On the Behavior of Serum Albumin in Acidic Perchlorate Solutions

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Oct., 1959

BEHAVIOR OF SERUMALBUMININ ACIDICPERCHLORATE SOLUTIONS

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ON THE BEHAVIOR OF SERUM ALBUMIN IN ACIDIC PERCHLORATE SOLUTIONS] BY JOHNR. CANN Contribution No. 86 f r o m the Department of Biophysics, Florence R . Sabin Laboratories, University of Colorado Medical Center, Denver, Colorado Received March 8 , 1969

When the p H of a bovine serum albumin (BSA) solution in 0.15 M NaC104 is lowered from 4.7 to about 4.1 no precipitation occurs, but on lowering the pH further to a value of 3.7 a heavy precipitate develops immediately. The recipitate redissolves progressively as the p H is lowered still further and is com letely redissolved a t p H 3.2-2.3. Folzwing dissolution of the precipitate, a new precipitate slowly forms at the low p f i values. Although the various precipitates redissolve a t p H ' s above about 4.5, i t has been shown that the protein suffers irreversible damage as a result of rolonged exposure to acidic perchlorate solutions. The implications of these findings for the structure of BSA in acicfc media are discussed. Precipitation a t p H 3.0 is readily reversed by heating when the reaction occurs a t 0" but is irreversible a t 36". The over-all rate of precipitation exhibits a strong minimum at about 26". The kinetics of precipitation are explicable in nC104- 3 P(C10,-' F! I + 1', where the symbol P denotes the "expanded" BSA terms of the reaction scheme: P imolecule and P(ClO4-),, a protein-perchlorate complex. he protein-perchlorate complex is assumed to undergo some structural change leading to a reversibly precipitated rotein I which in turn is converted irreversibly to another insoluble form 1', via a reaction occurring in the reci itate. $hereas the rate of reversible precipitation decreases with increasing temperature] the rate of the reaction mcreases. The action of perchlorate does not appear to be oxidative in nature.

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Introduction The sedimentation behavior of conalbumin and y-pseudoglobulin in acidic media exhibits a complex type of variation with changes in either pH or ionic strength of the solution.2-6 Under certain conditions (e.g., ionic strength 0.1 NaC1, pH changing from 7 to 3.1) these proteins undergo configurational changes which affect their sedimentation constants, but which usually are without influence on their molecular weights. With properly chosen type and concentration of salt, however, acid pH's also can cause aggregation. At pH 3 these proteins aggregate at high but not low NaCl concentration and a t lower concentrations of other supporting electrolytes. The effectiveness of univalent anions in aggregating the protein increases in the order C1-, Br-, NOs- and C104-. Bovine serum albumin (BSA) also undergoes configurational changes in acidic and it would be of interest to study possible aggregation of this protein in different supporting electrolytes. Preliminary experiments designed to study the sedimentation behavior of BSA in acidic perchlorate solutions revealed that under certain conditions of NaC104 concentration, the protein precipitates from solution when the pH is lowered from the isoionic point to a value less than about 4. The purpose of this paper is to present the results of a systematic investigation of this phenomenon and to discuss the implications of these observations for the structure of BSA in acidic media. (1) Supported in part by research grant No. E-1482 from the Nat,ional Institute of Allergy and Infectious Diseases of the National Institutes of Health, Public Health Service: and in part by the Damon Runwn Fund and the American Cancer Society. (2) R. A. Phelps and J. R. Cann, Arch. Biochem. Biophys., 61, 51 (1955).

(3) J. R. Cann and R . A. Phelps, Biochim. Biophys. Acta, 2 6 , 378 (1957). (4) J. R. Cann and R . A. Phelps, J . A m . Chsm. Soc., 7 7 , 4255 (1955). (5) R. A. Phelps and J. R. Cann, Biochim. Biophys. Acta, 23, 149 (1957). (5) J. T.Yang and J. F. Foster, J . A m . Chen. Soc., 7 6 , 1588 (1954). (7) C. Tanford, J. G. Bussell, D. G. Rands and S. A. Swanson, i b i d . , 77, 6421 (1955).

