The glycoprotein alpha-1-proteinase inhibitor (alpha-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 alpha-1-PI, a 60 kDa protein was obtained by serially subjecting ovine serum to 40-70% (NH4)(2)SO4 precipitation, Blue Sepharose, size-exclusion, and concanavalin-A chromatography. Extensive insights into the trypsin, chymotrypsin, and elastase interaction with ovine alpha-1-PI, point towards the involvement of Phe(350) besides the largely conserved Met 356 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 Met(356) and the strategically positioned Pro anchored peptide stretch. Further, human alpha-1-PI is more thermolabile compared to ovine alpha-1-PI, higher thermolability is mainly attributed to poorer glycosylation. The enzymatic deglycosylation of human and ovine alpha-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 alpha-1-PI using the human alpha-1-PI template has been used to explain the observed inhibitor-protease interactions. (C) 2007 Elsevier Inc. All rights reserved.