64
HERBERT MORAWETX AND WALTER L. H U O ~ SJR. ,
Vol. 56
THE INTERACTION OF PROTEINS WITH SYNTHETIC POLYELECTROLYTES. I. COMPLEXING OF BOVINE SERUM ALBUMIN. BYHERBERT MORAWETZ~ AND WALTER L. HUGHES, JR. Univereiiy Labmatoty of Physical Chemistry Related to Medicine and Public I€@,
Earvard Unwersi&, Boston, Maas.
The reci itation of bovine serum albumin by four synthetic pol electfolytes was studied as a function of H, ionio stren tg, po&mer/protein ratio and the degree of polymerization of d e r e m . Bovine ssum albumin recoveref from its compfex with polymethacrylic acid had the same optical activity, solution viscosity and crystallizability as the original material and seemed therefore, to be in its native state. Anion binding, characteristic of serum albumin, was consldered to cause the shift of the precipitation curves to lower pH in the presence of chloride or thiocyanate and to account for the high stability of albumin complexes with a copolymer of styrene and maleic anhydride. It wm shown that oxyhemoglobin can be separated from serum albumin in the interisoelectricrange by precipitahon with polymethacrylic acid.
first to be investigated, since its high solubility in I. Introduction It has long been recognized that the coprecipita- water would put particularly stringent requirements tion of proteins a t pH values lying between their on an efficient precipitating reageat. isoelectric points presents problems in any scheme II. Experimental
of protein fractionation.2 Green suggested3 that the separations could be enhanced by approaching the precipitation zone from the direction where all the proteins bear charges of the same sign. More recently a fractionation scheme has been described4 which takes advantage of protein-protein interaction for the separation of groups of proteins. The value of the method depends on the presence of specifically interacting proteins in the original mixture. This principle can be extended t o the isolation of individual proteins from binary mixtures by the addition of a new charged component. On looking about for such charged components one is immediately attracted to the synthetic polyelectrolytes6t6since they are readily available and may be tailored to fit the problem. Furthermore, in many cases they are known to form highly insoluble precipitates with specific counter-ions, thus suggesting a method for their eventual removal from the protein system. The synthetic materials can be made very cheaply and in great variety by copolymerization of a large number of readily available monomem or by chemical modification of polymers and copolymers. Since they can be made with a much greater density of ionizable groups than that characteristic of proteins, it may be expected that they will displace proteins from their salt-like complexes, permitting a more quantitative separation of normally interacting proteins. Before attempting protein separations a study of the interaction of polyelectrolytes with pure individual proteins seemed indicated, and bovine serum albumin' was chosen as the (1) Publio Health Service Research Fellow of the National Heart Institute. (2) E. J. Cohn, J . am.Phya'ol., 4, 697 (1922). (3)A. A. Green, J . Am. Chcm. Soc., 60, 1108 (1938). (4) E. J. Cohn, el d.. ibid., TS, 465 (1960). (5) Naturdy occurring polydectrolytea such 8 8 protamines''* nucleic acids' and gum arabic,1°have frequently been shown to preeipitate proteins under suitable conditions. (6) Synthetio polyelectrolyteshave also been used lately for the precipitation of bacterial suspensions.1* (7) F. Haurowits, KoUoid-2.. 74,208 (1936). (8) A. Klecskowsld, Biochem. J.. 40, 677 (1946). (9) K,B. Bji5rneaj6, and T.Teorell. Arkiv Kim. Mineral Qsol., Al9, NO. a4 (1945). (10) H.G. Bungenberg de Jong. "Colloid Science," Vol. 11, Elsevier Press,Inc.. New York, N. Y.. 1949,Chapter 10. (11) E. Katchabki, in preparation.
