The isolation of carcinogen-binding protein from livers of rats given 4-dimethylaminoazobenzene

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Abstract
Three azo-dye-binding proteins were identified in the soluble cell supernatant fraction from livers of rats that had received 4-dimethylaminoazobenzene by intraperitoneal injection. One is basic and was highly purified. It has an isoelectric point of pH 8. 4 in bar-bital-sodium chloride buffer, I0[center dot]1, and S20, W value of 3[center dot] 5s and a molecular weight determined by Sephadex chromatography of 45000. It does not have N-terminal amino acids with free [alpha] -amino groups. Digestion with Pronase gives rise to a single azo-dye-bound peptide, which on hydrolysis is shown to contain glycine, alanine, serine, threonine, glutamic acid and aspartic acid. The amino acid that binds the azo-dye was not identified. On starch-gel electrophoresis the basic protein separates into a double band, indicating microhetero-geneity. The other two proteins were partially purified and occur in a fraction together. They have isoelectric points near neutrality and a molecular weight as determined by Sephadex chromatography of 13800. The absorption spectra in formic acid of both the basic and the low-molecular-weight proteins are similar. The azonium ion has an absorption maximum at 518m [mu] and another adsorbed chromogen is present with an absorption maximum at 395m [mu].