Purification of a protein with trypsin-inhibiting activity from Rhizoma Pinelliae and analysis of its N-terminal amino acid sequence / 中草药

ABSTRACT

Objective To purify a Rhizoma Pinelliae protein,determine its inhibiting activity and mechanism to trypsin,and analyze its N-terminal amino acid sequence. Methods Active Rhizoma Pinelliae protein was purified from the 40% (NH_4)_2SO_4 sediment of the crude extracted protein from Rhizoma Pinelliae by affinity chromatography of trypsin-Sepharose CL 4B and gel filtration of Sephadex G-50;12% of SDS-PAGE was used for determining the purity of the purified Rhizoma Pinelliae protein and estimating its molecular weight;N-terminal amino acid sequence of active Rhizoma Pinelliae protein was analyzed by Edman degradation. Results The purified active Rhizoma Pinelliae protein showed a sin-gle band on SDS-PAGE gel,which estimated molecular weight was about 1. 4?10～4,its specific activity was 1 059. 012 U/mg,and the yield was about 24.40%;The N-terminal sequence of the preceding six amino acid residues of active Rhizoma Pinelliae protein was DPVVDG;The quality inhibiting ratio of active Rhi- zoma Pinelliae protein to trypsin was 14. 72,and the Ki value ,the inhibition constant was about 7. 17 ?10～(-6) mol/L. Conclusion Active Rhizoma Pinelliae protein is a kind of serine protease inhibitor,a com-petive inhibitor to trypsin.