Experimental Materials.-The bovine serum albumin was Armour's crystallized bovine plasma albumin, Lot No. 568004. Reagent grade chemicals were used. Methods.-Concentrated stock solutions of BSA were prepared by dissolving the protein in water and then dialyzing us. water for about 18 hours at 7'. Portions of the stock solution were diluted with concentrated NaC104 solutions to give the desired protein and perchlorate concentrations. The p H was adjusted with 0.5 M HCl. The p H of the solutions were measured at room temperature with a Beckman Model G p H meter. The solutions were then brought to the desired temperature in a constant tempera' . Below 26', ture bath. A crushed ice-bath was used at 0 the temperature was maintained constant to *0.1' or better during the entire time-course of precipitation. At 26" and above, the temperature was constant to AO.1 during measurements of initial rates of precipitation, but temperature fluctuations as large as f 0 . 5 ' sometimes occurred during long-term experiments. After precipitation had occurred for the desired length of time, the reaction mixture was clarified by rapid centrifugation. Since the precipit,ates formed at low temperatures redissolved on raising the temperature, care was taken to centrifuge the sample at the temperature of precipitation. Samples precipitated at other temperatures were centrifuged at room temperature. During early stages of precipitation at 34 and 36" difficulty was encountered in completely clarifying the solutions since precipitation continued during centrifugation. Accordingly, in all cases, the reaction time was taken as the time that elapsed from the start of the experiment to completion of centrifugation. The supernatants were diluted with ionic strength 0.1 phosphate buffer a t p H 7.1 and analyzed for their protein content. In those cases where the supernatants were turbid after centrifugation, the solutions clarified on dilution with buffer. Protein concentrations were determined spectrophotometrically at 2800& with a Beckman model D U spectrophotometer. A solution of BSA which had been precipitated from 0.3 M NaC104 at about p H 3 and then redissolved at about p H 6, gave an optical density which was the same, within experimental error, as the value expected for a solution of BSA never exposed to low p H ' s . In control experiments BSA did not precipitate from it: solution in 0.15 M NaCl, p H 3.0, during 3 hours at 26.2 and 2 hours a t 1.O". Viscosityomeasurements were made at 25.1 iz 0.01' and 1.0 f 0.01

.

Results and Discussion Precipitation Curves.-In our first set of experiments, the pH's of different samples of.0.94% solution of BSA in 0.3 M NaC104 were lowered from the isoionic pH of 5.56 to values in the range 5.15 to

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JOHN R. CANN

2.05. Precipitation did not occur between the isoionic pH and pH 4.3, but when the pH was lowered further a precipitate formed immediately. After aging for 0.5 hour at room temperature, the various samples were clarified by centrifugation and the supernatants analyzed for their protein content. A precipitation curve was then constructed by plotting the per cent. of protein precipitated vs. the pH. As shown in Fig. 1, the precipitation curve rises sharply from zero precipitation at pH 4.3 to a maximum of essentially complete precipitation at about pH 3.6. The per cent. precipitated then decreases very slightly as the pH is lowered still further to a value of about 2.0. Acid-precipitation curves were also determined for other concentrations of NaC104, and the nature of the curves was found to be strongly dependent upon the perchlorate concentration. Thus, while the curve obtained in 0.2 M NaCIOl was similar to that in 0.3 44 NaC104 (depending upon the pH about 10 to 30% less precipitate was formed a t the lower perchlorate concentration), no immediate precipitation occurred in 0.1 M NaC104 even at pH 2.2 and only a strong Tyndall effect developed when a solution at pH 3.0 was aged at room temperature for 24 hours. The curve obtained in 0.15 M NaC104 is of particular interest since it differs markedly from those at other perchlorate concentrations in several important respects. When the pH of a BSA solution in 0.15 M NaC104 is lowered from 4.66 to 4.06 no precipitation occurs, but on lowering the pH further to a value of 3.7 a heavy precipitate develops immediately. In contrast to the behavior in 0.2 and 0.3 M NaC104, this precipitate redissolves progressively as the pH is lowered still further and is completely redissolved at pH 3.15-2.32.* Consequently, as shown in Fig. 1, the immediate-precipitation curve is approximately bell-shaped. A second interesting feature of the precipitation behavior in 0.15 M NaC104is the slow development of a new precipitate at low pH values following dissolution of the original precipitate formed at high pH’s. Thus, for example, when a solution of BSA in 0.15 M NaC104 was acidified with HC1, the precipitate that formed on passing through the pH range 4 to 3.7 completely redissolved on passing from pH 3.7 to 2.99. Within about a minute following dissolution of the precipitate, the pH 2.99 solution once again assumed a bluish hue and precipitation ensued. During the next 3 hours 44% of the protein precipitated out of solution and in 23 hours, 68%. The rate of this second precipitation is dependent upon pH. Thus, in the pH range 3.7 to 3.3, partial dissolution of the precipitate, formed a t higher pH values, is followed by reprecipitation a t such a rapid rate that quantitative determination of the immediate precipitation curve in this region was impossible. This accounts for the broken-line portion of the bell-shaped precipitation curve shown in Fig. 1. It appeared desirable as a first step toward the elucidation of this complex behavior t o construct a precipitation curve for aged acidic solutions of protein, SGmples of a solution of BSA in 0.15 M (8) If the solution ia held at 8 p H value between 3.8 and 3.5 for too long a time before lowering the pH t o 3.0, the precipitate does not completely redissolve.

