超遠心機によるプロタミンの研究

概要

論文の詳細を見る
Ultracentrifugal characteristics of protamines, mainly of salmine. were investigated using a Spinco Model E Ultracentrifuge. Two kinds of buffer solution, i. e. a phosphate buffer of pH 7.7 at ionic strength of 0.2 and a NH_3-NH_4Cl-NaCl buffer of pH 8.6 at ionic strength of 0.2, were used as solvent, and their effects on the sedimentation behavior were compared with each other. Sedimentation diagrams of salmine always showed a single boundary without faster and slower components, but no definite conclusion was arrived concerning the slower components. Although the accuracy of homogeneity test was not enough owing to low sedimentation constant and rather high diffusion constant, it was concluded that salmine was ultracentrifugally fairly homogeneous. Taking the previously reported results of electrophoresis at 0℃ into consideration, protamine seems to be a rather homogeneous protein as far as molecular weight and electric charge are concerned. The sedimentation constant (s_<20>,_w) of salmine was almost constant over the concentration range from 0.18 to 0.70% in the NH_3-NH_4Cl-NaCl buffer, and the extrapolated value to infinite dilution was calculated to be 0.6_2S. In the phosphate buffer, however, the sedimentation constant increased with the increase of the concentration and became constant for concentrations above ca. 0.5%, the value at infinite dilution being 0.5_8 S and the mean value above 0.5% 1.0_0 S. The former value coincides with that in the NH_3-NH_4Cl-NaCl buffer. The abnormal sedimentation constant-concentration relationship in the phosphate buffer was accounted for by the reversible monomer-dimer equilibrium between salmine molecules in the presence of phosphate ions. The presence of dimer molecule was suggested by the results of molecular weight determination described below. It is also possible to explain the fact that only a single boundary appeared always in the experiments, if the rate of association-dissociation process is assumed to be far greater than the rate of ultracentrifugal separation. Reduced viscosity of salmine in the phosphate buffer decreased upon increasing concentration in contrast to that in the NH_3-NH_4Cl-NaCl buffer, in which the value increased slightly with concentration as usual. This also supported the above-mentioned assumption. The possible role of phosphate ions for the dimer formation of salmine is discussed from the point of view that a number of phosphate ions combine with salmine as was already shown by electrophoresis. From the sedimentation constant at infinite dilution and the diffusion constant previously reported by Iso, Kitamura and Watanabe, the molecular weight of salmine (monomer) was calculated to be about 5,000. If it is assumed that the molecule is a non-hydrated ellipsoid of revolution, the salmine molecule will be an elongated ellipsoid having an axial ratio of about 10. These results are consistent with those derived from diffusion and viscosity data. For the evaluation of the molecular weight of salmine at higher concentration in the phosphate buffer, a diffusion study was carried out foming a boundary between 1.00 and 0.50%o salmine solutions. The diffusion constant obtained in this case was 10.9×10^<-7> cm^2/sec., and in conjunction with the sedimentation constant for above 0.5%, 1.0_0 S, it follows that salmine exists as a dimer (M=9,000) at higher concentration in the phosphate buffer. Dimerization seems to occur side by side. Clupeine was also ultracenitrifugally homogeneous, and could not be distinguished from salmine in the ultracentrifuge. More precise and extended study using a synthetic-boundary cell for instance, is desirable to solve the mechanism of the aggregation of protamines.
宇宙航空研究開発機構の論文
1955-02-25

著者

宇井信生
東京大学理工学研究所

超遠心機によるプロタミンの研究

スポンサーリンク

概要

著者

関連論文

スポンサーリンク