Preferential Solvent Interactions between Proteins and Polyethylene

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 256. No. 2, Issue of January 25, pp. 625-631, 1981 Prrnted in U.S.A.

Preferential Solvent Interactions between Proteins andPolyethylene Glycols* (Received for publication, June4, 1980, and in revised form, September 12, 1980)

James C. Lee+ and Lucy L. Y. Lee From the E. A . Doisy Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104

Preferential solvent interactions betweenpolyethyl- romolecules from solution by the addition of salts or organic ene glycols and five proteins were investigatedby den- solvents (McPherson, 1976a). sity measurements and analyzed by the multicompoWhile most proteins arerelatively stable in high concentranent theory. These measurements were conducted as a tions of salts, many proteins are denatured by organic solvents function of concentration and molecular size of the (Schrier and Scheraga, 1962; Herskovits et al., 1970); hence, synthetic polymer at different pH values. The results the choice of organic solvents to serveas precipitating agent showed that proteins are preferentially hydratedunder is critical and limited in number. One of the organic solvents the experimental conditions employed, Le. polyethyl- employed successfully as crystallizing solvent is 2-methyl-2,4ene glycol is excluded from the protein domain. The pentanediol. Recently, however, McPherson (1976b) reported introduction of protein thermodynamically destabilizes that polyethylene glycols induce crystallization of 13 proteins the solvent system. The magnitude of instability inout of 22 tested with 6 being crystallized for the first time. It creases with increasing concentrationof the polymer. was concluded that PEG’ may be the best initial reagent for Furthermore, systemsof polyethylene glycols of higher molecular weight are more destabilized. A linear rela- crystallization of proteins for x-ray diffraction analyses. Although these organic solventshave been successfully tionship was observed between the magnitude of destabilization and average hydrophobicity of the proteins employed as crystallizing solvents for many proteins, little is employed with the exception of tubulin. The system is known about the basic principles involved. It is only recently through the work of Timasheff and co-workers (Timasheff et moredestabilized in the presence of proteinswith 1978) that the mechanism higher content of hydrophilic residues indicating that al., 1976; PittzandTimasheff, the interaction between polyethylene glycol and ion- through which protein crystallized in the presence of organic ized residues is thermodynamically unfavorable with asolvents is becoming evident.By studying the preferential negative ion being more so than a positive one. After solvent interactions between proteins and organicsolvents correcting for the contribution of ionic effect on the and by application of multicomponent thermodynamic theory instability of the systemit was found that atleast for a (Casassa and Eisenberg, 1964) it was shown that proteins are protein of average hydrophobicity of 1000 cal/residue preferentially hydrated, i.e. exclusion of solventfrom the the mass of the protein contributes to the instability protein domains. Addition of proteins to the mixed solvent also. It may, therefore, be concluded that ina polyethyl- evidently leads to anincrease in the chemical potential of the ene glycol system the presence of protein leads to un- system (Timasheff et al., 1976). In the case of 2-methyl-2,4favorablethermodynamicinteraction which in turn pentanediol, this unfavorable thermodynamicinteraction leads to phase separation. The causes of such unfavorleads to phase separation, probably caused bylocal salting out able interaction include the charges residing on the of the organic molecule by the charges on the surface of the protein. protein molecule (Pittz and Timasheff,1978). Besidesinducing crystalformationand precipitation of proteins, PEG can also enhance protein self association in One of the basic thrusts of protein chemistry andenzymol- solution. Recently Herzog and Weber (1978) reported that ogy is to elucidate the mechanism of protein function and PEG can enhance the in vitro reconstitution of brain microregulation at the molecular level. Among the techniquesavail- tubules. In an effort to elucidate the mechanism of solvents able, x-raydiffraction is one of the morepowerful tools forthe which enhance microtubule formation the effects of PEG of elucidation of macromolecular structure and thus makespos- varying molecular weights on in vitro microtubule reconstisible an attempt to correlate structure withfunction. In a tution and tubulin precipitation were studied in this laboragreat many cases, however, such investigations of proteins tory (Lee and Lee, 1979). It was shown that the presence of have been thwarted due to failure to obtain crystalline sam- tubulin again leads to a destabilization of the system. The ples. The impetus for biomacromolecules to crystallize is the thermodynamic instability, however, is reduced by the forminimization of free energy, a state achievable by slowly mation of microtubules. The enhancement of microtubule bringing the system toward a state of minimum solubility. formation is, therefore, a consequence of the system trying to Many approaches havebeen employed to achieve such a state achieve a state of stability. It is not clear, however, if such of minimum solubility, and one of these is to force the mac- analysis appliesto otherproteins, and themechanism of PEG* This work was supported by National Institutesof Health Grants protein interaction is still not known. In an effort to elucidate the mechanism of crystallization of proteins induced by PEG NS-14269 and AM-21489 andthe Council forTobaccoResearch, the preferential solvent interactions of several proteins with U.S.A., Inc. The costs of publication of this article were defrayed in part by the payment of page char:es. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.