The bovine serum albumin (BSA) was a crystallized sample obtained from h o u r and Company. A stock solution of 1% was made up and the albumin concentration determined by ultraviolet a b tion measurement using the optical, density data of Cobn,%ughes and Weare.I2 The solution was kept froeen at -17 in small ampules and melted only immediately before use. The oxyhemoglobin was prepared by Miss Vir inia Gossard according to the method described by brabkin." Its concentration was calculated from the optical density at 350 mp Using the data reported by Sidwell, Munch, Barron and Hogness.14 Polyme+c@c ?id (MA) .was prepared from glacial methacryhc acid whch was b a d at reduced pressure under nitrogen and kept frozen at -17". The monomer was mixed with five parta by weight of benzene, benzoyl peroxide catslyst added (1.5% of the weight of the monomer) and the polymeriaation was canied out by heating for two days a t 60'. The resin suspension was washed with acetone and ether and vacuumdried at 70". The yield was 98%, the intrinsic viscosity in methanol 1.05. Some of the polymer was fractionated by precipitating twice from aqueous solution at pH 6.8 and a calcium acetate concentration of 0.05 M. The preci itate was dissolved in 5% sodium hydroxide, dialyzed t&ee days against M/100 hydrochloric acid, the solution froxn and vacuum evaporated. Polymethacrylic acid samplea of a range of different molecular weights were prepared a t 60" from solutions of 10 ml. of glacial methacrylic acid ( R o b and H m ) in 40 ml. of methyl ethyl ketone with different amounta (29 mg., 83 mg., 165 mg., and 328 mg.)of azo-bis-iibutyronitrile cab alyst. After 24 hours the polymeridion was essentially complete. The precipitated resin waa washed.with ether, dissolved in water, dialyzed for two day^ and dned from the frozen state. Intrinsic Viscoeties in methanol were 1.58, 1.11,0.79and 0.57. Methacrylic acid-vinylpyridine copolymer waa prepared from 2-vinylpyridine (Reilly Tar & Chem. Co.) distilled a t reduced pressure under nitrogen immediately before use. A monomer mixture containing l .5 mole per cent. vinylpyridine was polymeriwd in meth 1 ethyl ketone solution at 60" to a conversion of 12%. Tie copolymer had m absorption coefEcient 27.0 at 265 rnp in solution of
pH 4.0. Polyvinylamine hydrobromide (PVA) prepared b the method of Re olds and Kenyonu was supplied by the!l&tman Kodak It analyzed ?.27% bromine and 11.34 nitrogen. The intrinsic wcoslty m 0.15 N sodium chlong (based on the conoentrationof pol ylamine) was 1.12. Maleic anhydride-styrene c o p o E e r (MAS)containing
E.
(12) E. J. Cob,W.
L.Hughes. Jr.. and J. H.Weare, J . Am. Chan.
Soc., 69,911 (1947).
(13) D. L. Drabkin. J. Bid. C?ma., 164, 703 (1946). (14) A. E. %dwell, Jr., R. E. Munch, E. 8. 0. Barron and T. R. Hosneas. tW, lW.336 (1938). (15) D. D.RaynoIda and W. 0. Kenyon, J . Am. Clum. Soc., 69,911 ( 1947).
