) Both prot,ein solrltion and solvent viscosities were determined at 25°C. in an OstwaldFenske capillary visrometer with :L water flow time of 168 sec. Protein solut.ion viscosities were measured after overnight dialysis against. ImfYered solw tionr of urea. I)ensities were obtained with :LII A. H. Thomas Co. spec4fic gravity pip1 (1 ml. capacity). Sedimentation const:tnls were t.hen cwrrcctcd to 20” and *olution in w-at,er (Sye, ,). wcording to the equat~ions of Svedherg ant1 Pcdrrsen (9). The vnlidit,y of the correction of srdimentatjiou rates of three-component systems hy this method was discussed in :i previous paper (10). .I11 ultracent riftgcx 1‘1111s w(‘re made at room temperat uro. 11 l!xliLTS
\Vhett BI’A was cw~Muged at pII 4.5 in the :~hset~c~eof urea, 95 % of t hc protxin seditncnled with art &, ,, of 1.1 Svctlbcrg units, 3 % moving faster, with an &,, ,, of about (i. How~vw, itt t*hc: presetwr of 4 ;1[ utwt, at pII 4.5, three wtnpottcxtts wcr(~ swtt. ‘l’hc Anvest moving ~~~npottvttt (LX % of the total) had att S,(,, ,, of 4.1, 26 7~ moved :tt XII S,(,, ,,, of (i.3, :tttd 3X % had :ttt S,,), ,> o f !).!I. I’igttrc IO S~O\\S the rw~lrtt.iott of it fast -moving cwtnpotwtt ft~~tn thv tnaitr pvali 20 mitt. after 1tic> ult r:twt~trifugc rw&~d top spred. l’igtw II), t:tlwtr :rfter 82 mitt, l)\- \\-hiclt titnc the f:t.*itwt seditnettt,ittg peak had movc~l out of thv tirld of visicttt, slto\vs that the slowc~r tttaitt pt~tli h:ttl het~t~ furthw resol\wl ittt o t LVO i~otttpoitcttts. So ptwipita&t of protciti oc~urrc~l. Similar twults \wrt’ ol~i:tittc~l wit,h :I differcttt s:itiiplt~ of cqst:tllittc~ Armout~l~l’,~ (lot, (&3Y2i aiicl \vith l~utn:ut scrutii :tll~utititt (I*‘r:tctiott \‘, I<4 (‘rws; 1 :tlt,h0ugtt iit wch ww tjhcw \va:: lws of the hw\+~st cwttipottc~ttt t11a11itr t 11th13P.2 11wt1 t’or t.hv c~~tnpl~~t~~study. 111‘I‘:rl)le 1 arc prcwtttc4 the twult~s itt 4 111ttrw :tt pH 4.5 ;tttd :I SI~III-. tttaty of ~hc rrsults ol)t:titted when revc~t~11 of the clffwts of -1 alf UW:I :uld lo\\- pH K:IS ;ttttwtpted. lkdysis at pH 4.5, to r~ttto~~~ t,hc urw, ti~r~sc~l thc~ atiiouttt of f:Aest st~ditnetttitig ~~tnpotrr~ttt (:tlthough its Lo, ,, \\-:td IIO\Y higher), whcrws dialysis at pH 8.0 clitniti:itc~d Otis c*otiipottettt~. llaisittg the pH to X.(i, \vhilr ritltc~r tltaittt;tittittg the urc;t c+ollcclltr:ttiolr 01 aftw its t~~w~ov:~l at pIT 1.3, rc~lucwl th(h proportiott of hrwviest c*otnpottetltS t 0 al)out 10 YZ of 1hr tot~ttl protciti. X0 protein Itrt’c’il)if;tt~iott oc~~ttrrcd tluritrg any of thcw pt~wdurc~.
a
FIG. 1. Sedimentation di:qyxns of I3L’A after reaching full speed; (b) 82 min. dt,er proceeds from left t,o right.)
