ARCHIVES
OF BIOCHEMISTRY
Interaction
AND
BIOPHYSICS
178, 425-434 (1977)
of Tobacco Mosaic Virus and Tobacco Protein with Bovine Serum Albumin’ MAX A. LAUFFER
University
of Pittsburgh,
Virus
RAGAA A. SHALABY
AND
327 Clapp Hall,
Received
Mosaic
Received
Pittsburgh,
Pennsylvania
15260
May 24, 1976
Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.
In 1947 and 1948, Lauffer reported that tobacco mosaic virus (TMV)’ is separated from solution in the form of mesomorphic fibers upon addition of bovine serum albumin (BSA) when the pH is above 5, where both have negative charges (1, 2). The amount of albumin required to crystallize the virus was inversely related to the ionic strength and, at constant ionic strength, the amount of albumin needed for precipitation was greater at pH 5.2 than at pH 5.8. The lowest concentration of BSA at ’ This is a publication from the Biophysical Laboratory of the Department of Life Sciences, University of Pittsburgh. This work was supported by U.S. Public Health Service Grant GM 21619. 2 Abbreviations used: TMV, tobacco mosaic virus; BSA, bovine serum albumin; TMVP, tobacco mosaic virus protein; CD, circular dichroism; OD, optical density. 425 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
which a precipitate was obtained was reported to be 0.5%. The mesomorphic precipitates were found to be composed largely if not entirely of virus. Edsall and Wymann (3) point out that salting-out effects can be produced by dipolar ions and also that a realistic interpretation of salting-out is that water molecules crowd around ions and squeeze out the molecules of the solute being saltedout. The possibility is suggested, therefore, that the precipitating action of BSA on TMV under conditions of like charge is something akin to the salting-out of proteins by simple ions. Dudman (4, 5) described studies on the precipitation of TMV.at pH 7 by a number of macromolecules, including BSA. His result with BSA is qualitatively in accord with the previous finding but quantita-
426
LAUFFER
AND
tively grossly at variance. Dudman (5) reported that a concentration of 6.35% BSA is required to precipitate TMV at pH 7 and made no mention of any effect of ionic strength or pH. He explained his findings in terms of a simple variant of excluded volume theory. According to his view, at a BSA concentration of 6.35%, practically all of the space in the solution is occupied by BSA and the virus is thereby crowded out. Dudman (4) reported further that the concentration of macromolecules required to exclude TMV from solution is independent of virus concentration. The problem of the interaction between TMV and BSA has been reinvestigated in an attempt to analyze the two alternative theories of the mechanism of action critically and to provide an explanation for the large quantitative differences between the findings of Lauffer and of Dudman. It is well known that the endothermic, entropy-driven polymerization of TMV protein (TMVP) is facilitated by simple electrolytes; an increase in ionic strength shifts the equilibrium in the direction of polymerization. This effect of electrolytes has been explained in terms of salting-out theory (6). It is logical, therefore, to expect that if BSA behaves like a salting-out agent with TMV, it will facilitate the polymerization of TMVP. It does; the present contribution describes our findings. It is also logical to expect that other dipolar ions, such as glycine or glycylglycine, will behave like salting-out agents, both in precipitating TMV and in polymerizing TMVP. They do; the effect on TMV solubility is presented herein and the results on TMVP polymerization will be described in a future publication. MATERIALS
AND
METHODS
The common strain of TMV was isolated by differential centrifugation and its protein was extracted by the acetic acid method, both in the manner described by Shalaby and Lauffer (7). The virus was stored as a solution in double-distilled water at a concentration of 27 mg/ml at pH 7.1. The protein was dissolved in dilute KOH to a final pH of 8 and clarified by centrifugation at 40,000 rpm for 3 h. The concentrations of both virus and protein were determined spectrophotometrically, as described by Smith and Lauffer (8).