.-
COMPLEXING OF BOVINE SERUMALBUMIN
Jan., 1952
50 mole per cent. of each monomer was obtained from Car'bide and Carbon Chemicals Co. (Resin SYHM). The intrinsic viscosity in methyl ethy lketone was 0.31. This resin is soluble in water only after hydrolysis of the anhydride groups (achieved rapidly by treatment with dilute alkali and more slowly by stirring in water at 80'). Methacrylic acid-diethylaminoethyl methacrylate copolymer (DEMMA) was prepared aa descnbed rn a prewous communication.'@ It contained 54.6 mole per cent. methacrylic acid, had an isoelectric point of 8.0 and an intrinsic viscosity in pyridine of 1.5. Ground glass stoppered tubes were used for mixhg the albumin and polyelectrolyte solutions, using a total volume of 10 ml. for each experiment. When it was desired to vary pH at low ionic strength, stock solutions of the polyelectrolytes were made up to different degrees of neutralization and mixed in varying proportions so as to avoid exposing the protein to local concentrations of acid or alkali. Acetate buffers were used when it was desired to keep the pH a t a given value while investigating the effect of another variable. nts precooled to 0", the stoppered After introducing all r tube was inverted seveytimes and left standing at 0', for at least three hours although e uilibriup-was apparently attained much more rapidly. %e recipitate was separated in a refrigerated centrifuge and t i e supernatant was used for analysis and pH determination on a glass electrode a t 25". Analyses were carried out by determinations of the optical density with a Beckman spectrophotometer using l c m . uartz cells to hold the appropriately diluted samples. ince many of the supernatants from protein precipitations to be added to produce clear wereslightly hazy, buffeers solutions before the absorption measurements. In such cases the blanks contained the same buffer concentration. Supernatanta from preci itation with anionic polymers were diluted with an equafamount of M/10 djsodium phosphate unless they contained alkaline earths cations, in which case a mixture of five parts M / 4 trisodium citrate and ono part M/4 citric acid was used. Su rnatants from polyvinylamine precipitations were diluterwith acetate buffer of H 4.00 and ionic strength 0.8. Polymers MAS and PVA tad characteristic W absorption maxima and the concentrations of albumin and polymer could be obtained from optical density measurements at two wave lengths (sec Fig. 1). The results obtained from analyzing the supernatants from a series of experiments involving precipitation with MAS were checked by dissolving the precipitate in disodium phosphate solution and analyzing in a similar manner. The amounts of protein and polymer found in the two phases added up to the quantities known to be present within an average error of 6%. Copolymer DEMMA had an absorption coefficient E:?,,,. 0.55 at 280 mp due probably to light scattering from the very high molecular weight material. The resin had no charactcristic absorption maximum and no attempt was made to interpret optical densities in terms of protein concentration. Polymcthacrylic acid had E:?,,,,. 0.385 at 280 mp and its contribution to the optical dcnsitics of supcrnatants from albumin precipitation was ncglectcd.
.
65
Viscosity measurements were carried out in an Ostwald type viscosimeter a t 25'. Optical activity was determined using a 20-cm. polarimeter tube and allowing 15 minutes after mixing the reagents before taking a reading. Two samples were prepared by mixing partially neutralized resin solutions with a concentrated solution of BSA, so that no precipitation occurred. One sample was made by precipitating BSA by polymethacrylic acid a t pH 4.0 and redissolving the precipitate by addit,ion of disodium phosphate. The protein concentration was calculated from the optical density at 280 mp, after allowing for the absorption of the resins.
111. Results A. Polymethacrylic Acid.-The effect of the resin/protein ratio on the precipitation of BSA by MA in buffered solution a t various pH values is shown in Fig. 2. Resin addition beyond an optimum amount inqeases albumin solubility. Holding the resin/protein ratio constant and studying
.6
8-
0
05
10
15
Polymethocryhc Acid Added, in qmfliler.
Fig. 2.-Preci itation of bovine serum albumin by polymethacrylic a c i z effect of resin/protein ratio at constant H; r/2 = 0.04 (accbtate buffer); BSA concn. = 1.82 g./ Iter. the albumin precipitation &s a function of pH at differcnt concentrations of acct8atebuffer and in the presence of thiocyanate, the data plotted in Fig. 3 werc obtained. Acetate 20
1
0% Concenlrolion a I 0 2 gdliler e o
No added so11 OWN N a C
D
010 N Nook
004N NaOAc t 005 N KCNS 022 gms MAlgrn 0SB e
'6
Ub
40
4: PH
Pig. 3.-Precipitation of bovine scrum albumin by polyincthacrylic acid: effect of acetate and thiocyanate.
ZM
ZM
zm
280 290 330 Wwcknplh h m&.
w)
rn
so
340
350
Fig. 1.-Ultraviolet absorption spectra of bovine serum albumin a t pH 4, maleic anhydride-styrene copolymer and polyvinylamine. (16) T. Alfrey. Jr., R. hl. Fuoas, preparat.inn.