a1 pH rexhing
b 4.5 in 1 .I1 urea. (a) 28 min. full speed. iSedimentation
It \\-a~ found that when BI’A was exposed to 1 X urea overnight in :L 0.1 p \~cronal buffer at pH 8.8, with 110 previous exposure at pH 4.5, no sggregatiou occurred. This fitlding led to a study of the effect of 3 N urea up011 BPA OXTCI the pH riltlgc 3.G8.8. The result,s are sumnmrized in Table II. At pH values below the isoelectric point, NaCl colwxt,ration was increased to minimize charge effcck III the pH range of 3.4-5.3, in 4 N urea,, three compoueut~s were observed in the ultracent,rifuge. At higher pH wlucs, only a single component with a rlormal S,,, ,,. (about 4.0) n-as found. At, pH 4 and below, the &I, ,,. of the slowest-moving component decreased with decreasing pfl. Harrington cd al. (II), wheu investigating BPA in 0.1 p buffer in t,he absenceof urea, observed a similar decreasein ~$0, 9 below pH 4, as well as a decreasein diffusion coefficient end increase in the frictional ratio. Table III shows the effect of urea concentration at pH 4.5. Prom 0 t,o
I 9;
198
GCTTER,
PETKRSOS
TAHL15 pH Dependence
of Sedimentation
AXI)
SOBER
II
of BPA
in 4 Jl
Solvent”
PH
Sro,,,. X 10’3
3.9
3.s
0.25
JI SaCI-0.02
df NaOA-0.28
.lI HOrIc
4.1
0.25
Jf SaCI-0.02
Jf NaOAr--0.15
.lf HOAc
4.3
0.25
df SaCI-0.02
dl NttOAc-O.OY
M H0.4~
4.5
0.15
X SaCI-0.02
JI NaOA-0.06
311 H0.4~
0.06
-11 SaCI-0.04
31 ?;aOAr-0.013
4.9
r .I3 0.
- r a. n 5.7 7 3 s.x
I’rea
.I1 HO.Sc
3.0 5.9 10.1 3.4 5.1 9.5 3.8 6.3 Y Y 4.0 6.3 10.0 4.1 6.3 !) Y 4.1 6.2 10.6 3.0 5.7 9.5 4.2 ‘4.2 3.Y 3 .7
Rel.
‘;
44 22 34 47 41 12 42 29 2Y 40 10 50 36 26 3x 44 “0 :(6 5X “8 14 100 100 100 I 00
merit with those of Keurath and &turn (12) and of Neur:tth cutal. (13) for horse serum albumin. In I;ig. I redwed viscaity at, 1 % protein wncentrntion is plotted agninat, urfx molnrity in the present study, and this is compared with results obtained for HI’A by Frensdorff ct a/. (It) and for horse serum albumin by iYeurst.h and &urn (1%). There was :m increase in the slope of the curve in t,he 4-5 X urea region, with I gradual leveling off at, urea concentrations nbovc 5 X. The implication of these findings with regard t.o protein swelling, and their rcla,tion to the drop in A%, ,,, of the slowest component when urea concentration was raised from 4 to 5 M (Table III), will be discussed later.
SEDIMESTATION
BEHAVIOR
OF
BPA
.\ pnrtjial fractionation of the three components oht:ained from HPA iI1 1 JI urea at pH 1.5 \v;L:: made ty heat precGpit:rtioll, following :L prowdurc similar t>o that employed t)y L’utllnm \vith Benc~e-Jones prottk (I, p. 844). After the urea had been removed hy dialysis against 0.01 JI S:lC’l at pII 5.2, about 40 ‘3Gof the RI’A xas prwipitated ty heutilg the> solution for 10 min. at 5R”C. There WLS no prwipit,ation when BPAI, mlexposed to urea was similarly heated. ITltrac.elltrifil~~ll uualysis of t hc superuatnut from the heat. tre:itme:llt sho\vrd that the f:wt,cst-moving cv)mponent had txw1 removed, leaving only the t \vo more ~lo\vly eedimc>litiug peaks. 1>1scuss10s
AIany conflicting report’s have appeared concerning t,he effect of COIIwlltrat cd urea upon RP.1. L)oty and Katz (15) interpreted t,heir light-
a
b
Frc:. 2. Sediment:ttion dLgr:tms of ljP.4: (n) In 7.5 t.tzken 155 min. nfter centrifuge had reached top speed. uren 11y dialysis, keeping pH at 4.5. Picture taken 75 reached top speed. (Sedimentation proceeds from left
