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Zonal Electrophoresis of the Soluble Proteins of Liver and Tumor in Azo Dye Carcinogenesis^*

( The Institute for Cancer Research, Philadelphia, Pa.)

A procedure has been developed for the zonal electrophoretic resolution of the soluble proteins of rat liver with the use of a modified Porath column of ethanolyzed cellulose. The mild method, with 750 mg. or less of protein, has been applied to various studies. An initial degree of resolution reproducibly separates the soluble liver proteins into all their principal charge classes with high recovery. Further electrophoretic expansion achieves a very high degree of resolution of the subcomponents of the relatively basic (h) proteins.

The method has been applied to the separation of the soluble liver proteins of normal rats and those fed the strongly hepatocarcinogenic 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), the weakly carcinogenic 4'-methyl-4-dimethylaminoazobenzene, the noncarcinogenic 2-methyl-4-dimethylaminoazobenzene, or the control diet. The azoprotein profiles of whole extracts and their h subcomponents are also presented. Azo metabolites are bound to the soluble liver proteins with a relatively high degree of specificity. The carcinogens produce more of the principal azoprotein (slow h₂) than does the noncarcinogen. The degree of electrophoretic homogeneity of this azoprotein matches those of certain species of individual enzymes, suggesting a single function for its normal protein analog. In addition to the slow h₂, there exist two small families of soluble azoproteins, one of which appears to be ribonucleoazoproteins in which dye is bound to the protein moeity, rather than to nucleic acid. Profiles of the soluble liver ribonucleoproteins are also presented, showing that they are localized in one electrophoretic class.

The soluble proteins of rat liver tumors induced by feeding 3'-Me-DAB have likewise been zonally resolved. The amount of the relatively basic (h) proteins, in particular the slow h₂, is markedly reduced in tumor compared with that of normal and azo preneoplastic livers. In contrast to the preneoplastic liver, proteins of the tumor have no covalently bound azo dyes (Miller and Miller). In extension of that finding, the various soluble tumor proteins contain only very small quantities of loosely combined dyes. In particular, the small amount of h₂ proteins present has only a trace of such, if any.

Finally, this report summarizes the various parallel involvements of the h₂ proteins of rat liver in hepatocarcinogenesis by aminoazo dyes and by 2-acetylaminofluorene, and of the h₂-like proteins of mouse skin in skin carcinogenesis by polycyclic hydrocarbons.

^* Aided by a Grant No. E-73 from the American Cancer Society.

Received 1/ 7/63.