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Fig. 1.-Precipitation curves for BSA in acidic perchlorate solutions: 0 , 0 . 3 M NaC104, aged 0.5 hour at room temperature; 0,immediate precipitation in 0.15 M NaC104 (points at pH 4.06, 3.94 and 3.70 obtained within 0.5 hour after adjusting pH); a, 0.15M NaC104, aged 4 days at room temperature.

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Fig. 2.-Comparison of immediate precipitation curve for BSA in 0.15 M NaCIOl with intrinsic viscosities [ v ] in 0.15 M KC1: G, precipitation curve; 0, intrinsic viscosities of Tanford and co-workers.’

NaClOd were adjusted to the desired pH values and allowed to age a t room temperature for 4 days. The samples were then centrifuged and the supernatants analyzed for their protein content. The resultant precipitation curve, Fig. 1, shows three pH regions of interest: (a) the pH range 4.66 to 4.18 in which no precipitation occurs; (b) the pH range 4.18 to about 3.3 in which precipitation passes through a maximum; and (c) the range from 3.3 to about 2.3 in which precipitation increases continuously when the pH is lowered. These results indicate that, depending upon pH, BSA can exist in at least three distinct forms. In the pI1 range 4.7 to 4.2 the protein exists in a form which is soluble in 0.15 to 0.3 M NaC104. At a pH of about 4.2 the protein apparently undergoes some structural alteration which changes its solubility properties such that the protein now precipitates in 0.15 to 0.3 M N,zC104. A second structural alteration occurs when the pH is further lowered t o a value of about pH 3.7. This third form of BSA is initially soluble in 0.15 M NaC104 but slowly undergoes some further change in structure which results in slow precipitation of the protein. (The possibility that the bell-shaped, immediate precipitation curve in 0.15 M NaC104 simply represents isoelectric precipitation seems to be eliminated by the fact that BSA does not show isoelectric precipitation in 0.15 M KC1.)

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Oct., 1959

BEHAVIOR OF SERUM ALBUMIN IN ACIDIC PERCHLORATE SOLUTIONS

These three forms of BSA may correspond to the compact isoionic form, the “expandable” form and the “expanded” form of the protein. The existence of these latter three structurally different forms of BSA is indicated by viscosity, optical rotation and light scattering measurements on solutions of the protein in NaC1-HC1 and KC1-HC1.6s7 It is of interest to compare the immediate precipitation curve of BSA in 0.15 M NaC104 with the viscosity data of Tanford and his co-workers’ on the same protein in 0.15 M IIC1. This comparison is made in Fig. 2. The viscosity data have been interpreted as follows’: between pH 10.5 and 4.3 BSA exists in a compact form held together by a network of bonds involving the side-chain groups of the protein. Near pH 4.2 the titration of some of the side chains results in transition to an “expandable” form. This form undergoes continuous expansion, increasing with charge and decreasing with ionic strength, so as to reduce the electrostatic free energy. The comparison made in Fig. 2 suggests that the compact form is soluble in 0.15 to 0.3 M NaC104; the “expandable” form is insoluble in these media, while the “expanded” molecule is soluble. However, the “expanded” molecule must undergo some further change in structure which results in precipitation in perchlorate solutions. This change proceeds slowly in 0.15 M NaC104 but rapidly in 0.3 M NaC104. Tanford and his co-workers’ have also observed a slow, molecular change in the “expanded” molecule as evidenced by a slow increase in viscosity of its solutions in 0.15 M KC1. On the Reversibility of Precipitation.-The protein which precipitates from acidic perchlorate solutions redissolves a t higher p H values. For example, about 99% of the protein precipitates from its solution in 0.3 M NaC104 when the p H is adjusted to pH 2.9. When the p H of the acidic solution and its precipitate was readjusted to a value of 4.5 by the addition of 0.5 M NaOH, the precipitate redissolved completely to give a clear solution. Furthermore, the protein could be precipitated and redissolved in this manner several times in succession. While all the precipitates formed under the various conditions described above redissolved when suspended in p H 7.1 phosphate buffer of ionic strength 0.1, fresh precipitates dissolved much more readily than aged ones. Sometimes the aged precipitates gave solutions a t p H 7.1 which showed a Tyndall effect but the bluish hue gradually disappeared on standing a t room temperature. Precipitates formed a t higher temperatures (34-36”) also dissolved in pH 7.1 buffer with some difficulty. Despite the fact that all of the various precipitates formed below pH 4 redissolved a t higher pH’s, it can be shown that the protein suffers some irreversible damage as a result of exposure to acidic perchlorate solution for a prolonged period of time. This is illustrated by the following experiment: a sample of BSA in 0.15 M NaC104 was adjusted to pH 3.0. As in previous experiments, the precipitate formed in the pH range 4 to 3.7, redissolved a t lower pH’s. The clear, pH 3.0 solution was aged for 5 hours at 26” during which time about

