rodents the clotted semen forms a "copulation plug" which occludes the vagina, while in man the coagulated semen quickly liquifies due to the presence of pro-

teolytic enzymes in the seminal plasma.5 Previous experiments showed that four major and additional minor protein constituents could be separated from crude guinea pig vesicular secretions by chromatography on Sephadex CM-50 ion exchangers.6 One of these bulk proteins was basic in character, and devoid of tyrosine, proline, and cyst(e)ine. This basic protein appeared to be a precursor of the seminal clot formed by the action of vesiculase on vesicular secretion. We describe here a simple method for the preparation of the basic clottable protein of guinea pig vesicular secretion in a homogeneous form. Some physiocochemical properties of this protein are discussed in relation to the mechanism of its coagulation by vesiculase. Manyai and his co-workers have recently described the isolation and characteristics of a similar basic protein present in the seminal vesicle secretions of rats, mice, and guinea pigs.7-9 Methods and Materials.-Isolation of basic vesicular secretion protein: Guinea pig vesicular secretion was expressed manually from ligated glands into an iced medium containing 0.15 M NaCl, 0.002 M disodium ethylenediaminetetraacetate (EDTA), 0.04 M Tris-HCl of pH 7.9. After stirring, the solution was centrifuged at 12,000 X g for 20 min and insoluble material discarded. Two volumes of redistilled acetone (-20?) were added slowly and with stirring to the supernatant fluid. The precipitate of protein was collected by centrifugation at 2? and washed three times with acetone (-20?). The proteins were dried in vacuo over NaOH at 230, and stored at -20? in the presence of dessicant. The acetone powder was extracted with 10-20 vol of 0.01 M Tris-HCl of pH 7.5 for 1 hr at 23?. The solution was dialyzed overnight at 40 against 4 liters of the same buffer. Insoluble material was removed by centrifugation. The basic vesicular secretion protein was isolated by chromatography on O-(diethylaminoethyl) cellulose (DEAE-cellulose); a typical protocol is as follows. The dialyzed solution (62 ml containing approximately 800 mg of protein) was layered on a column (5 X 48 cm) of DEAE-cellulose. (The DEAE-cellulose was previously washed successively with 0.5 N NaOH, water, 0.5 N HC1, water, and finally equilibrated against 0.01 M Tris-HCl of pH 7.5.) An additional 400 ml of 0.01 M Tris-HCl of pH 7.5 was passed over the column. A linear gradient varying from 0 to 0.25 M NaCl in 0.01 M Tris-HCl of pH 7.5 was then applied. The column was operated at 230, and 10-ml fractions were collected in a refrigerated apparatus. The first major and symmetrical peak of protein material eluted was found in fractions 80-105, which contained the basic protein. Additional solid NaCl was added to the pooled fractions 84-101, inclusive, to give a final concentration of 0.5 M. To this solution (180 ml) was added 540 ml of acetone (-20?) with stirring at 4?. After standing for 30 min, the precipitate was removed by centrifugation at 12,000 X g for 30 min. The insoluble protein was washed three times with cold acetone, dried in vacuo, and stored at -20? over dessicant. The yield of protein was 120 mg. It may be mentioned that two other major peaks of material containing protein were later eluted from the DEAE-cellulose column, following application of the gradient of NaCl, in fractions 130-160 and 170-194. Amino acid analyses: Samples of protein (0.3-0.7 mg) were hydrolyzed in 3 ml of constant 1991