Enzymatic Modifications of the Protamines. II. Separation and Characterization of Phosphorylated Species of Protamines from Trout Testis

Abstract

Substantial quantities of highly phosphorylated protamines were prepared from hormonally induced trout testes at the early protamine stage of development. Purified protamines from testes induced to mature by injection of salmon pituitary extracts were resolved into eight fractions on long carboxymethyl cellulose columns by eluting with a gradient of LiCl in the presence of 6 M urea; only two major fractions were found in protamine extracted from naturally maturing testes. Each fraction was not homogenous but consisted of a mixture of several related protamines. Analysis of radioactivity in in vivo32P-labeled protamine indicated that six of the eight fractions were phosphorylated. Amino acid analysis, phosphate determinations, and starch gel electrophoresis indicated that three of the phosphorylated peaks correspond to the mono-, di-, and triphosphorylated derivatives of the first fraction (three serines) of protamine, while the other three correspond to a similar series of the second fraction (four serines) of protamine. These protamines with differing levels of phosphorylation may be useful for in vitro studies of the interaction of phosphoprotamines with DNA or chromatin.