91 IIIWL nt pH 4.5. I’icture (b) After remowl of 7.5 M min. after centrifuge hnd to right.)
1
0 0
I 05
I 1.0
1.5
I 20
,
CONCENTRATION
FIG. 3. R.educed viscosity of Bl’A as a function of urea expressed in g./lOO ml. Conditions: concentration (ttbscissn) 2nd containing: NaOAc, adjusted t,o pII 4.5 II-ith HOhc, urea; l 4 M nren; 0 no urea. 200
concentration. Protein 112 0.15 112 N&I-0.02 (> 7.5 X urea.; 0 5 112
0
2
3 UREA
4 5 MOLARITY
6
7
8
scsttering results as indicating t,hnt the albumin n~olwulc had undergone approximately isotropic swelling in concentrated urea solutioiis. I&y and Edsall (IG) found no prot’ein aggregation by light-scnt,tering when bovine mercaptalbumin WIS dissolved iu 8 AU urea solutiow, apart from the mercury dirnerizutiou reaction. However, t’herc have been several reports of aggregation of serum albumin iu the preseuce of collceutrated urea (14, 17, 18, 3, 4, 19). &uz~uaru~ (L(3), from optical rotation and viscosity studies of RI’;1, has inferred &at the aggregation which he observed in t,he prescncc of urea iuvolvcd an exchange reaction between :L sulfhydryl group of one molecule aud :I disulfide board of mother, as originally post&ted by Huggius cf al. (‘LO). 111a later paper, IGuzrnauu and L)ouglns (21j discussed t,he various forms in which unfolded serum slburniu might mist’ in cwweut,mted urea, depending upon conditions of pH md protein c~onccutration. They stated th;it, the exchange rwctioll previously cited (20) does uot occur at an appreciable rata below pII 3 aud that’ the disulfidc cross liuks are not disturbed. Kay and Ed&l (l(i) repwtcd t,hat “at pII \-alucs of 8 or below, secondary :Lggreg:Ltionreac-
202
GUTTER,
PETERSON
ANI)
SOBER
tions, due perhaps t,o sulfhydryl-disulfide interchange, are virtually absent.” Our sedimentation studies, however, demonstrate that aggregation does occur at acid pH at certain urea concentrations. The partial reversal of this aggregation by changing pH, or complete reversal by increasing urcn concentration, is in accord with previous reports that at acid pH there is little or no sulfhydryl-disulfide rearrangement. One would not expect the disulfide bonds in protein molecules to be susceptible t,o cleavage by urea, and this supposition is supported by t,he work of Anfinsen et al. (22) wibh ribonuclease, and of Greenstein (23) with insulin. Bot)h proteins remained active in concentrated urea, suggesting strongly t,hnt the disulfidr bonds, necessary for activit,y, were not affevted. S:lroff cd al. (24) have reported other da,t:t which suggest that the aggregnt,ion they observed in serum albumin at low and very high pII values \vns not t,he result of intermolecular covalent, bonding. At, pH 3.5, they found heavy components in human and bovine swum albumin, 1vit.h srdimentatioll weffi(Gnt,s of :lpproximatjely 6 and I9 Svedberg units, at high salt) ~onc~crltr:tt’iorls or after long periods of storage in 0.1 X salt. These effects at Ion- pH, which occurred in the absence of urea, were reversible by dialysis at :I higher pII. We have confirmed their rwults and ha\-<, show1 t,hat) in addition to t’he vhangcs they ohserred at low pH, considel:My mow :tggrcg:lt,ion owurred in the presrwe of 3 ~11 urw. The e\-idence suggests that, t,he aggregation reported here arises from rearr:lllgcment.s in hydrogen-~)onding or rlect,rost,atic linkages. In our study, the challgcs in reduwd viscosity (l;ig. 4) and \,alues for int.rinsic viscosity obtjained from I’ig. 3 could be interpreted as a marked irwrcase in swelling of the El’=2 molecule &en urea concentrat,ion was increased frorn 4 to 5 ,21, followed 1,~ a more gradual s\z-elling as urea caoncentration was further increased. This would explain the drop in the component (Table III) when going from 1 *c ?O,\r of the slowest,-moving to 5 31 urea, :uld indicates t,hat the vornponent, having an S,O, w of 1. I X 10-l” in 4 111 ure;~ corresponds to that, showing an I!$“, qr of 2.9 in 5 JI urea and 2.5 in 7.5 JI urea. This identity is further suggested by the work of l’ut,n:lm ct al. (2) 011the molecular lveight of RPA in 8 &i’ urcn from Although a molecular weight \&osity and diffusion measurements. somewhat, higher than normal was reported in that study, c~ombination of our value of 2.4.5 )( 10-l” for t,hc Sze, ,v of I3P.A in 8 M urea [cxtrapolated from our wlues at 5, 6, and 7.5 111, and ill fairly c~losc agreement &,h that of CJh:irl\\-ood (3)] with Putnam’s value of 1.01 X IO-’ for the
diffusion coefficient in 8 M urea (2) gives a molecular in reasonable agreement with that for native BPA.
the
We are indebted to Dr. viscosity measurcmenl,s
William R. Carroll for and t,heir intjerprctntion. ~'oTE
ADDED
Is
helpful
weight
discussions
of 59,000,
conrcrning
PmoF
Suhsequcntly to submission of this paper for publication we have learned, in a personal communication from I)r. Joseph F. Foster of l’urdlle University, that some aggregation of BPA occurs in the duralumin ultracentrifuge cell because of contamination with trace amounk of aluminum. However, further work showed that ure:i-induced aggregation occurred to fhe same extent, in the Kel-F plastic cell as in t,he duralumin cell.