The abbreviations used are: PEG, polyethylene glycol; PEG 400, PEG 1000, and PEG 4000, PEG with an average molecular weight of 400, IOOO,and 4000, respectively.

625

626

Interaction Glycol-Proteins Polyethylene

PEG were studied by density measurements. The effects of PEG on the structure of these proteins were probed. T h e results of such studies are reported in this paper. A preliminary report of this work has been presented earlier (Lee and Lee, 1980). EXPERIMENTALPROCEDURES

Materials Spectrapor membrane tubing with molecular weight cutoffof 6,000 to8,000 and 12,000 to 14,000, and PEG 400,1O00, and 4000 were purchased from Fisher Scientific Company and were used without further purification. The following proteins and polypeptides were obtained from Sigma Chemical Co.: lysozyme (lot 57C-8025); ribonuclease A (lot 58C-8072);bovine serum albumin (lot 16C-7201); plactoglobulin (lot 106C-8072); poly-L-lysine of 90,000molecular weight (lot 89C-5054),240,000 (lot 89C-50793,and 500,000 (lot 49C-5050); and poly-L-glutamate of 70,000 molecular weight (lot 77C-5028). Chymotrypsinogen A was purchased from Worthington Biochemical Corp.

The superscript" indicates infinite dilution of protein; U, is the partial specific volumeof component 3 (PEG);po is the density of the solvent; &' and &* is the apparent partial specific volume of the protein measured at constant chemical potential and constant molality of is the preferential interacPEG, respectively; and = (ag3/ag2)T.pI,s,, tion parameter. the absolute solvation (total amount Having determined (ag3/ag2), of component 3 "bound"' to protein) or the absolute hydration of the protein can be calculated, since (Inoue and Timasheff, 1972)

where Aa is the absolute solvation, A , is the absolute hydration, and gRis the solvent composition. Component 3 may be excluded from the protein domain; hence, an exclusion term may be incorporated into Equation 2 (Reisler et al., 1977; Kupke, 1973) yielding