SHALABY Crystalline BSA was obtained from the Sigma Chemical Company. It was dissolved in water and clarified by centrifugation at 10,000 rpm for 30 min. Concentration was measured spectrophotometrically and adjusted to 10% and stored in the cold. All chemicals were certified ACS grade obtained from Fisher Scientific Company. In experiments on the interaction of BSA with TMV, series of l-ml solutions were prepared, each solution containing identical amounts of TMV and buffer and variable concentrations of BSA. The gradation of BSA concentration was 0.25% in critical experiments and 1% in exploratory experiments. End-points were then recorded as a range between the highest BSA concentration producing no turbidity and the lowest producing turbidity. The invariable result was that the end-point decreased with time, initially rapidly, then slowly after several days, after which the turbid solutions precipitated. Both the turbid solutions and precipitates exhibited clearing completely upon thixotropic behavior, shaking. Such clear solutions became turbid again and formed precipitates much more rapidly than initially. The presence of turbidity was determined by visual observation. All experiments on TMV were carried out at 22 r l”C, except when stated otherwise. Temperature-induced polymerization of TMVP in the presence of BSA was investigated as described by Smith and Lauffer (8) and Shalaby and Lauffer (7). The extent of polymerization was estimated from turbidity measurements at 320 .nm using a Beckman DU spectrophotometer equipped with thermospacers which allow the sample compartment to be maintained at any desired temperature. The actual temperature was measured by introducing a thermistor probe which measured the sample temperature to 10.05”C into the blank cuvet. The results were strictly reversible in that the extent of polymerization followed the same path upon increasing as upon decreasing temperature. With each experiment, a control, consisting of protein in phosphate buffer at the particular pH and ionic strength, was run simultaneously. The results are represented as plots of (OD - OD,) vs temperature. OD, is the optical density at the lowest temperature, where the polymerization has not started. The reason for subtracting OD, is twofold. First, the initial low-temperature optical densities were found to vary under the same experimental conditions, probably resulting from slight denaturation of TMVP. In most cases, they were found to be higher than the values calculated for turbidity for particles of the initial size presumed to be present. Second, in presence of high concentrations of BSA (up to 4%), the initial optical density was manyfold greater than the subsequent contribution from the polymerization, reflecting the contribution of BSA. It was discovered that TMVP must be equili-
TMV TABLE
AND
TMV
PROTEIN-BSA
I
CONCENTRATION OF BSA (AS A PERCENTAGE) REQUIRED TO PRECIPITATE 0.8 mg/ml OF TMV IN SODIUM PHOSPHATE-EDTA-SODIUM AZIDE BUFFER Time (h) Ionic strength 1 18 96 0.25 0.17 0.10 0.05
5-6 5-6 5-6
4-5 4-5 5-6
3-4 4-5 4-5
3-4
OF BIOCHEMISTRY
Interaction
AND
BIOPHYSICS
178, 425-434 (1977)
of Tobacco Mosaic Virus and Tobacco Protein with Bovine Serum Albumin’ MAX A. LAUFFER
University
of Pittsburgh,
Virus
RAGAA A. SHALABY
AND
327 Clapp Hall,
Received
Mosaic
Received
Pittsburgh,
Pennsylvania
15260
May 24, 1976
Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.
In 1947 and 1948, Lauffer reported that tobacco mosaic virus (TMV)’ is separated from solution in the form of mesomorphic fibers upon addition of bovine serum albumin (BSA) when the pH is above 5, where both have negative charges (1, 2). The amount of albumin required to crystallize the virus was inversely related to the ionic strength and, at constant ionic strength, the amount of albumin needed for precipitation was greater at pH 5.2 than at pH 5.8. The lowest concentration of BSA at ’ This is a publication from the Biophysical Laboratory of the Department of Life Sciences, University of Pittsburgh. This work was supported by U.S. Public Health Service Grant GM 21619. 2 Abbreviations used: TMV, tobacco mosaic virus; BSA, bovine serum albumin; TMVP, tobacco mosaic virus protein; CD, circular dichroism; OD, optical density. 425 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
which a precipitate was obtained was reported to be 0.5%. The mesomorphic precipitates were found to be composed largely if not entirely of virus. Edsall and Wymann (3) point out that salting-out effects can be produced by dipolar ions and also that a realistic interpretation of salting-out is that water molecules crowd around ions and squeeze out the molecules of the solute being saltedout. The possibility is suggested, therefore, that the precipitating action of BSA on TMV under conditions of like charge is something akin to the salting-out of proteins by simple ions. Dudman (4, 5) described studies on the precipitation of TMV.at pH 7 by a number of macromolecules, including BSA. His result with BSA is qualitatively in accord with the previous finding but quantita-
426
LAUFFER
AND