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55% of the protein precipitated from solution. The sample then was centrifuged. The precipitate was redissolved in an appropriate volume of p H 7.1 phosphate buffer, ionic strength 0.1, dialyzed overnight us. buffer, and then dialyzed vs. water. This material will be referred to as the “precipitated portion.” The supernatant obtained by centrifugation of the aged acidic sample was adjusted to p H 6 with NaOH, dialyzed us. water, concentrated by pervaporation and dialyzed once more us. water. This material will be referred to as the “non-precipitated portion.” The acid-precipitation behavior of a 1% solution of each of these materials in 0.15 M NaC104was determined. When the pH of the solution of “precipitated portion” was lowered to a value of about 3.7, heavy precipitation occurred immediately. However, in contrast to BSA, which had not been exposed p r e viously to low pH’s, the precipitate did not redissolve, a t least to any great extent, when the p H was lowered further to a value of 2.7. This shows that the protein suffered irreversible damage during its first acid-precipitation. I n contrast, the “nonprecipitated portion” behaved similarly to, although not identically with, BSA which had not been exposed previously to low pH’s. Thus, when the pH of the perchlorate solution of the “non-precipitated portion” was adjusted to about 3.7, immediate precipitation occurred. The precipitate redissolved when the pH was lowered further to a value of 3.0, and a new precipitate then appeared. However, the initial rate of the new precipitation was about three times that observed with BSA never exposed previously to low pH’s. It must be concluded, therefore, that even the portion of BSA which has not had time to precipitate a t pH 3.0 in 5 hours has nevertheless undergone some irreversible change in structure during that time. Kinetics of Precipitation.-In order to gain insight into the mechanism of precipitation of BSA from its acidic perchlorate solutions, it seemed advisable to investigate the kinetics of precipitation. The rate of precipitation of the protein from its solution in 0.15 M NaC104 a t pH 4 to 3.7, is very rapid. However, the precipitation which occurs a t p H 3.0 following dissolution of the precipitate which formed as the pH of the solution passed through the range 4 to 3.7, is sufficiently slow to permit kinetic studies. The rate of precipitation a t pH 3.0, 0.15 M NaC104, shows a curious dependence upon temperature. Whereas the rate of precipitation increases with increasing temperature in the range 26 to 36.3”, the rate increases with decreasing temperature in the range 26 to 0”. Furthermore, while the precipitate formed at 36.3” does not redissolve when the temperature is lowered to 26”, the precipitate formed at 0” largely redissolves at 15.5”. The kinetics of precipitation in each of these temperature ranges will be considered separately and then a possible reaction scheme will be discussed. The results of measurements on the rate of precipitation at 26.0 and 36.3” are presented in Fig. 3 which is a semi-logarithmic plot of the fraction of protein remaining in solution us. the time in hours. Clearly, the precipitation process does not follow

JOHN R. CANN

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simple first-order reaction kinetics. Although the semi-logarithmic plots are linear, within experimental error, during early stages of precipitation (during precipitation of about 30y0 of the protein a t 26” and about 65% at 36.3”), they deviate markedly from linearity during the further course of precipitation. During this latter stage the rate of precipitation becomes progressively smaller than the rate expected for a first-order r e a c t i ~ n . The ~ question naturally arises as to whether the decrease in the logarithmic rate of precipitation with time is simply an artifact arising from a change in p K during the course of reaction. The reaction mixtures were unbuffered, and the rate of precipitation is sensitive to pH. However, this possibility is eliminated by the observation that the pH of the precipitating mixture does not change during 17 hours at 26”. A second possibility is that BSA is heterogeneous with respect to its rate of precipitation from acidic perchlorate solutions. The following experiment was designed to test this explanation: a sample of BSA in 0.15 M NaCIOa, pH 3.0, was allowed to precipitate for 5 hours. The sample then was centrifuged. The supernatant was adjusted to pH 6, dialyzed v8. water, concentrated by pervaporation and then dialyzed once more vs. water. This is the “non-precipitated portion” of BSA discussed in the section “On the Reversibility of Precipitation.” If the “non-precipitated portion” were a slowly precipitating fraction of BSA, then one would expect this material to precipitate very slowly during a second exposure to 0.15 M NaC104 at pH 3.0. Furthermore, the time-course of precipitation should be more nearly linear than that obtained with the original BSA. Actually, precipitation of this material was rapid and the rate curve had the same shape as observed with the original BSA. However, the situation is complicated by the fact that the initial specific rate of precipitation of the “non-precipitated portion” was about three times the rate shown by the original BSA. Nevertheless, these results would seem to eliminate the possibility that BSA is heterogeneous with respect to precipitation rate. One plausible explanation of the shape of the rate curves is that the protein precipitates in accordance with a scheme such as