where E3 is the exclusion of component 3 per unit component 2, and E3 are independent of solvent composition. assuming AI, AR, Preferential interaction is also a reflection on the perturbation of Methods the chemical potential or activity coefficient of PEG by the presence Solvents Used in theDetermination-The partial specific volumes of proteins since (Timasheff, 1973; Pittz and Timasheff, 1978; Lee et of the proteins were determined in the following solvents: poly-^- al., 1979) lysine; lo-' M sodium acetate, pH 4.7; poly-L-glutamate, 10" M NaCl, M NaOH, pH 11.0; lysozyme, 2 X IO-* M KC1, lo-' M HC1, pH 2.0, 0.1 M KCl, M HC1, pH 3.0, 2 X M KCI, lo-* M acetate,pH 5.0, 2 X M KCl,10" M phosphate, pH 7.0, and lo-' M glycine, pH 10.0; bovine serum albumin, 0.2 M NaCI, pH 7.0; p-lactoglobulin, M HC1, pH ribonuclease A and chymotrypsinogen A, 0.1 M NaC1, The conformation of proteins was monitored by circular dichroism 3.0. using a Cary model 60 spectropolarimeter equipped with a model Protein Concentration Determination-The protein concentrations were determined by measuring the absorbance of an aliquot 6001 attachment. The spectra were routinely recorded from 350 to 185 nm. Overlapping spectra were obtained with 0.1 and 0.01-cm which had been gravimetrically diluted with diffusate. The values of fused silica cells.All runs were performed at 22-23°C. the absorbances used were: ribonuclease A, 0.738 ml/(mg.cm) at 278 nm (Scott and Scheraga, 1963); lysozyme,2.635 ml/(mg.cm) at281.5 RESULTS nm (Sophianopoulos et al., 1962);chymotrypsinogen A, 1.97 ml/(mg. cm) at 282 nm (Jackson and Brandts, 1970); bovine serum albumm, In an attempt to understand the basic mechanism involved 0.658 ml/(mg.cm) at 278 nm (Noelken and Timasheff, 1967); p- in protein crystal formation induced byPEG, the interaction lactoglobulin, 0.96 ml/(mg.cm) at 278 nm (Townend et al.,1960).The extinction coefficients of these protein solutions in PEG were deter- of the solvent components at the protein surfacewas studied mined by UV spectroscopy. Aliquots of native protein stock solutions, by measuring the preferential solvent interaction parameter. 10 to 20 mg/ml in concentration, were diluted volumetrically to T h e effects of PEG on lysozyme were studiedas a function of identical extehts with the low salt buffers and with PEG solvents. The different polymer sizes and at different polymer concentraUV spectra of these dilute solutions were recorded with a Cary 118 tions. Resultsof such studies are summarized in Table I. The spectrophotometer. Knowing the extinction coefficient of these pro- polymer species employed in this study include PEG 400, teins in low salt buffers and the absorbance ratios, Ahuffer/APw, PEG 1000, and PEG 4000. It is evident that under the experresulted in the extinction coefficients of these proteins in PEG solutions. The concentration of poly-L-lysine and poly-L-glutamate was imental conditions the preferential solvent interaction paramassumes a negativevalueregardless of the estimated by biuret measurements using the dry weights of these eter, (ag3/agz), polymer size. A negative value implies a preferential enrichpolymers as standard. Density Measurements-Interaction between solvent components ment of water at the protein domain or exclusion of PEG from and proteins was monitored by density measurements using previ- t h e same domain. The extent of such exclusion as shown in ously published procedures (Lee and Timasheff, 1974; Lee and Lee, column 4, Table I, increases with increasing PEG concentra1979). The densities of the solvents and the protein solutions were measured with a Precision density meter DMA-O2D (Mettler/Paar). tion as evidenced by a more negative value for aga/agn at All measurements were made at 20°C with the cell compartment higher PEG concentrations. The data from preferential solmaintained at this temperature with a refrigerated and heated circu- vent interaction studies were further analyzedin accordance lating bath from Hotpack. with Equation 3, as shown in Fig. 1.A linear relation between Density measurements were carried out with protein samples at ag3/ag2and g3 was obtained for both PEG 1000, 4000, a n d both constant molality and constant chemical potential of PEG. The higher concentrations of PEG 400. The linear extrapolations preparation of samples was carried out using published procedures t o infinite dilutions of PEG concentration yield values of (A3 (Lee et al., 1979; Lee and Lee, 1979).Special precautions were taken -0.19, -0.07, and -0.1 g/g for to ascertain that constant chemical potential was indeed attained by - E 3 ) .The values obtained are dialysis. Cellulose dialysis tubings with molecular weight cutoff at PEG 400, PEG 1000, and PEG 4000, respectively. Since agn/ 6000 to 8000 were used. Protein solutions were made up by dissolving ag, decreases with increasing PEG concentration, E3 must salt-free lyophilized protein in the PEG-buffer system of choice and outweigh ASand the valuesof (A3 - E3) most likely represent dialyzed against 500 ml of solvent at room temperature for 16 to 24 h. the minimum values of Eo. PEG is, therefore, excluded from Longer periods of dialysis were also employed, and the resulting density measurements did not yield different data within the usual the protein domain in all cases studied.The slope of the plots experimental precision observed. From density measurements carried in Fig. 1 should yield values of A I .It was calculated that A I out both at constant chemical potential and constant composition of assumes values of 0.44, 0.59, and 5.67 g/g for PEG 400,PEG solvent components, the preferential interaction between PEG (com- 1000, and PEG 4000, respectively. These values are larger ponent 3) and protein (component 2) in water (component 1) can be than those reported in the literature (Bull and Breese, 1968; calculated since It is a measurement of thermodynamic interactions without invoking any specificity, stoichiometry, and mechanism between the protein and the solvent component.

