Erythrina caffra Trypsin Inhibitor Retains Its Native Structure and Function After Reducing Its Disulfide Bonds

Abstract

Erythrina Trypsin inhibitor (ETI) the seeds of Erythrina caffra is a high-affinity inhibitor of trypsin, chymotrypsin and tissue plasminogen activator. Its 172 amino acid polypeptide chain is stabilized in its compact, native state by two disulfide bonds. In spite of their conservation in all trypsin inhibitors of the soybean trypsin inhibitor (STI-Kunitz) family, their state of oxidation is essential only for protein stability but not for inhibitory function. Reduction/reoxidation of ETI in the presence of glutathione reshuffling buffer (GSH/GSSG; pH 8·3) not only allows the inhibitor to be restored in its native structure, but also does not interfere with its binding affinity; carboxymethylation or carboxamidomethylation of the free thiol groups does not affect K1 significantly (for trypsin (K1)ETIcm = 2·3 nM, (K1)ETIcm = 1·9 nM; for chymotrypsin (K1)ETIcm = 30 μM, (K1)ETIcm = 25 μM). The two cystine cross-bridges in the native ETI lead to enhanced stability toward pH and cheotropic agents. As taken from intrinsic protein fluorescence at acid pH and varying ionic strength (pH <4, I = 0?01 to 0·15 M), the oxidized inhibitor retains its spectral properties, whereas reduced and carboxymethylated or carboxamidomethylated ETI undergo at least denaturation. At alkaline pH, the oxidized protein is stable up to pH 9·5, whereas the reduced protein undergoes structural alterations at pH >7, reaching a final plateau at pH 10·0 to 10·5. In the case of urea (U) or guanidinium chloride (GdmCl) denaturation at pH 7·0, structural transitions of the oxidized inhibitor show "hysteresis" with half-concentrations (cU) 1/2∼ 10 M and (cGdmCl)1/2∼4·5 M for denaturation, and (cU)1/2 = 47 M and (cGdmC1)1/2 = 1·5 M for renaturation. In contrast, the reduced (and chemically modified) inhibitors exhibit true equilibrium transitions at (cU)1/2 = 0?9 M and (cdmCl)1/2 = 0·5 M, respectively. Reduction/reoxidation in the absence and in the presence of denaturants (GdmCl) can also be applied to ETI covalently attached to a solid matrix.