Isolation of a Major Ultracentrifugal Class of Soluble Proteins from Rat Liver; Localization of Soluble Protein-bound Aminoazo Dyes Therein
Abstract
1. The slowest sedimenting class of soluble, nonparticulate proteins of rat liver has been isolated by the mild process of preparative ultracentrifugation, using the schlieren optical system for visual guidance during sampling. This class represents one-half of the supernatant proteins, or one-fourth of the proteins of rat liver. Nevertheless, they sediment in the analytical ultracentrifuge as a single component (labeled "3.6-component") with a sedimentation constant of 3.6 S at infinite protein dilution. From this and the presented dependence of the sedimentation rate on the concentration of protein, preliminary consideration indicates an approximate molecular weight of 50,000 for the average protein of this class. 2. These proteins contain the major portion of the soluble, nonparticulate, protein-bound aminoazo dye derivatives resulting from the feeding of the hepato-carcinogens, 4-dimethylaminoazobenzene or 3'-methyl-4-dimethylaminoazobenzene; the very weak hepato-carcinogen, 4'-methyl-4-dimethylaminoazobenzene; or the noncarcinogen, 2-methyl-4-dimethylaminoazobenzene. Electrophoretic analysis of these proteins in sodium veronal buffer, 0.10 µ, pH 8.60, reveals the presence of at least two components. One of them, the "h" component, previously found to contain the bulk of the soluble protein-bound dye derivatives, represents approximately one-fourth of these proteins. 3. No significant difference in the ultracentrifugal and electrophoretic properties of the proteins of the 3.6-component was observed after feeding the stock, basal, or any of the above aminoazo dye diets for lengths of time needed for their approximate maximum binding to liver proteins. 4. After feeding these diets, no significant difference was observed in the ultracentrifugal composition of the total supernatant proteins of rat liver. * Supported by grant 228-2 from the Damon Runyon Memorial Fund for Cancer Research and an institutional grant from the American Cancer Society.