40 744

Interaction Glycol-Proteins Polyethylene

627 TABLEI

g/100ml ofsolution g/g

ml/g

rnol/mol

PEG 400 0.0 2.8 5.6 11.2 22.4 33.6 44.8

0.702 & 0.002 0.704 0.705 0.705 0.704 0.706 0.705

-0.03 f 0.02 -0.16 -0.24 -0.32 -0.39 -0.50

-1

0.713 f 0.002 0.717 0.729 0.733 0.738

0.704 f 0.002 0.705 0.705 0.704 0.704

-0.06 f 0.02 1.9 -0.08 1.2 -0.16 0.8 -0.22 -0.31 0.6

-1 -1 -2 -3 -4

0.705 f 0.002 0.698 0.699 59.44 40.56

-0.07 & 0.02 -0.21 -0.26 8.5 5.8

-0 & 1

-6 -9 -11 - 14 -18

PEG IO00

0.732

2.5 5.0 10.0 20.0 30.0 PEG 4000 0.716 0.5 1.25 2.5 3.75

*1

0.704 f 0.002 0.708 0.730 0.740 0.744 0.748 0.752

f 0.002

-1

-1 -1

*1

g,g

0.09 f 0.6 2.7 1.9 1.1 0.8 0.7

2.3 1.5 1.5 0.9 0.8 13.7 16.9 10.2 7.0

(cal/(mol in lo00 g of H20)')

0.7 f 0.5 2.3 1.6 1.o 0.7 0.6

1.3

10.9 13.8

(cal/(g in lo00 g of H20)',

0.49 1.61 1.12 0.70 0.49 0.42

1.33 0.91 0.84 0.56 0.42 76.22 96.50

10% of the value of RT/m3 (Robinson and Stokes, 1959). At all solvent compositions studied, (ap3/amz) assumes a positive value indicating that the chemical potential of the system is increased by the introduction of protein, z.e. the system is b destabilized thermodynamically. Apparently the PEG 400 and i PEG 1000 systems are equally destabilized as indicated by the c similar magnitude observed for (ap3/am2). The PEG 4000 a system, however, is much greater destabilized, an observation ? similar to that reported previously for tubulin (Lee and Lee, 1979). The preferential solvent interaction between PEG and protein has so f a r been studied in two protein systems; however, in an attemptto achieve an understanding of the basic mechanism involved, the study was expanded to include other 1 protein systems which wereselected to represent systemswith 0.2 04 06 different physical properties, e g . molecular weight and chem9, h Vnl9m ical composition. The preferential interaction of these proteins FIG. 1. Relation between the preferential solvent interaction with 10% (w/v) PEG 1000was studied, and the results are parameters, (aga/dg2), and solvent composition, g3.0, data for shown in Table 11.The values of (dgddgz), as shown in column PEG 400,0, X, data for PEG 1000 and 4000, respectively. 4,are negative in all protein systems tested. It may, therefore, be concluded that PEG is excluded from protein domains, and Kuntz, 1971; Tanford, 1961);however, they areconsistent with such an observation represents a general phenomenon within the interpretation that PEG is excluded from the domain of the limited number of systems investigated. The magnitude lysozyme, with probably the higher molecular weight polymer of (dg3/&), however, is a function of the protein system more so than the smaller ones. Such observations are in employed. It ranges from -0.01 to -0.25 g/g. As it was in the qualitative agreement with those reported for tubulin (Lee case of lysozyme the systems were thermodynamically destaand Lee, 1979). At present no explanation can be offered to bilized by the introduction of proteins as indicated by the identify the cause of deviation from linearity for data points positive values of (dp3/dmz)calculated, as shown in column 7 at low concentrations of PEG 400. It may conceivably be due of Table 11. In order to facilitate comparison among protein to theassumptions involved in such data analysis, namely, A I , systems, the change in chemical potential of the solvent AS, and E3 are independent of solvent composition. There system is expressed as (dp3/dg2~,,p,p3, shown in column 8 of may, indeed, be changes intheseparameters. To include Table 11. These values vary from 0.08 to 1.36 cal/(g in 1000 g correction terms in these parameters at present may unnec- of H20)*. essarily complicate the issue without providing further insight An attempt is made to correlate the terms (dp3/amz)T,p,p( into the problems at hand. and average hydrophobicity, HI&, a term defined by Bigelow Preferential solvent interaction can be expressed as changes (1967) as the average free energy of transfer of the amino acid in the chemical potential of the solvent component in the side chains of a protein from an aqueous environment to a presence of protein (Timasheff, 1973; Pittz and Timasheff, nonpolar environment, based on the values given by Tanford 1978; Lee and Lee, 1979). The values of changes in chemical for ethyl alcohol (Tanford, 1962). In order to eliminate the potential of PEG calculated in accordance with Equation 4 variable of protein molecular size, (ap3/dmz)is normalized by are shown in Table I, column 7. In this calculation, (dp3/ dividing it by protein molecular weight to yield the term (+3/ am3)T.p,m, was approximated by RT/m3, since there is no data dgz) T,P,@:,.The relation between (dpdagz) measured in 10% (w/ on the variation of the activity coefficient of PEG concentra- v) PEG 1000 and H+avis shown in Fig. 2. With the exception tion. The error introduced by such an approximation is about of tubulin, there is a reasonable linear correlation between

