Studies on Soybean Trypsin Inhibitors

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Abstract
Cyanogen bromide treatment of soybean trypsin inhibitor (Kunitz) gives two peptide fragments; fragments ABC and D. The isolated fragment ABC or D alone shows no trypsin inhibitory activity. The activity, however, is largely regenerated on mixing the two components at neutral or weakly basic pH. The reconstituted inhibitor, STI-C, had nearly the full native inhibitory activity. The CD spectrum showed that the reconstitution of the inhibitor was almost complete 40min after mixing, that the half time for the reconstitution was about 3min, and that the basic conformation of native STI was essentially recovered. STI-C dissociated at acidic pH, or even at neutral or weakly basic pH in the presence of 4 m urea. Lyophilization of the fragments appears to produce species incapable of reconstituting the active inhibitor due to aggregation of the fragments. STI-C which had been concentrated by lyophilization showed “temporary inhibition.” One tryptophan residue in STI-C was oxidized by hydrogen peroxide in 10% dioxane with an oxidation curve similar to that of native STI. At 0.8 mole of tryptophan oxidized, STI-C retained about 80% of the native inhibitory activity. After separation of the oxidized STI-C into two fragments by gel filtration in the presence of 4M urea, the modified tryptophan residue was identified as tryptophan-117 in fragment D. Isolated intact fragments ABC and D were individually oxidized with hydrogen peroxide and mixed at weakly basic pH with an equimolar amount of intact D or ABC, respectively. In both cases, complexes between ABC and D were formed. The CD spectra showed that the complex with tryptophan-117 oxidized had recovered almost the same conformation as H,2O2-oxidized STI-C, while the complex with tryptophan-93 oxidized had a rather different conformation. Trypsin inhibitory activity was found for the complex with tryptophan-117 oxidized, but not for the complex with tryptophan-93 oxidized, indicating that tryptophan-93 plays an important role either in the maintenance of the native structure or in the inhibitory activity of STI, while tryptophan-117 plays a minor role both in the tertiary structure and in the inhibitory activity of STI. This paper is concerned with the relationship between the inhibitory activity and the conformation of various derivatives of STI-C on the basis of their CD spectra.

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