EFFECT OF DISULFIDE BONDS ON THE STRUCTURE, FUNCTION, AND STABILITY OF THE TRYPSIN TPA INHIBITOR FROM ERYTHRINA-CAFFRA - SITE-DIRECTED MUTAGENESIS, EXPRESSION, AND PHYSIOCHEMICAL CHARACTERIZATION/

Erythrina trypsin/tPA inhibitor (ETI) from the seeds of Erythrina caff ra retains its native structure and inhibitory function after reducing its two disulfide bonds. In order to elucidate the specific role of t hese crosslinks, alanine residues were substituted for cysteines after cloning the gene in Escherichia coli. Expression of the recombinant i nhibitor and the substitution mutants, C83A, CC39,83AA, and CC132,139A A, led to inclusion bodies. After solubilization in guanidinium-chlori de (GdmCl)/dithiothreitol and oxidation in glutathione buffer, activit y could be recovered at yields up to 80%. The mutant proteins exhibit full inhibitory function without detectable alterations of their nativ e structure. However, their stability is reduced: at acid pH, where th e oxidized natural inhibitor retains its native structure, the reduced wildtype protein and the mutants undergo at least partial denaturatio n, reflected by decreased pH ranges of stability: pH 5-7 for the reduc ed inhibitor, pH 2.5-8.5 for CC132,139AA, and pH 3.5-8.5 for C83A and CC39,83AA. Urea and GdmCl denaturation at pH 7 show hysteresis for bot h the oxidized inhibitor and the double mutant CC132,139AA. In contras t, the reduced protein and the other mutants exhibit true equilibrium transitions at pH 7, with urea half-concentrations of 0.9 M and 1.9 M and GdmCl half-concentrations of 0.5 M and 1.0 M, respectively. The st ability of Erythrina trypsin/tPA inhibitor follows the sequence: oxidi zed ETI > CC132,139AA > CC39,83AA and C83A > reduced ETI.