Preparative Fractionation of Protamines into Their Components

Preparative Fractionation of Protamines into Their Components 

Fractionation of Whole Clupeine into Y and Z Fractions by Column Chromatography on Buffered Alumina (Homogeneous Clupeine Z) 

Elution chromatography on alumina, introduced by Scanes and Tozer (1956) for the fractionation of clupeine, was slightly modified to give better resolution by eluting the protein from a buffered alumina column (Ando and Sawada, 1960). 

The specimen of clupeine sulfate (200.9 mg) was adsorbed on a column (1.88 x 46 cm) of alumina buffered with 0.48 M phosphate buffer, pH 9.0—9.2, and eluted with the same buffer at 25 °C at the flow rate of 14 ml/20 min/tube. Two main peaks, Y and 2, were obtained. The latter was tentatively divided into two parts, 21 and 2II as shown in Fig. VII-4. Each of these three fractions gave a single peak at the expected position after rechromatography on a buffered alumina column as shown in Fig. VII-5 a, b, c (Ando and Sawada, 1961). 

Thus the 2 fraction was thought to be homogeneous and used for the structural analyses, described later, which further confirmed the homogeneity. 

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B. Preparative Fractionation of Protamines into Their Components... 

Fig. VII-8. Column chromatographic fractionation on preparative scale of protamines into all or some of their components using CM-Sephadex C-25 or Bio-Gel CM-2. A. Protamine ca. 25 mg of whole clupeine sulfate (from Clupea pallasii) in a small amount of water. Column 0.9 X 130 cm of Bio-Gel CM-2 (high capacity). Elution 0.05 M acetate buffer, pH 5.8, containing 1.5 M NaCl at room temperature at a flow rate of 2.4 ml/h. B. Protamine ca. 50 mg of whole salmine sulfate (from Oncorhynchus ketd) in a small amount of water. Column 0.9 X 150 cm of Bio-Gel CM-2 (high cap.). Elution 0.05 M acetate buffer, pH 5.8, containing 1.0 M and 1.5 M NaCl at room temperature at a flow rate of 10 ml/h. C Protamine ca. 600 mg of whole iridine sulfate (from Salmo irideus) in as small as possible amount of the eluting buffer containing 0.5 M NaCl. Column 3.0 x 146 cm of CM-Sephadex C-25. Elution 0.05 M acetate buffer, pH 5.8, containing 1.5 M NaCl at room temperature at a flow rate of 85 ml/b (Reproduced from Ando, T., and Watanabe, S., 1969)...

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