EXAMINATION OF DISSOCIATION OF MULTICHAIN PROTEINS IN GUANIDINE HYDROCHLORIDE BY MEMBRANE OSMOMETRY

A number of proteins have been examined in dilute aqueous buffers and in concentrated guanidine hydrochloride. Sedimentation coefficients, intrinsic viscosities, and second virial coefficients of these proteins in 6.0 M guanidine hydrochloride-0.5 m mercaptoethanol suggest that all of the proteins are random coils and molecular weight measurements demonstrate that those with subunits are fully dissociated. Numberaverage molecular weights, determined by membrane osmometry for the native and guanidine hydrochloride dissociated proteins, are: bovine serum albumin, 68,320 ± 600 and 67,790 ± 1100; ovalbumin, 44,620 ± 300 and 46,530 ± 600; horse liver alcohol dehydrogenase (EC 1.1.1.2), 86,000 ± 1750 and 40,790 ± 300; rabbit muscle enolase (EC 4.2.1.11), 82,550 ± 800 and 36,500 ± 200; beef heart lactate dehydrogenase (EC 1.1.1.27), 136,290 ± 1400 and 36,180 ± 800; bovine methemoglobin, 63,720 ± 1100 and 15,840 ± 800; and rabbit muscle aldolase (EC 4.2.16), 156,500 ± 1000 and 42,400 ± 300. These results indicate that the proteins have one, one, two, two, four, four, and four subunits, respectively. For the native proteins weight-average molecular weights obtained by sedimentation equilibrium centrifugation agree well with those obtained by osmometry. The use of sedimentation equilibrium to determine the weight-average molecular weights of the guanidine hydrochloride dissociated proteins is complicated by the necessity of assuming a value of the partial specific volume, [formula omitted], for the polypeptide chains in a dissociating medium. However, molecular weights were obtained by this method which were consistent with the results from the membrane osmometry. © 1968, American Chemical Society. All rights reserved.