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Reductive unfolding and oxidative refolding of a Bowman–Birk inhibitor from horsegram seeds (Dolichos bif lorus): evidence for ‘hyperreactive’ disulf ide bonds and rate-limiting nature of disulf ide isomerization in folding.

Rajesh Singh, R. and Appu Rao, A. G. (2002) Reductive unfolding and oxidative refolding of a Bowman–Birk inhibitor from horsegram seeds (Dolichos bif lorus): evidence for ‘hyperreactive’ disulf ide bonds and rate-limiting nature of disulf ide isomerization in folding. Biochimica et Biophysica Acta, 1597. pp. 280-291. ISSN 0167-4838

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Abstract

Horsegram protease inhibitor belongs to the Bowman–Birk class (BBIs) of low molecular weight (8–10 kDa), disulfide-rich, ‘dual’ inhibitors, which can bind and inhibit trypsin and chymotrypsin either independently or simultaneously. They have seven conserved disulfide bonds. Horsegram BBI exhibits remarkable stability against denaturants like urea, guanidine hydrochloride (GdmCl) and heat, which can be attributed to these conserved disulfide bonds. On reductive denaturation, horsegram BBI follows the ‘two-state’ mode of unfolding where all the disulfide bonds are reduced simultaneously resulting in the fully reduced protein without any accumulation of partially reduced intermediates. Reduction with dithiothreitol (DTT) followed apparent first-order kinetics and the rate constants (kr) indicated that the disulfide bonds were ‘hyperreactive’ in nature. Oxidative refolding of the fully reduced and denatured inhibitor was possible at very low protein concentration in the presence of ‘redox’ combination of reduced and oxidized glutathiones. Simultaneous recovery of trypsin and chymotryptic inhibitory activities indicated the concomitant folding of both the inhibitory subdomains. Folding efficiency decreased in the absence of the glutathiones and in the presence of denaturants (6 M urea and 4 M GdmCl), indicating the importance of disulfide shuffling and the formation of noncovalent interactions and secondary structural elements, respectively, for folding efficiency. Folding rate was significantly improved in the presence of protein disulfide isomerase (PDI). A 3-fold enhancement of rate was observed in the presence of PDI at molar ratio of 1:20 (PDI/inhibitor), indicating that disulfide bond formation and isomerization to be rate limiting in folding. Peptide prolyl cis–trans isomerase (PPI) did not affect rate at low concentrations, but at molar ratios of 1:1.5 (PPI/inhibitor), there was 1.4-fold enhancement of the folding rate, indicating that the prolyl imidic bond isomerizations may be slowing down the folding reaction but were not rate limiting.

Item Type: Article
Divisions: Protein Chemistry and Technology
Uncontrolled Keywords: Bowman– Birk inhibitor; Disulfide bond; Reductive unfolding; Oxidative refolding
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 29 Protein Chemistry
600 Technology > 08 Food technology > 22 Legumes-Pulses
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 26 Jun 2007 12:29
Last Modified: 28 Dec 2011 09:28
URI: http://192.168.1.42/id/eprint/1267

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