Polyethylene Glycol-Proteins Interaction

628

TABLE 11 Preferential interactions ofproteins andpoly-L-amino acids with solvent components in water-l0% (w/v) PEG lo00 On'

+2

'

ml/g

Lysozyme 0.7290.705 f 0.002 Chymotrypsinogen A0.732 0.743 albumin serum Bovine 0.7380.743 P-Lactoglobulin 0.750 0.752 RNase A 0.692 0.723 Tubulin" 0.736 0.773 Poly-L-lysine 0.890 0.003 90,000 MU> 240,000 0.790 0.890 500,000 Mu 0.790 0.890 Poly-L-glutamate 70,000 M,< 0.490 0.700

*

'I

+- 0.002

0.790 -C 0.003

-99

(Jg,/Jg,)T,,,,

(Jmdam2)T,p,.p,(dgl/d@)T+,+,(Jp3/Jm2)Tf*1 10x

g/R

g/g

mol/mol

-0.16 -0.07 -0.03 -0.01 -0.21 -0.25

-2 -2 -2 -0 -3 -28

1.5 0.6 0.3 0.08 0.1 1.9 2.3 1.36

3.63-0.68 3.63-0.68 -0.68 3.63

32.7-61 -162 87.2 -338 181.7

6.2 6.2 6.2

-1.42

53.1

13.0

(Jpa/3g,)T,pp,

(cal/(mol in I O 0 0 (eaZ/(g in loo0 gofH?O)') gofH?O/')

1.3 1.1 1.3 0.3 1.5 15.0

0.91 0.43 0.20

1.12

7.58

Data from Lee and Lee (1979).

