Studies on Protamines XII. Determination of the Carboxyl-terminal Structure of Clupeine and Salmine Using Enzymatic Procedures*

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Abstract
1. The amino acid sequences of the carboxyl-end of clupeine-1 (a protamine prepared from Clupea pallasii), clupeine-2 (Clupea harengus) and salmine (Oncorhynchus keta) were investigated by applying alternate action of highly purified porcine pancreatic carboxypeptidases B and A [EC 3. 4. 2. 2 and 3. 4. 2. 1]. It is concluded that only the stepwise release of amino acids from the carboxyl-end of the protein along the peptide chain has occurred. 2. From clupeine-1 and −2, 4 moles of arginine followed by 0.9−1 mole of alanine, together with trace amounts of glycine, serine, and threonine, were released per mole of protamine by the procedure. The results are interpreted to mean that both clupeine specimens mainly have the same carboxyl-terminal sequence of… Ala4.OH. 3. When the remaining clupeine−1 “core” was subjected to the second cycle of alternate enzymatic action, arginine (3.4 moles per mole of the “ core”), and serine (0.67 mole) followed by valine (0.75 mole) were released. The results and informations obtained from the sequence study of tryptic digests of a clupeine specimen, suggest that a molecular species with the following carboxyl-terminal sequence, …(Arg). Val. Ser.Arg4.Ala.Arg4.OH, composes 70–80% of clupeine-1. 4. In the case of salmine, only 3 moles of arginine were released by the alternate digestion. When hydrazinolysis-DNP-procedure was applied to the salmine “core”, net 0.69 mole of di-DNP-ornithine per mole of the “core” was recovered. It is suggested that the salmine specimen has the carboxyl-terminal sequence of …(Pro?).Arg.Arg3.OH. 5. Analyzing the way of tryptic hydrolysis of the ester bond of clupeine-l and salmine methyl esters by using hydroxamate-ferric ion colorimetry, it is concluded that, in both protamines, the arginine content at the carboxyl-end amounts to at least 95%.

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