H. Pinner and H.Morswetn, i n
has little effect on the cornplcx formation but thiocyanate producr:o a considerable shift of the prccipitation curvc to lower pH values. Figure 4 shows that both the molecular weight of the polymcr and the precipitation temperature arc only of secondary importance. Result8 obtained in a study of the interaction bctween alkaline eart,h cations and a copolymer of methacrylic acid with a small amount of vinylpyridine were used in selecting the optimum conditions for polyniethacrylic acid precipitation. The pyridine residues facilitated the determination of the polymer by optical density measurement. at 265 nip and wcrc not cxpcctcd to produce an important, chrrngr in the
HERBERT MORAWETZ AND WALTER L. HUGHES, JR.
66
r7.1
1.6
and pH 5.75 the o timum MA/oxyhemoglobin ratio was found to be 0.15. 8nder these conditions 91% of the oxyhemoglobin was precipitated w h l e all of the albumin remained in solution. B. Maleic Anhydride-Styrene Copolymer.-Copolymer MAS had a characteristic ultraviolet absorption spectrum and it was,therefore, possible to follow the concentration of both components in equilibrium with resin-albumin precipitates. The effect of varying the amount of MAS added to a BSA solution is shown in Table 11. These data cannot
Temp. PG)
0 0
o 0.57 o 1.50
1.05
25
U 2 = 0.04 BSA conc. ~1.82 gm/liter
1.2
L
C
TION
0.8
Y)
THE
Initial
L
2 \
a v)
TABLE I1 RESIN/PROTEIN RATIOON THE PRECIPITAOF A 0.31% BOVINE SERUM ALBUMIN SOLUTION BY MALEICANHYDRIDE-STYRENECOPOLYMER
EFFECTOF
2 0
n
Vol. 56
g. M AS/g.
BSA
Supernstant BSA MAS, &/I. &/I.
PH
Precipitate, g. MAS/g. BSA
0.4-
E
0
MA added, grn/liler.
Fig. 4.-Precipitation of bovine serum albumin by polymethacrylic acid : effect of temperature and molecular weight (- [VI ) of resin. propcrtios of the polyelectrolyte above pH 5. It waa found that the efficiencyof the precipitation increased in the order Mg++ < Ca++ < Bat+, the polymer salt being most insoluble in the pH range 6.0 to 6.5. As little as 0.01% MA is flocculated froin 1%BSA solution by 0.04 M barium chloride, and no protein is carried down with the polymer. Three criteria were used for the absence of protein denaturation: o tical activity, solution viscosity and crystallizability. Tatle I lists results of optical activity measurements. Since some of the readings were as low as 2", the differences between the optical activities found for the various samples are within the probable experimental error. They are also in satisfactory agreement with the value of 78' f 2" found by Cohn, Hughes and Weare.12
To(alSP,
3.
-------- -
I
'4
TABLEI EFFECTOF POLYELECTROLYTES ON THE OPTICAL ACTIVITY OF BOVINESERUM ALBUMIN BSA
concn., g./l.
29.0 28.5 13.8 14.2
G. resin/g. BSA
Resin
..
