ESTIMATION OF MOLECULAR WEIGHTS IN MIXTURES OF 2 PROTEINS BY MENISCUS DEPLETION METHOD

Sedimentation equilibrium experiments with mixtures of two proteins in different proportions have been performed at speeds high enough to deplete effectively the meniscus region of protein in an attempt to establish what conditions are necessary before the molecular weights and proportions of two proteins can be estimated with reasonable accuracy using this technique. The mixtures used were 20% ovalbumin plus plasma albumin, 5, 10, and 20% ribonuclease plus ovalbumin and 5, 10, and 20% ribonuclease plus plasma albumin. Molecular weights were evaluated from the slopes of In j vs. r2 plots, from extrapolation of reciprocal plots of Mw and Mn to infinite dilution and from “two species plots.” The possibility of estimating the proportions of the two proteins present in the original solution by extrapolating Mn to the cell bottom was investigated. Experiments with the individual proteins are also reported. It is concluded that if σ(σ = w2M (1 - vp)/RT)≃ 5 for the species of lower molecular weight, the two species plot gives values for the molecular weight, Mi, of this species to better than 7% for the ribonuclease-plasma albumin mixtures (M2/M1 = 5) and within about 10-15% for the ribonuclease-ovalbumin mixtures (M2/M1 = 3.4) and is considerably better than the other methods tried for evaluating M1. M2 was obtained to better than 7% accuracy for all combinations above 5% ribonuclease-ovalbumin by using the two species plot. Attempts at estimating the proportions of the two species present were unsuccessful even in the mixture of 20% ribonuclease and plasma albumin because of the large errors in Mn in solutions of low concentrations. It is thought that these errors were probably due to adsorption of protein on the walls of the centerpiece. © 1969, American Chemical Society. All rights reserved.