Partial Characterization of a Basic Protein from Bull Sperm Heads

Isolation and Partial Characterization of a Basic Protein from Bull Sperm Heads 

It has been proved impossible to extract basic protein from mammalian spermatozoa by the usual methods. This has led to the sponge hypothesis advanced by Bril-Petersen and Westenbrink (1963), according to which the whole nucleus of the mammalian spermatozoon is regarded as a sponge consisting of a keratinoid network, held together by numerous disulfide bonds and associated with DNA to make the protein resistant against extraction or destruction. 

Recently the isolation of the nuclear protein has been successfully performed after disruption of all the disulfide bridges in the postulated keratinoid network (Coelingh etal. 1969 Coelingh, 1971). 

The isolated heads of bull spermatozoa were reduced in a strongly denaturing medium (5 M guanidinium chloride, 0.28 M 2-mercaptoethanol, 0.005 M EDTA, and 0.5 M Tris -HCl, pH 8.5), leading to complete dissolution of the nucleus. Reoxidation of the released sulf-hydryl groups was prevented by blocking them with ethylenimine. DNA was then removed from the cooled solution by acid precipitation (5M HCl and 2.5 M guanidinium chloride). After dialysis, fractionation of the dissolved head proteins by gel filtration on Sephadex G-75 yielded two fractions, as shown in Fig. VII-11. 

The minor fraction (25%, peak I in Fig. VII-11) was of cytoplasmic origin and had no basic character. The major fraction (75%, peak II) was of nuclear origin and was rich in arginine and half-cystine. After further purification of fraction II by chromatography on Amberlite CG-50 (2x10 cm, 0.2 M acetic acid, then 0.05 M HCl), 

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