pH
.... . 5.33 0.00 .47 MA, 45% neutralized 6.00 .49 MAS, 53%neutralized 6.46 .45 MAJaddedatpH4ppt. 5.28 redissolved by NazHPOl
Speaific op$aal activlty
-75" -78 -75 -76
The BSA used had an intrinsic viscosity of 0.43 and could be crystallized at -5' from a 20% solution containing0.1 M acetic acid, 0.5 M sodium acetate and 40% by volume of ethanol. It was found that small amounts of polymethacrylic acid raise substantially the intrinsic viscosity and inhibit completely the crystallizability of the rotein. These two criteria were used, therefore, to provee!t separation of the pure albumin from its complex with the polymer, as well 8s the preservation of the protein in the native stfate. Thc RSA was recipitated at pH 4.71 by 0.4 g. of fractionated polymetKacrylic acid per gram protein. The precipitate was redissolved a t pH 5.38 and the resin precipitated by making the solution 0.05 molar in barium chloride. After centrifugation, the supernatant was filtered through fritted glass, dialyzed for two da s and dried from the frozen state. The recovered protein cad an intrinsic viscosity of 0.44 and crystallized under similar conditions as the starting material with comparable yields. That proteins can be separated in the interisoelectric range by use of polyelectrolytes, as postulated, has been demonstrated by the separation of oxyhemoglobin and BSA. At 1.9% BSA, 2.5% oxyhemoglobin, ionic strength 0.08
t 'I
/SA
/
.-c.-
.5r
3
$ 0
'
0.05
015
025
035
Degree of Resin Neutralizalion .
Fig. 5.-Precipitation of bovine serum albumin by maleic anhydride-styrene copolymer: effect of the degree of resin neutralization. versely, the variation of the resin/protein ratio in buffered solutions a t pH 4.77 and 5.16 gave the data shown in.Fig. 6. Any MAS added beyond a given amount remained In solution and the addition of excess resin increased the albumin solubility from it5 minimum to a new characteristic level. The insolubility of the resin-protein complex in sodium chloride solution is apparent from the data of Table 111. Although the alburmn precipitate w t h MAS dmolved on raising the pH of the solution, the complex proved to be very stable and all attempts at its dissociation failFd. The presence of the resin increased the protein solubihty above its isoelectric point a t low ionic strength and high ethanol concentration. I n fact, a solution containing 3 g.11. BSA, 1.3 gJ1. of half-neutralized MAS and 30% by volume of ethanol, could not be precipitated by addition of 95% ethanol or anhydrous acetone. Calcium acetate solution added
COMPLEXING OF BOVINE SERUM ALBUMIN
Jan., 1952
1.6
1.2
4E
0.8
I\. I \
II
Rlole sodium hydroxide/equivalent yolyvinylamine hydrobromide
r/2 =0.04 Original BSA conc.: 1.82gmlliler
--- BSA
MAS
D
0.4
0
TABLE IV PRECIPITATION OF SERUMALBUMINWITH POLYVINYLAMINE 3.1 g./L BSA, 1.6 g./l. PVA
PH 477 o 5.16
-
.I45 ,239 .333 .427 .521 .630 .738 .846 .955
.OB .05 .05 .05 .05 .05 .05 .05 .05
//
1.6
1.2
Fig. 6.--l-’recipitittion of bovine serum allwmin by maleic anhydride-styrene copolymer: cffect of resin/protein ratio a t constant pH. in the pH raiigc: 6.2 to 6.4 prccipitatcd all the protcin with the rosin. Tho precipitation became progressively less complete a t higher pH values, but the resin/protein ratio was essentially the same in the precipitate and the supernatant. Optical activity measurements (Tatdo I) indicate that no protcin denaturation is involved in this complex Formation. TABLE 111 EFFECT OF IONIC STRlGK.OTH ON THB I’RECIPITATION OF BOVINESERL.hI ALBUMINBY hlALEIC ANHYDRIDESTYRENE COPOLYMER 3.1 g./l. BSA, 2 g./I. MAS, degree of resin ~icutralizntion 0.1OG Supernatant BSX, g./l. MAS, g./l.
0.19 .38 .37 .20 .21 .19
O.OO0 ,017 ,033 ,050 .067 ,083
1.29 0.68 .54 .45 .38 .33
of
sodiii 111 chloride
0.00
0-
08
Normality
0.145 .333 .521 .630 .738‘ .846 .955
MAS added, gm/liter.
Norinality of sodiuin chloride
67
.oo .oo .00 .00
.oo .oo
PH
Supernatant HSA, PVA, &/I. &/I.
