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

The glycoprotein α-1-proteinase inhibitor (α-1-PI) is a member of the serpin super family that causes rapid and irreversible inhibition of redundant serine protease activity. A homogenous preparation of ovine α-1-PI, a 60 kDa protein was obtained by serially subjecting ovine serum to 40-70% (NH4)2SO4 precipitation, Blue Sepharose, size-exclusion, and concanavalin-A chromatography. Extensive insights into the trypsin, chymotrypsin, and elastase interaction with ovine α-1-PI, point towards the involvement of Phe350 besides the largely conserved Met356 in serine protease recognition and consequent inhibition. The N-terminal of C-terminal peptides cleaved on interaction with elastase, trypsin, and chymotrypsin prove the presence of diffused sub-sites in the vicinity of Met356 and the strategically positioned Pro anchored peptide stretch. Further, human α-1-PI is more thermolabile compared to ovine α-1-PI, higher thermolability is mainly attributed to poorer glycosylation. The enzymatic deglycosylation of human and ovine α-1-PI results in diminished thermostability of the inhibitors, with sharp decrease in thermal transition temperatures but retaining their inhibitory potency. Homology modeling of the deduced amino acid sequence of ovine α-1-PI using the human α-1-PI template has been used to explain the observed inhibitor-protease interactions.