Characterization of the zinc-metalloprotein nature of rat spermatidal protein TP2

Abstract

Spermatidal transition protein, TP2, was purified from rat testes by Hg-affinity chromatography. The present study reports the details of the zinc-metalloprotein nature of TP2 by employing the ⁶⁵Zn-blotting technique. Chemical modification of cysteine by iodoacetic acid, and histidine by diethylpyrocarbonate, resulted in a near complete inhibition of ⁶⁵Zn-binding to TP2. The ⁶⁵Zinc-binding was localized to the V8 protease-derived N-terminal two-third polypeptide fragment. Circular dichroism spectroscopy studies of TP2 (zinc pre-incubated) and its V8 protease-derived polypeptide fragments revealed that the N-terminal fragment has a Type I-β -turn spectrum, while the C-terminal fragment has a small but significant a-helical structure. EDTA altered the circular dichroism spectrum of TP2 and the N-terminal fragment (zinc binding domain) but not that of the C-terminal fragment.