3.95 4.76 5.70 6.58 7.53 8.25 8.67
3.05 3.05 2.97 2.62 1.94 0.86 .22
1.60 1.61 1.53 1.58 1.33 0.98 .46
4.51 4.93 5.42 5.82 6.42 6.73 7.18 7.50
2.76 1.75 1.37 1.03 0.82 .58 .44 .47 1.63
1.48 1.28 1.18 1.10
8.iO
.145 .239 .333 .427 .630
.17 4.74 0.31 .17 5.22 .31 .31 .17 5.63 .17 .42 6.00 .17 6.60 .79 2.44 .I7 7.44 .i38 15ffect of 32 Volume % Ethanol 0.521 0.00 0.55 $738 .OO .OY .955 -00 .04
0.98
.85 .i2 .63 .91 0.80 .76 .iO
.72 .i 2 1.41 1.00 0.86 .30
the flocculent precipitates observed with the anionic polymers. Addition of chloride results in a surprisingly large shift of the precipit>ationcurve toward lower p H values and an even greater cffect is produced by thiocyanate (see Table I\’ and Figs. 7 and 8). Ethanol reduces the soluhilit,yof the USA complex with PVA. CoKenlroticn BSP = 182 p /Iller 055 grams WA/grom 8SA , No Salt Added b 0 0 5 N NaCl 1.2 0 0 5 N KCNS
14
I
C. Polyviny1amine.-The precipitation of BSA by 1’VA leads to the forniatiori of a precipitate which has the appearance of a highly viscous second liquid phase in contrast to
MISNaOH/Equ~volenf Palyvtnylomine Hydrokcmide.
\. 4
5
Fig. 8.--Precipitr~tio11 of bovine seruni :tllwniii l)y pi)lyvinylamine as a function of the degrce of neutralization.
7
6
e
OH.
Fig. ’I.--Precipitation of bovine serum albumin by polyvinylamine as a function of pH: effect of sodium chloride.
All the albumin could be precipitated by alcohol in the presence of PVA, but some of the resin was carried down with it. At 1.8 g./l. BSA, 1 g./l. PVA, 40% ethanol by volume, pH 4.77, ionic strength 0.04 and - 5 O , the precipitate contained all the BSA with 30% of the PVA. Converselv, the precipitation of polyvinylamine by sulfatc in the ph range 5-6 did not precipitate any of the protein. I t was found, howcver, that at high protein/resin ratios the polyvinylamino sulfate tended to remain in colloidal suspcnsion and could not be satisfactorily separated.
I~EI~BEHT MORAWETZ AND WALTPR L. HUGEES,JR.
68
D. Diethylaminoethyl Methacrylate-Methacrylic Acid Copolymer.-When 1% solutions of the amphoteric polyelectrolyte DEMMA and of BSA were mixed a precipitate was formed, the amount depending on the ratio of the solutions taken. The precipitation waa always very incomplete &s can be seen from the optical densities of the supernatants plotted in Fig. 9. The preci itate formed only at extremely low ionic strength and issolved in 0.005 N sodium chloride. 5.6
65
70 pH'
72
735
2.41
O'C. Protein Concentration. 3.1 gm/liter
0%
0.41
01 0
0.5
IO
15
20
Gm'Resin/gm B S A
Fig. 9.-Interaction of bovine serurn albumin with a methacrylic acid-diethylaininoethyl methacrylate copolymer. Extinction at 280 nip is plotted vs. resin/protein ratio.
