INTERPRETATION OF EQUILIBRIUM SEDIMENTATION MEASUREMENTS OF PROTEINS IN GUANIDINE HYDROCHLORIDE SOLUTIONS . PARTIAL VOLUMES, DENSITY INCREMENTS, AND MOLECULAR WEIGHT OF SUBUNITS OF RABBIT MUSCLE ALDOLASE

被引:162
作者
REISLER, E
EISENBERG, H
机构
[1] Polymer Department, Weizmann Institute of Science, Rehovot
关键词
D O I
10.1021/bi00839a051
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The partial specific volume ̅v20 at 25° of bovine serum albumin was found to decrease from 0.734 in 0.2 m NaCl to 0.728 in 6 m Gu.HCl-0.1 m β-mercaptoethanol; the apparent volume Φ′, defined by (∂p/∂c2)μ0 = 1 - Φ′p° (where (∂p/∂c2)μ0 is the density increment at constant chemical potential μ of diffusible solutes and p° is the density of the solvent), equals 0.721 in the latter solvent. For rabbit muscle aldolase in water we found ̅v20 to be 0.737 and 0.739 at 20 and 25°, respectively. In 3, 4.5, and 6 m Gu.HCl at 25°, ̅v20 was found to be practically unchanged 0.733, 0.732, and 0.733, respectively; Φ′ increased from 0.703 to 0.714, 0.725, and 0.730 in 3, 5, 6, and 7 m, respectively, of Gu-HCl, 0.1 m β-mercaptoethanol, at 25°. We show that the density increments in Gu.HCl solutions (in conjunction with published values of the equilibrium distribution in the ultracentrifuge) yield the correct molecular weight for bovine serum albumin and molecular weights corresponding to four subunits per native aldolase. We discuss preferential interaction parameters ξ30 = (∂w3/∂w2)μ0 (subscript 3 refers to Gu-HCl and 2 to the protein component) and show that, in the case of aldolase, ξ30 is positive, but decreases significantly with increasing Gu.HCl concentration. We also present kinetic data on denaturation of bovine serum albumin and aldolase in 6 m Gu.HCl, both in the absence and presence of variable concentration of β-mercaptoethanol, as followed by changes of solution viscosity with time. The sedimentation constant s20,w of aldolase in 0.15 m NaCl (pH 6) was found to be 7.43 in the limit of vanishing enzyme concentration (lowest concentration measured 0.02 mg/ml). © 1969, American Chemical Society. All rights reserved.
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页码:4572 / +
页数:1
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