Purification and biochemical characterization of ovine α-1-proteinase inhibitor: Mechanistic adaptations and role of Phe350 and Met356

摘要: The glycoprotein alpha-1-proteinase inhibitor (alpha-1-PI) is a member of the serpin super family that causes rapid and irreversible inhibition redundant serine protease activity. A homogenous preparation ovine alpha-1-PI, 60 kDa protein was obtained by serially subjecting serum to 40-70% (NH(4))(2)SO(4) precipitation, Blue Sepharose, size-exclusion, concanavalin-A chromatography. Extensive insights into trypsin, chymotrypsin, elastase interaction with point towards involvement Phe(350) besides largely conserved Met(356) in recognition consequent inhibition. N-terminal C-terminal peptides cleaved on elastase, chymotrypsin prove presence diffused sub-sites vicinity strategically positioned Pro anchored peptide stretch. Further, human alpha-1-PI more thermolabile compared higher thermolability mainly attributed poorer glycosylation. enzymatic deglycosylation results diminished thermostability inhibitors, sharp decrease thermal transition temperatures but retaining their inhibitory potency. Homology modeling deduced amino acid sequence using template has been used explain observed inhibitor-protease interactions.