IV. Discussion The solubility studies here reported include so many complicating factors that no more than a qualitative interpretation of the results is justified. In principle, the complexing of proteins with polyelectrolytes may be regarded as an extension of the use of polyvalent ions for protein precipitation'. Previous studies have indicated that protein precipitation by such naturally occurring polyelectrolytes as gum arabic1° and protaminess can take place only a t low ionic strength. Among the synthetic polyelectrolytes used in the present investigation only the ampholyte DEMMA formed such easily dissociable complexes with bovine serum albumin, while the albumin precipitates with the cationic and anionic polymers were surprisingly stable a t relatively high salt concentrations. This stability of the complex may be related to the flexibility of the polymer chain, permitting a close approach of its ionic charges to the opposite charges of the protein. It was generally found that a critical ratio of resin to protein was required for maximum precipitation (Figs. 2 and 6). The solubilization of the precipitate on addition of either component implies the formation of soluble complexes, as observed in antigen-antibody reactions. Maximum precipitation would be expected to coincide with the formation of isoelectric complexes and Bungenberg de JonglO has shown that this is the case for the gelatin-gum arabic system. Computing the charge of polymethacrylic acid from titration data obtained in the absence of albumin, maximum pro-
Vol. 56
tein precipitation is found to correspond to about 20 anionic polyelectrolyte charges per BSA molecule, a t pH 4.02, 4.78 and 4.96 while a higher ratio of anionic charges is required at pH 5.12. This is more than the albumin charge a t any but the lowest of these pH va1ues,17 but the proximity of the oppositely charged colloid reduces the electrical free energy of ionization and undoubtedly increases the charges of both components to values which are difficult to estimate. The effect of the resin/protein ratio on complex formation a t constant pH could be studied particularly well with copolymer MAS, since both components could be determined spectrophotometrically in the supernatant (Figs. 5 and 6). The data indicate that with increasing amounts of MAS added to a given amount of albumin, the resin/protein ratio in the precipitate increases to a maximum and any further resin remains entirely in solution. The constant albumin solubility in the presence of large amounts of resin may be interpreted as characterizing the solubility of the resin-albumin complex of this limiting composition. The results resemble somewhat those obtained by Bjornesjo and TeorellDin their extensive study of the precipitation of ovalbumin by thymonucleic acid. The precipitation equilibria were almost independent of the molecular weight of polymethacrylic acid (Fig. 4) ,as would be expected, since it is known that for any but very low molecular weight polyelectrolytes the electrical field due to the ionized chain molecules is almost independent of their degree of polymerization.'* While the precipitation of BSA with the anionic polymers was reversed rather sharply in the neighborhood of the isoelectric point of the protein, polyvinylamine added a t low salt concentration became an effective precipitant only above pH 7 (Fig. 7). The low tendency of serum albumin to complex with basic colloids has been ascribed by Haurowitz' to a steric configuration in which most of the anionic groups are located in the interior of the molecule. The precipitation of BSA with polyvinylamine was shifted to much lower pH values when chloride or thiocyanate were added to the solution (Figs. 7 and 8). The effect to be expected from such salt addition could be ascribed in general to a combination of the following factors: (a) Reduced electrostatic interaction of the oppositely charged colloids due to increased counter-ion density. (b) Increase in the negative charge of the albumin due to anion binding. (c) Increase in the polyelectrolyte charge due to a reduction of the electrical free energy of ionization. The experimental data indicate that factors (b) and (c) outweigh the effect of increased counter-ion density. The observation that thiocyanate is more effective than chloride is in accord with the relative affinity of serum albumin for these two anions. Scatchard, Scheinbeg and Armstrong found19 that isoelectric human serum albumin (17) C. Tanford. J . Am. Chcnr. Soc.. ?441 I,(1950). (IS) A. Katchalski and P. Spitnik. J . Polumer Sci., I, 432 (1947). (19) G. Sratchard. I. H. Scheinberg and S. H. Armstrong, Jr.. J . Am. Chcm. Soc..
PP, 536,540 (1950).