1. Treatment of BPA with 3-5 111urea at’ pH 4.5 resulted in the appearance of three ultracentrifugal components. Only one component n-as seen below 3 N and above 6 X urea. 2. Three peaks, mit’h &o, w values of approximately 1, 6, and 10 Svedberg units, were observed in 4 Al urea over the pH range 3.G5.S. Only one component, with a normal S x), 1,.of about 4, appeared at pH \;alues of 5.5 and higher. 3. A considerable increase in the intrinsic viwosit’y of BYA occurred at urea concentrations above 4 M, particularly in t,he int’erval between 4 and 5 ,M. The reduction in Sz,,, 1Vat 5 JI from that seen in 4 M urea was interpreted as being due to a marked increase in swelling of t,he albumin molecule. 4. Possihlc mechanisms have been discussed for t,he aggregation of BPX in urea observed by sedimentation techniques, and conditions under which either partial or complete reversal occurred have been presented. REFEIZENCES 1.
E‘. w., in “The Proteins", (13. iYeurat,h and Ii. Bailey, cds.), Vol. I, B. Academic Press, New York, 105:s. 2. PI:TN.IM, F. Iv., !&ICKSON, J. O., VOLKIX, Ii:., AKI) NEIRATH, H., J. &IL. Physiol. 26, 513 (1943). 3. CHARLWOOD, P. A., Cnn. .I. Che~x. 33, 1013 (1!)55). 4. MPKXXZIE. H. A., SMITH, M. B., AKI) WAKE, 11. (;., A'ntrc~ 176, 738 (1955). 5. I~~ELEs,G., AND GYTTER, F.J.,J. Am. Chem.Soc.73,3770 (1051). 1'1-TNAZI,
Part
6. PICKELS,E.G.,HARRINGTON, W.F., ANU SCHACI-IMAN,H.K.,P~OC.N~~~.A~~~. Sci. U. S. 38, 043 (1952). 7. WA~:GH, 1). F., AND YPIIANTIS, 1). A., i&v. Sci. I&r. 23, 609 (1952). 8. BIANcaERI.4, A., AXI) KEOELES, G., J. .lm. Chem. Sot. 76, 3737 (1954). 9. SVEDBERO, T., ANI) PEDERSEN, K. O., “The Ultracentrifuge.” Clarendon Press, Oxford, 1940. 10. GUTTER, F. J., SOBER, H. 4., AND PETERSON, E. A., Arch. Biochem. Biophys. 62, 427 (1956). II. HARRJSGTON, W. F., JOHNSON, l'., AND OTTEWJJ,L, IL H., B&hem. J. 62, 569 (1956). 12. SEURATH, H., AND SAUM, A. %I., J. Biol. C"hew7. 128, 347 (1939). 13. ~EURATIX, H., COOPER, G. It., ANI) ERICKSON, J. O., J. Rio!. Chem. 142, 240 (1942). l-1. FRENSDORYF, H. Ii., WATSON, IM. T., AND KAI~ZI\IANN, W., J. ilrt~. Cke,,r. Sot. 75, 5167 (1953). 15. I)OTY, P., AND KATZ, S., Abstr:~cts 118th I\lecting, americ:m Chemical Society, p, I4C. Chicago, Ill., 1950. 16. KAY, C. &I., AN) I':IMAI,L, J. T., birch. Biochem. Biophys. 65, 354 (1956). 17. KAVZMANN, W., in “The Mechanism of Enzyme Action" (W. I>. McElroy and B. Glnss, eds.), p. 105. Johns Hopkins Press, Baltimore, 1954. 18. HOSPELNORN, V. L)., CROSS, B., .~ND JENSEN, E. V., J. Arrl. Chem. Sot. 76, 2827 (1954). 19. KAUZMASX, W., J. Celllrlar Con~p. Physiol. 47, Suppl. 1, 113 (May, 1956). 20. HCCXXNS, C., TAPLEY, I>. F., AND JENSEX, E. V., Natwe 167, 592 (1951). 21. KAUZMANN, W., AXD I~OCGLAS, R. G., JR., 4rch. B&hem. Biophys. 65, 106 (1956). 22. AXFINSEX, C. B., HARRJNCZTON, W. F., Hvrwr, Aa., LINDERSTR~M-LANG, K., OTTESEN, M., AND SCHELLMIIN, J., Biochim. et Biophys. Acta 17, 141 (1955). 23. Quoted as Ref. 167 of NEURAT~I, H., GREENSTEIN, J. P., PI-TNAM, F. W., AND ERICKSON, J. O., Chwn. k?os. 34, 15’7 (1944). 24. SAROFF, H. ,4., LOER, G. I., AND SCHERAGA, H. A., J. Am. ChevL. Sot. 77, 2908 (1955).