(11 where the symbol P denotes soluble protein; I, reversibly precipitated proteins; and 1’, irreversibly Precipitated protein. It is assumed that the first precipitate to form can reconvert to soluble protein, and that this reversibly precipitated protein is in turn transformed irreversibly into a structurallydifferent insoluble form. The last reaction is assumed to occur in the precipitate. With the proper choice of rate constants such a scheme will give a semi-logarithmic rate curve similar in shape to those shown in Fig. 3 and describable by a sum of two exponential rate terms with constant coefiP Z 1 4 I ’

(9) Precipitstlon does not follow a second-order rate law, and the absence of an induction period seems to eliminate the possibility that aggregation of the protein in solution is rate limiting at this p H . (There might be an induction period at lower p H s , but this possibility has not been investigated systematically.)

Vol. 63

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cients. Although our rate datalare not adequately represented by such a kinetic expression (a sum of three exponential rate terms with constant coefficients does give a reasonable fit), it must be remembered that the soluble protein suffers irreversible damage during prolonged exposure to acidic perchlorate solution so that departures from a simple reaction scheme such as that shown above are to be expected. If the precipitation process does indeed involve a reversible step, then it is conceivable that some of the precipitate formed at a given temperature might redissolve a t a lower temperature, Actually, this is not the case. None of the precipitate formed during 11 minutes a t 36.3’ (about 61% of the protein) redissolved when the temperature was lowered to 26” even after remaining at the lower temperature for 1 hour. I n fact, during this time precipitation continued but a t the much reduced rate expected a t the lower temperature. Of course, the reaction scheme given above is not necessarily inconsistent with this observation. Whether or not any reversibly precipitated protein present at the higher temperature redissolves a t the lower one, would depend upon the effect of temperature on the equilibrium constant for the reaction P G I. If the lower temperature shifts the equilibrium position in favor of I, then reversibly precipitated protein would not redissolve. Direct experimental evidence for such an equiIibrium will be presented shortly. The initial stage of precipitation in the temperature range under consideration has been examined in some detail. Within experimental error, the initial specific rate of precipitation a t 26” (ie., the initial rate of precipitation per unit concentration of protein) is independent of the protein concentration over the range 0.2 to 2.4%. This indicates a first-order reaction with respect to protein concentration. In contrast, the specific rate of precipitation is extremely sensitive to perchlorate concentration. Thus, at 26”, BSA does not precipitate from its 0.1 M NaC104 solution a t pH 3.0 in 3.5 hours, and only a strong Tyndall effect develops in 24 hours. On the other hand, precipitation occurs too rapidly in 0.2 M NaC104 to permit kinetic measurements. In fact, at this perchlorate concentration it is impossible to separate the various processes