It is apparent that PEG is excluded from the domain of lysozyme in the pH range of 2.0 to 7.0. At pH 10.0, however, 0 there is no preferential interaction, hence, no exclusion of PEG. The net number of charges on lysozyme is changed from 18 to 4 under these conditions (Tanford and Roxby, 1972). , the net number of The relation between (ap3/am2)T , P , ~ ,and charges on lysozyme is shown in Fig. 3. The system is destabilized when lysozyme has a net charge of 9 to 18 and thereis not muchdifference in the magnitudeof destabilization. With a net chargeof 4,however, the situationis altered significantly. The system is now stable, as indicated by the fact that (dpa/ dmz) assumes a value of zero under suchconditions. Attempts were made to obtain a linear correlation withthe total number of charges on lysozyme and were not successful. The presence of charged groups apparently contributes to the instability of the solvent system, although the magnitude of such aneffect FIG. 2. Relation between (aps/dgz) and averagehydrophobicity of proteins, Hcp.,. 0, data for tubulin. -, best fitting of data i s not known at present. The exclusion of PEG from the proteindomain may also be by linear regression. related to thethermodynamically unfavorable interaction bethese two parameters. Apparently the proteins with higher tween PEG and the charges on the surface of the protein. content of hydrophobic residues do not induce as much instaIngham (1977) reported the results of a solubility study of amino acids in PEG. It was shown that most of the amino bility in the solvent system. Theslope of such a plot is -3.15 acids tested are less soluble in PEG, i.e. the free energy of X residues/(gin 1000 g of H20)'. In aneffort to resolve the contributionof protein molecular transfer of amino acids from water to PEGis unfavorable. In size and charge distribution to the destabilization of the sol- relation to this, a phase separation in the PEG-water system vent system, the preferential interaction between polyamino induced by inorganic salts was observed.The phase separation acids and 10% (w/v) PEG 1000 was studied. Poly-L-lysine and as a function of PEG concentration was examined in MgSO, poly-L-glutamic acid of similar molecular weight were chosen in a similar fashion reported by Pittz and Timasheff (1978) system. The results, as hoping that these systems would yield information on the for the 2-methyl-2,4-pentanediol-water effect of charge on the parameter (dp3/dg2).The respective shown in Fig. 4,indicate that the PEG systems are thermosolvent systems, namely at pH 4.7 for poly-L-lysine and pH dynamically destabilized by the presence of MgSO, as evidenced by phase separation. The concentration of MgS0, 11.0 for poly-L-glutamate, werechosen so that both these polypeptide chains were in random coil conformation (Adler required toinduce phase separationdecreases with increasing size of PEG leading to the conclusion that at the sameconet al., 1973). Poly-L-lysine samples of differentmolecular weights were also employed hoping to yield information on centration of MgS04 the order of instability of the solvent the effect of molecular size on the parameter (dp3/ag2). The system induced by the presence of salt is PEG 4000 > PEG results of these studies are shown in Table 11. The values of 1000 > PEG 400. Such observation is in good agreement with (dp3/dg2)again are positive, indicating a destabilizationof the that of protein-PEG-water system (TableI). The effect of PEG on the conformation of proteins was solvent system asin the case of proteins. The poly-L-glutamate system yielded a more positive value of (dpS/dgz) than thatof investigated by circular dichroism. Fig. 5 shows the CD specpoly-L-lysine. It indicates that the system is more destabilized tra of lysozyme at various pH values in the presence and in the presence of negative charges than positive ones. The absence of 10%(w/v) PEG1000. The farUV spectra, as shown in the presence of such a high density of charges in the polyamino in Fig. 5A, indicates that there is no major perturbation acid system may not yield information reflecting that of a secondary structure of lysozyme in the presence of PEG in protein-PEG system. In order to provide more information the range of pH tested. The nearUV spectra of lysozyme, as the effects of varying thenetchargeson lysozyme were shown in Fig. 5B, indicate that some of the chromophores investigated. The preferential interaction parameter was de- were perturbed at pH 10.0 in the absence of PEG. In the termined in the lysozyme-PEG 1000 system as a function of presence of PEG, however, such perturbation was not obpH. The resultsof such a study are summarized in Table 111. served. Conceivably PEG may preventlysozyme from assum-

Polyethylene Interaction Glycol-Proteins

629

TABLEI11 Preferential interactions of egg white lysozyme with solvent components in water-lG% (w/u) PEG lo00 as a function o f p H PH

0.731 0.729

h'

2.0 3.0 5.0 7.0

0.709 0.705 0.704 0.700 0.699

f 0.002

0.733 0.723 0.699

10.0

(d&/dgz)r,,,,

@2*

* 0.002

(dm3/Jmnj~,J.,,,

(@dJ&jTm~~

-2 -2 -3 -2 0

-0.14 -0.16

-0.20 -0.15 0.0

1.4 0

0

A

2.0

1.2 1.3 1.6 1.3 0.0

1.3 1.5 I .8

0.84

0.91 1.12 0.91 0

so

I -

P

"150

4

+ t

ZK)

0

IO

20

z FIG: 3. Relation between the preferentialsolvent interaction parameter, (ap,/am2), and net charge onlysozyme,2.

-

224

WAMLL)IOTH250 In

nm.

27u

FIG.5. Circular dichroism spectra of lysozyme at 22-23°C as a function of pH in the absence and presence of10%(w/v) PEG 1000. The symbols and experimental conditions are: -, at pH 2 to 7 in the absence of PEG; - - -, at pH 10.0 in the absence of PEG; and . . . , at pH 2 to 10, in the presence of PEG.