Jan., 1952
COMPLEXING OF BOVINE SERUM ALBUMIN
bound six chloride ions and sixteen thiocyanate ions when the free anion concentration was 0.05 N. On the other hand, no specific anion binding is observed with soluble synthetic polyelectrolytes.16 Thiocyanate shifts also the BSA-polymethacrylic acid precipitation curve to lower pH values, (Fig. 3) but the effect is much less pronounced than in PVA precipitation. This may be due to a competition of the polymeric acid for the cationic centers at which thiocyanate ions can be bound to albumin. The anion binding characterizing serum albumin is no doubt responsible for the striking stability of the BSA complex with copolymer MAS containing a phenyl group for every two carboxyls. The precipitation range extends in this case well beyond the isoelectric point of the protein and the complex seems t o be stable even in solution a t higher pH, as indicated by the failure of all attempts to separate the components. It is significant, that copolymer MAS can be separated from y-globulin,mwhich does not exhibit anion binding. This interpretation is supported by Ballou, Boyer, Luck and Lum reporting the relative afEnity of serum albumin for acetate and phenylacetate. 21 Similar conclusions were reached by Klotzi in comparing the albumin binding of succinate and the anions of various aromatic acids.22 The present studies of the interaction of serum albumin with synthetic polyelectrolytes were undertaken to evaluate their utility as reagents for protein fractionation. The removal of added reagents and the recovery of the protein in its native state are prerequisites of any fractionation process. Polymethacrylic acid and polyvinylamine could be precipitated quantitatively from albumin solutions by barium and sulfate ions, respectively, provided the polyelectrolyte was freed of its low molecular weight fraction. Bovine serum albumin recovered from its precipitate with polymethacrylic acid was found to be unchanged with respect to intrinsic viscosity, specific optical rotation and crystallizability. Similar tests for denaturation could not be carried out with albumin recovered from its polyvinylamine complex, because of the lack of available PVA. However, the albumin recovered in these experiments also appeared grossly unal(20) E. Alarneri, private oommunication. (21) G. A. Ballou, P. D. Boyer, J. M. Luck and F. G. Imn, J . Clinical Inueetigation,93,454 (1944). (22) I. M. Klotz, J . Am. Chem. aoc., 68, 2299 (1946).
60
tered. The optical activity of serum albumin has frequently been shown to increase sharply on denaturation2a-2Kand it is therefore significant that even the albumin complex with maleic anhydride-styrene copolymer retained the specific optical activity characteristic of albumin in the native state, although the forces binding the protein to the polyelectrolyte were too strong to allow separation of the components. Preliminary studies with .M. J. Hunter in this Laboratory have also shown that liver esterase retained its activity following coprecipitation with serum albumin and polymethacrylic acid. In confirmation of these results may be mentioned prior studies on the interaction of enzymes with nucleic acids and protamines. Thus Warburg and Christian used nucleic acid to precipitate enolase and recovered it without loss of activity." Krebs showed that the inactivation of phosphorylase by salmine or polylysine is reversed by precipitating the basic colloid with insu1in.n Large organic anions can also be used under proper conditions as protein precipitants without resulting denaturation.2s The application of polyelectrolytes for protein fractionation was illustrated by the separation of albumin and oxyhemoglobin in their interisoelectric range, using polymethacrylic acid reagent. In the usual procedure the interaction of the proteins is avoided by carrying out the separation at a pH below the isoelectric point of albumin, although oxyhemoglobin is rather unstable in this region. The avoidance of extreme pH values may be of advantage in other separations of relatively labile proteins. For blood proteins, whose isoelectric points are usually acid to the pH of optimal stability, the use of basic resins would appear preferable. The phenomenon of anion binding by serum albumin indicates that polyelectrolytes may even prove useful in albumin separations from other proteins of the same isoelectric point. This might be effected either by making the albumin more negative by addition of a strongly bound anion, or by use of such polyelectrolytes as copolymer MAS containing groups known to enhance anion binding. (23) H.W. Aten, C. J. Dippel, K.J. Keuning and J. Van noren, J . Colloid Sei., 3, 65 (1948). (24) W. Pauli and W. Koelbl, Koll. Bei., 41, 417 (1935). (25) R. B. Simpson, Doctoral thesis, Princeton, 1949. (26) 0.Warburg and w. Christian, Biochem. z., 810, 384 (1942). (27) E. G. Krebs, private communication. (28) T. Astiup and A. Biroh-Anderson, Nature (London), 160, 637 (1947).