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BEHAVIOR OF SERUM ALBUMININ ACIDICPERCHLORATE SOLUTIONS

Oct., 1959

1549

ments at 15.5, 7.0 and 0" are presented in Fig. 5, "Initial" specific rates of precipitation a t these temperatures are shown in Fig. 4. As indicated, the rate shown for 15.5" is subject to considerable uncertainty, and those at 7.0 and 0" must be considered as minimum values since precipitation proceeds so fast a t these temperatures that the actual initial rates cannot be measured. In any event, the data reveal several important features of the precipitation process. The first of these is the negative temperature coefficient a t low temperatures. Thus, as the temperature is lowered from 26 to 0", the rate of precipitation increases by at least a facFjj tor of 20. The rate a t 0" is at least 3 times the rate I I I Y I I 1 1 1 1 I ,*, sm ,n ,m at 36.3". As found for higher temperatures, the rate of pre,b ;, o: : cipitation at 0" is extremely sensitive to perchlorate Fig. 4.-Semi-logarithmic plot of initial specific rates of preconcentration. Whereas precipitation is very rapid cipitation us. temperature. in 0.15 M NaC104, no precipitation occurred during 24 hours in 0.1 M NaCIOr. An important property of precipitation at 0" is 0 15.5.C its reversibility with respect to temperature. This ," t---fB = is illustrated by the broken-line curve, ABC, shown in Fig. 5. About 87% of the protein precipitates during the first 15 minutes a t 0" (point A). If a t this time the temperature of the reaction mixture is raised to 15.5", approximately two-thirds of the L I \ I precipitate dissolves rapidly. After 10 minutes a t 15.5' only about 30% of the protein remains as a ' I precipitate (point B). If a t this time the tempera' I ture is once again lowered to 0" the protein rapidly reprecipitates and after 15 minutes at 0" (point C) the amount of precipitate is about the same as obtained during the initial 15 minute exposure to this temperature. The amount of precipitate remain" " ' 30 I ' I ' 50 J ing after the reaction mixture a t 0" is warmed to 10 20 40 Time lmin.), 15.5" appears to be significantly lower than the amount expected from the rate measurements at the Fig. 5.-Time-course of precipitation in 0.15 M NaCIOa at pH 3.0. The broken-line curve is explained in the text. higher temperature. This is attributed to some irreversible precipitation a t 0". These results indiwhich occur as the pH of the solution is lowered cate that the shape of the rate curves at low temfrom the isoionic point to pH 3.0. Despite this peratures reflects a rapid approach to equilibrium strong dependence of the rate on perchlorate con- complicated by slow formation of some irreversibly centration, it was possible to get some idea as to precipitated material. Furthermore, the equilibthe order of the precipitation process with respect rium between soluble and precipitated proteins to perchlorate concentration by measuring the ini- shifts in favor of precipitate when the temperature tial specific rates at 36.3' in 0.13 M and in 0.15 M is lowered from 15.5 to 0". These findings supNaC104, pH 3.0. It was found that the initial spe- port our interpretation of the shape of the rate cific rate approximately doubles on increasing the curves obtained in the temperature range 26 to 36", perchlorate concentration by even this small but it must be concluded that only a relatively small amount. This indicates that, within the experi- fraction of the high-temperature precipitate is of the mental error of these measurements, the process is reversible type formed a t 0". fourth or fifth order with respect to perchlorate. The question naturally arises whether the action Finally, the rate of precipitation in 0.15 M NaC104 of perchlorate ions on BSA is simply oxidative in is very sensitive to temperature. The initial rate at nature. Experiments with other anions seem to 36.3" is almost seven times the rate a t 26". Initial eliminate this possibility. Thus, when the pH of specific rates a t various temperatures are presented a BSA solution in 0.3 M NaCNS was lowered from in Fig. 4. An activation energy of about 34 kcal./ the isoionic point to about pH 3, a heavy precipimole was calculated for the temperature range 26 to tate formed rapidly. No immediate precipitation 36". Such a large activation energy suggests that occurred when the pH of a BSA solution in 0.15 M precipitation occurs as a result of profound changes NaCNS was adjusted t o pH 2.7. However, a preoccurring in, the. structure - o f Lthe protein mole- cipitate developed slowly on standing a t room temcule. perature: the solution was very turbid after 3 hours The kinetics of precipitation at temperatures be- and gave a heavy precipitate in 18 hr. At 36", both low 26" me in striking contrast to those described precipitation and gelation occurred. The protein for the temperature range 26 to 36". Rate measure- also precipitated from its solution in 0.3 M NaI,

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pH 3.0. The rate of precipitation a t room temperature was faster than in 0.15 M NaC104, and the rate was greater at 36” than a t room temperature. The simplest reaction scheme which can account for the kinetics of precipitation over the entire temperature range 0 to 36”, appears to be P,+ nC10,P(ClO,-), I +I’ (2) A

