STUDIES ON PROTAMINES

II. DETERMINATION OF THE END GROUPS OF CLUPEINE AND SALMINE AND THEIR MOLECULAR WEIGHT

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1. Methylesters of clupeine and salmine were prepared from purified sulfates and crude picrates of the protamines (derived from Clupea pallasii and Oruorhynchus keta, respectively) by two methods, in which esterification was carried out (A) by allowing them to stand at room temperature for prolonged periods of time in methanol containing 0.1 N hydrochloric acid, or (B) by short time treatment under cooling in methanol saturated with hydrogen chloride.
2. Methoxyl contents of the purified ester sulfates of both protamines were found to be 0.40-0.41 per cent in the products obtained by method A when esterified thoroughly, and 0.39-0.46 per cent by method B. Since these methoxyl groups are considered to be introduced only into C-terminal carboxvl groups of the protamine molecules, molecular weight of 6, 000-6, 200 (or 5, 400-6, 400 by method B) was calculated for both protamines from the result.
3. Free amino groups (N-terminal groups) of clupeine and salmine were determined by manometric Van Slyke method. Consideration was given to an amount of contribution from the guanidyl groups to the results. Thus clupeine was found to have an 0.5-0.9 equivalent of the amino group per molecule (molecular weight=6, 000), the remaining part appearing to be an imino group. Salmine was found to contain almost none of the amino group, hence it was supposed to have mainly an imino group in the N-terminus.
The authors are indebted to Drs. S. Ishii and M. Yamasaki for their valuable discussion and to Dr. C. Hashimoto for the methoxyl determination. The expense of this work was partly aided by the Scientific Research Grant from the Ministry of Education, to whom our thanks are also due.