.

ing a different conformation although it does not induce any significantconformationalchanges by its presence. These observations are in agreement with those reported previously for tubulin (Lee and Lee, 1979). The CD spectra of chymotrypsinogen A, P-lactoglobulin, RNase,and bovine serum albumin in the presence and absence of 10% (w/v) PEG 1000 showed that the presence of PEG does not significantly alter the structures of these proteins. DISCUSSION

1

1.0

I

20

[NOSG] In M

FIG.4. Relation between the concentration ofPEG solution and theconcentrationof MgS04 required,toinducephase separation. 0, PEG 4000;0, PEG IOOO; A, PEG 400.

Previous study on the tubulin-PEG-water system indicated that the introduction of tubulin to PEG solvent destabilizes the system(Lee and Lee, 1979). The enhancedself association and eventualprecipitation of tubulin observed inthe presence of PEGrepresent a means for thesystemtoreducethe thermodynamic instability. It is not known, however, if such thermodynamicrationale can be applied tootherprotein systems. This investigation was initiated to gain information on the interaction of PEG and proteins,with the goal to elucidate the mechanism involved in inducingprotein crystallization by PEG. The results of this study show that PEG does not induce any significant structural changesin proteins. Yet it helps to maintain the native conformation under conditions in which the protein might otherwise assume structures other than the native form. Such conclusion is based on the resultsof CD studiesof tubulin and lysozyme at high pH. The introduction of proteins into all PEG-water systems induces destabilization of the solvent system. In no case is there a stabilization of the system observed. Apparently the presence of PEG in any proteinsolution would always provide the driving force to generate phase separation so that the system may return to a thermodynamically more favorable state. The magnitude of such driving force is a function of polymer concentration and themolecular size of the polymer. McPherson (1976a) reported that the optimum PEGsizes for

630

Polyethylene Interaction Glycol-Proteins

inducing protein crystallization are 1O00,4000, and 6000 although PEG 400 may also yield protein crystals. The concentration of PEG required to drive a protein from solution seems to decrease with increasing PEG size.If one accepts the concept that the term (ap3/am2)reflects the driving force of the system, then based on the results of this investigation (Table I, column 7) PEG 4000 should be a better crystallizing agent than either PEG 1O00 or 400, and lower concentration of PEG 4000 would be required to induce crystal formation. Such aconclusion is consistent with the observation reported by McPherson (1976a). Having established that crystallization of protein is the result of an unfavorable interaction between PEG and proteins, an attempt hasbeen made to examine the cause of such interaction. The results on the effects of net number of charges on (apdam~)for lysozyme (Table 111)indicate that charges on protein may play a role. Apparently a protein with a smaller net charge may not perturb the thermodynamic stability of the system as much. The results on the polyamino acids indicate that a negative charge may actually induce greater instability in the solvent system than a positiveIy charged ion. Such an observation is in good agreement with that on the study of the effectiveness of ions on the precipitation of PEG from solution (Bailey and Callard, 1959).It was reported that anions are the more effective ions with cations being less. Although the homogeneity of the identity of charge present in the polyamino acids may help to differentiate the effect of these charges on preferential solvent interactions, the high density of such charges renders these synthetic polymers as poor models for proteins. The study onthe interaction of PEG and other proteins was, therefore, initiated. The proteins were chosen to represent different molecular sizes and average hydrophobicity. There is an apparentcorrelation between the ability to destabilize the solvent system and the content of hydrophilic residues, the more hydrophilic the protein the more destabilization induced in the solvent system. In another word, PEG is expected to be a better crystallizing solvent for a highly charged protein than a protein that has a higher content of hydrophobic residues. At present one may only speculate on the factor involved in causing tubulin to behave differently from the rest of the proteins tested. Lu and Elzinga (1978) and Ponstingl et al. (1979) have shown that the a and /3 subunits of tubulin are extremely acidic with 11 glutamic residues in the first 25 amino acids from the COOH-terminal. This cluster of negative charges may behave like poly-L-glutamate andinduce much greater destabilization on the solvent system than the If&., parameter indicates. Furthermore, tubulin is a larger molecule than the standard proteins employed in this study. It is conceivable, though unlikely, that larger protein molecules may interact with PEG differently, Although such rationalization is reasonable it is purely a conjecture at present. Having determined the relation between (ap3/dg2) and average hydrophobicity of the proteins tested, an attempt is made to establish a relation between (+3/ag2) and the molecular size of these proteins. A plot of log (ap3/ag~)uersus log (molecular weight of protein) should yield a plot of zero slope if molecular size does not contribute to the parameter. However, a slope of -1.2 was obtained, indicating that factor(s) which may include the molecular size of protein is involved. Remembering that (aps/dg2) is influenced by the chemical composition of proteins the contribution of H&., to ap3/dg2 is removed by a normalization procedure so that all proteins have a value of1000 for H&". Knowing the actual value of H&, A, and that the slope of the (ap3/agz)versus H#*" plot (Fig. 2) is -3.15 X lo-', the contribution of H& to the term (ap3/ag2)can be estimated by