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where the symbol P denotes the “expanded” BSA molecule and p(c104-)., a protein-perchlorate complex. The protein-perchlorate complex is assumed to undergo some structural change leading to a reversibly precipitated protein I which in turn is converted to another insoluble form I’ via a reaction occurring in the precipitate. The observed temperature dependence of the rate of precipitation can be understood qualitatively by assuming that the change in enthalpy for binding of perchlorate ions by the protein is negative and that the reacI’ has a large activation energy. At low tion I temperatures, equilibrium B in eq. 2 is established rather rapidly but the reaction I --+ I’ occurs very slowly. At higher temperatures, equilibrium A is shifted in favor of P and consequently the rate of formation of I will be decreased. At still higher temperatures, the reaction I -+ I’ becomes increasingly important and consequently the over-all rate of precipitation passes through a minimum with increasing temperature. lo One possible explanation of these data involves binding of perchlorate ions to a t least two different kinds of sites on the protein. Experiments carried out at 36.3” indicate that n is about 4 or 5. This finding presents certain problems since the average number of perchlorate ions bound by a molecule of BSA a t pH 3 must certainly be very much larger than 4 or 5. Measurements on the binding of perchlorate ion by serum albumin are limited to isoionic proteinl1J2 but some idea as to the probable extent of binding at p H 3 can be obtained from data on the binding of other ions. Thus, isoionic serum albumin, either bovine or human, in 0.15 M salt solution binds an average of about 10 chloride, 20 perchlorate or 23 thiocyanate ions per molecule while serum albumin a t pH 3.0-3.2 binds an average of about 30 chloride or GO thiocyanate ions per molecule.11-16 From these data one would expect BSA a t pH 3 to bind an average of about 50 (10) Another reaction scheme which has been considered, postulates that in 0.15 M NaClOd at p H 3.0, BSA can exist in two structurallydifferent interconvertible forms which precipitate by different mechanisms. A rapidly established, temperature dependent equilibrium between the two forms was assumed. It also waa assumed that the low-temperature form precipitates reversibly and the high-temperatufe form, irreversibly. This scheme lost its attractiveness when we observed that the reduced apeoific viscosities of 0.8-0.9% BSA solutions in 0.15 M KC1 at pH 3.0 were the same, within experimental error, at 1.0 as at 25.1’. This result indicates that the configuration of the “expanded” BSA molecule is insensitive to temperature. Of course, this does not exclude the possibility that BSA undergoes configurational changes in 0.15 M NaClOd, p H 3.0, as a result of changing the temperature. (11) G. Scatchard and E. S. Black, THISJOURNAL, 63, 88 (1949). (12) C. W. Carr, Arch. Biochim. Biophgs., 40, 286 (1952). (13) G. Scatchard, I. H. Scheinberg and 8. H. Armstrong, Jr., J . Am. Chem. SOC., 72, 535 (1950). (14) G. Scatchard, I. H. Scheinberg and 8. H. Armstrong, Jr., dbid., 72, 540 (1950). (15) 8. A. Alberty and H. H. Marvin, Jr., ibid., 73, 3220 (1951).

Vol. 63

perchlorate ions per molecule. If this is so, then practically all of the protein molecules should be in the form of complexes containing a t least 5 perchlorate ions. It is possible, of course, that BSA possesses several sets of perchlorate-binding sites, each set having a different affinity for the ion, and that a t least 4 or 5 ions must react with one particular set of sites in order for the precipitation process to occur. These sites would presumably have a very low affinity for perchlorate ion. On the other hand, it is not necessary to postulate special binding sites if it is assumed that all protein-perchlorate complexes can undergo a structural change leading to precipitation a t a rate which increases as the number of perchlorate ions in the complex increases. I n this event, the dependence of the rate of precipitation upon perchlorate concentration is not subject to the usual interpretation. Thus, for example, a t 0” where irreversible precipitation is of secondary importance, the “apparent order of the reaction with respect to perchlorate concentration” is, according to this method, equal to the difference between the average number of bound perchlorate ions in the activated states of the precipitation reactions and the average number bound by the soluble protein.’O I n either event, it is conceivable that perchlorate ions exert their effect by altering the charge distribution and thus the electrostatic forces within the “expanded” BSA molecule. Another possible explanation of the behavior of BSA in acidic perchlorate solutions is as follows. Binding of perchlorate ions to groups on the surface of the protein molecule in 0.15 M NaC104 a t first solubilizes the “expanded” form of BSA at room temperature, Figs. 1 and 2. It is supposed that the bound perchlorate ions then diffuse into the hydrophobic interior of the protein molecuIe, and as a result their solubilization effect is lost. Examination of the influence of various anions on the thermodynamic properties of aqueous solutions of non-electrolytes is pertinent to this explanation. It is particularly interesting that perchlorate ions salt-in argon into aqueous solution, whereas other anions salt-out argon. l7 Likewise, HC104 salts-in while HC1 salts-out benzene. The effectiveness of the anions of various sodium salts in salting-out benzene decreases in the order OH-, C1-, Br-, NO3-, Clod- and I-.18 Also, NaC104 and NaI salt phenylthiourea19 and y-butyrolactone20 into aqueous solutions, whereas electrolytes such as NaCl salt-out these substances. Apparently, perchlorate ions, like iodide ions which also cause slow precipitation of BSA at pH 3, prefer a relatively hydrophobic environment. Perchlorate ion is generally a poor complexing agent, and a t first glance it seems surprising that it is bound by proteins to such a large e ~ t e n t . ~ J l . Perhaps I~ this binding can be understood in terms of a tendency of perchlorate ions to (16) R . B. Simpson and W. Kauzmann, ibid., 76, 5139 (1953). (17) Personal communication from the late Professor John G . Kirkwood who suggested this explanation of the data. (18) W. F. RlcDevit and F. A. Long, J . A m . Chem. SOC.,74, 1773 (1952). (19) Merle Randall and C. F. Failey, Chem. Reus., 4, 285 (1927). (20) F. A. Long, W. F. McDevit and F. 8. Dsnkle, THISJOURNAL, 66, 813 (1951).

