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

Gupta, Vivek K and Appu Rao, A. G. and Gowda, Lalitha R. (2008) Purification and biochemical characterization of ovine a-1-proteinase inhibitor: Mechanistic adaptations and role of Phe350 and Met356. Protein Expression and Purification, 57 (2). pp. 290-302.

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Abstract

The glycoprotein a-1-proteinase inhibitor (a-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 a-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 a-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 a-1-PI is more thermolabile compared to ovine a-1-PI, higher thermolability is mainly attributed to poorer glycosylation. The enzymatic deglycosylation of human and ovine a-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 a-1-PI using the human a-1-PI template has been used to explain the observed inhibitor–protease interactions.

Item Type: Article
Uncontrolled Keywords: Trypsin; Chymotrypsin; Elastase inhibition; Serpin; Reactive site analysis; Homology modeling; Deglycosylation; Thermal stability
Subjects: 600 Technology > 08 Food technology > 16 Nutritive value > 03 Proteins
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 19 Aug 2008 07:27
Last Modified: 07 Dec 2016 13:04
URI: http://ir.cftri.com/id/eprint/8728

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