Purification and Characterization of a Proteinase Inhibitor from Field Bean, Dolichos lablab perpureus L. |
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Authors: | V R Devaraj N H Manjunatha |
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Institution: | 1. Department of Chemistry, Bangalore University, Central College, Bangalore, 560 001, India
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Abstract: | A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 107 M?1 and 3.1 × 107 M?1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively. |
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