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Oct., 1959

BEHAVIOR OF SERUM ALBUMIN IN ACIDIC PERCHLORATE SOLUTIONS

escape from a relatively unfavorable, aqueous environment. Finally, one is impressed by the similarities between the kinetics of precipitation of BSA from acidic perchlorate solutions and the urea denaturation of P-lactoglobulinZ1and ovalbumin.16 Further analysis is required to determine whether these agents produce their respective effects via mechanisms at least partially similar.

DISCUSSION C. T. O’KONSKI(University of California a t Berkeley).Before doing work at low pH, were your samples de-ionized? J. R. CmN.-Our BSA solutions were dialyzed us. water but were not de-ionized on a n ion-exchange column. C. T. O’KoNsKI.--I’d like to call your attention to an electric birefringence study of BSA, which includes measurements in acidic media using de-ionized samples (S. Kruse and C. T. O’Konski Abstracts, National Meeting, Am. Chem. SOC.,April, 1958; J . Am. Chem. Soc., in press). We observed aggregation at low pH in samples which were not de-ionized. I n the relatively stable de-ionized material, the rotary diffusion constant seems to increase when pH goes from 5 to 2.5. This suggests that the longest dimension of the (asymmetric) macromolecule is slightly decreased by acid, contrary to an earlier view that BSA is spherical and swells isotropically a t low pH. The explanation offered by Dintzis (Ph.D. Thesis, Harvard Univ., 1952) for the effect of de-ionization on the properties of BSA is that some fatty acid is removed by de-ionization. Perhaps the most remarkable indication of change is the fact that the slope of the ciirve of specific Kerr constant us. concentration changes sign upon de-ionization indicating a changed mode of interaction in dilute soludons. This may be very important in considering the kinetics of precipitation, and it appears that studies of the de-ionized material should be made. (21) L. K. Christensen. Compt. rend. Lab. Carlsberg, Sbr. chim., 28,

37 (1952).

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J. R. CANN.-I agree with Dr. O’Konski that our experiments on the kinetics of precipitation should be extended to BSA de-ionized by passage through a mixed-bed ionexchange column and, perhaps also, by electrodialysis. I suspect, however, that both of these materials may also show complex kinetics. Although de-ionized BSA is quite resistant to aggregation in acidic KC1 solutions of low ionic strength, it does aggregate a t high ionic strengths like those used in our ey eriments [ref. 7: M. J. Kronman, M. D. Stern and S. Timasheff, T H r s JOURNAL, 60, 829 (1956); S. N. Timasheff and R. J. Gibbs, Arch. Biochem. Biophys., 70, 547 (1957)l.

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Dr. O’Konski’s important observations on the configurational changes which BSA undergoes at low p H are consistent with the results of small-angle X-ray scattering experiments on BSA solutions (&I.Champagne, V. Luzzati and A. Nicolaieff, J. Am. Chem. SOC.,80, 1002 (1958)). Our changing views concerning the behavior of BSA in acidic solutions reflect the present state of knowledge of protein structure. I n any event, it is apparent that we are dealing with a three-stage process: a rapidly occurring configurational change a t about pH 4.2; a rapid change in configuration which occurs continuously over the pH range 3.7 to 2 and which is, in turn, followed by slow aggregation in high ionic strength KC1 solutions and measurably slow precipitation in 0.15 M NaC104. L. A. ROMO(du Pont Company).-To understand the stoichiometry and kinetics of the reaction, it would have been well to do the following: ( 1 ) Titrate and follow conductivity of the protein with perchloric acid as a function of pH. These measurements would have permitted you to establish the amount of Clod- bound by the protein and the charge distribution. (2)Back titrate the acid protein with a base and see whether there is hysteresis in the curve. This hysteresis should be related to the denaturation of the protein-also the viscosity data could be interpreted better with the results obtained by these experiments.

J. R. CANN.-I agree that measurements of this ty e will help elucidate the mechanisms of interaction of B S I with perchlorate ions in acidic media.