(loo0 -A) x (-3.15 x IO-')

= (ap3/ag2)H*

(5)

Having eliminated the term ( a ~ , / a g ~ )from ~ + the observed value of (ap3/ag2)0b"the residual is set to equal to the term (apdag2)". A plot of log (ap3/ag2)Ma"versus log (M,) shows a linear plot with a slope of zero indicating that there is no correlation between the destabilization of the solvent system and molecular size of the protein involved. A value of 0.63 f 0.10 is obtained for ( a p 3 / a g 2 ) ~ ~It 3 . may, therefore, be concluded that at least in10%(w/v) PEG 10oOthe destabilization induced in the solvent system can be resolved into two components, namely, mass of the protein and the H+," of the protein, i.e. (ap3/agdob"= (ap3/agdHm+

(6)

Since most proteins are characterized by H&, of lo00 k 200, (ap3/a&)H+would assume values of k0.6, a value of comparable which is (0.63 k 0.10). It seems, magnitude as (dp3/ag2)" therefore, both factors are important in contributing to the destabilization of the solvent system. The molecular mechanism which is responsible for @p3/ ag2)H+is most likely similar to that of 2-methyl-2,4-pentanediol proposed by Pittz and Timasheff (1978). The protein surface may be considered as covered by a mosaic of charges. At such an interface the interaction of PEG and these charges is thermodynamically unfavorable leading to exclusion of PEG from the protein domain. Such interpretation is consistent with the observations that 1)the PEG system is less destabilized byproteins with higher H&"; and 2) that lysozyme would destabilize the system less when it is near its isoelectric point. Ogston and co-workers (Ogston, 1958; Ogston and Phelps, 1961) have originally proposed that the mutual exclusion of proteins and large polymers may lead to precipitation of proteins. Experimental observations on precipitation of proteins by PEG have been reported to support such a hypothesis (Laurent, 1963; Juckes, 1971; Ingham, 1977,1978; Leeand Lee, 1979). The present observation that (dp3/@2)" assumes a constant value regardless of the size of the protein or synthetic polyamino acid (Table 11)seems to be incontradiction to the proposed mechanism. One mustremember, however, that precipitation and crystallization are processes which most likely are governed by different physical mechanisms. Crystallization is a thermodynamically controlled reaction. The driving force for phase separation resulting from crystal formation of proteins is most likely provided by the unfavorable interaction between the solvent system and proteins. At the PEG concentrations and within the narrow range of size of proteins employed in this study it is conceivable that the exclusion volume of PEG does not play a significant enough of a role to be detected by the present experimental measurements. On the other hand, precipitation is not only governed by the unfavorable interaction between proteins and PEG but also a physical exclusion byPEG. One of the consequences of such interpretation is that the concentration range of PEG employed for crystallization should be lower than that for precipitation. The reports by McPherson (1976a) and Ingham (1978) are apparently consistent with the present proposal. The molecular mechanism which leads to (+3/agZ)" is probably due to theenergy required to form a cavity in the solution to accommodate the protein molecule, a proposal similar to that reported by Sinanoglu and Abdulnar (1965). In conclusion, the present studyhas demonstrated that the induced phase separationof proteins from PEG-water system is due to theunfavorable thermodynamic interaction between protein and PEG. Such interaction can be at least partially attributed to the unfavorable interaction of PEG with charges residing on the surface of the protein. It is not known, however,

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