tively grossly at variance. Dudman (5) reported that a concentration of 6.35% BSA is required to precipitate TMV at pH 7 and made no mention of any effect of ionic strength or pH. He explained his findings in terms of a simple variant of excluded volume theory. According to his view, at a BSA concentration of 6.35%, practically all of the space in the solution is occupied by BSA and the virus is thereby crowded out. Dudman (4) reported further that the concentration of macromolecules required to exclude TMV from solution is independent of virus concentration. The problem of the interaction between TMV and BSA has been reinvestigated in an attempt to analyze the two alternative theories of the mechanism of action critically and to provide an explanation for the large quantitative differences between the findings of Lauffer and of Dudman. It is well known that the endothermic, entropy-driven polymerization of TMV protein (TMVP) is facilitated by simple electrolytes; an increase in ionic strength shifts the equilibrium in the direction of polymerization. This effect of electrolytes has been explained in terms of salting-out theory (6). It is logical, therefore, to expect that if BSA behaves like a salting-out agent with TMV, it will facilitate the polymerization of TMVP. It does; the present contribution describes our findings. It is also logical to expect that other dipolar ions, such as glycine or glycylglycine, will behave like salting-out agents, both in precipitating TMV and in polymerizing TMVP. They do; the effect on TMV solubility is presented herein and the results on TMVP polymerization will be described in a future publication. MATERIALS
AND
METHODS
The common strain of TMV was isolated by differential centrifugation and its protein was extracted by the acetic acid method, both in the manner described by Shalaby and Lauffer (7). The virus was stored as a solution in double-distilled water at a concentration of 27 mg/ml at pH 7.1. The protein was dissolved in dilute KOH to a final pH of 8 and clarified by centrifugation at 40,000 rpm for 3 h. The concentrations of both virus and protein were determined spectrophotometrically, as described by Smith and Lauffer (8).
SHALABY Crystalline BSA was obtained from the Sigma Chemical Company. It was dissolved in water and clarified by centrifugation at 10,000 rpm for 30 min. Concentration was measured spectrophotometrically and adjusted to 10% and stored in the cold. All chemicals were certified ACS grade obtained from Fisher Scientific Company. In experiments on the interaction of BSA with TMV, series of l-ml solutions were prepared, each solution containing identical amounts of TMV and buffer and variable concentrations of BSA. The gradation of BSA concentration was 0.25% in critical experiments and 1% in exploratory experiments. End-points were then recorded as a range between the highest BSA concentration producing no turbidity and the lowest producing turbidity. The invariable result was that the end-point decreased with time, initially rapidly, then slowly after several days, after which the turbid solutions precipitated. Both the turbid solutions and precipitates exhibited clearing completely upon thixotropic behavior, shaking. Such clear solutions became turbid again and formed precipitates much more rapidly than initially. The presence of turbidity was determined by visual observation. All experiments on TMV were carried out at 22 r l”C, except when stated otherwise. Temperature-induced polymerization of TMVP in the presence of BSA was investigated as described by Smith and Lauffer (8) and Shalaby and Lauffer (7). The extent of polymerization was estimated from turbidity measurements at 320 .nm using a Beckman DU spectrophotometer equipped with thermospacers which allow the sample compartment to be maintained at any desired temperature. The actual temperature was measured by introducing a thermistor probe which measured the sample temperature to 10.05”C into the blank cuvet. The results were strictly reversible in that the extent of polymerization followed the same path upon increasing as upon decreasing temperature. With each experiment, a control, consisting of protein in phosphate buffer at the particular pH and ionic strength, was run simultaneously. The results are represented as plots of (OD - OD,) vs temperature. OD, is the optical density at the lowest temperature, where the polymerization has not started. The reason for subtracting OD, is twofold. First, the initial low-temperature optical densities were found to vary under the same experimental conditions, probably resulting from slight denaturation of TMVP. In most cases, they were found to be higher than the values calculated for turbidity for particles of the initial size presumed to be present. Second, in presence of high concentrations of BSA (up to 4%), the initial optical density was manyfold greater than the subsequent contribution from the polymerization, reflecting the contribution of BSA. It was discovered that TMVP must be equili-
TMV TABLE
AND
TMV
PROTEIN-BSA
I
CONCENTRATION OF BSA (AS A PERCENTAGE) REQUIRED TO PRECIPITATE 0.8 mg/ml OF TMV IN SODIUM PHOSPHATE-EDTA-SODIUM AZIDE BUFFER Time (h) Ionic strength 1 18 96 0.25 0.17 0.10 0.05
5-6 5-6 5-6
4-5 4-5 5-6
3-4 4-5 4-5
3-4
