Purification and biochemical characterization of ovine alpha-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 alpha-1-proteinase inhibitor: mechanistic adaptations and role of Phe350 and Met356. Protein Expression and Purification, 57 (2). pp. 290-302. ISSN 1046-5928

PDF Protein_Expression_and_Purification,_Volume_57,_Issue_2,_February_2008,_Pages_290-302.pdf Restricted to Registered users only Download (1MB)

Abstract

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% (NH(4))(2)SO(4) 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.

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:	18 Jun 2009 04:21
Last Modified:	28 Dec 2011 09:35
URI:	http://ir.cftri.res.in/id/eprint/1820

Actions (login required)

View Item