Title: Soybean trypsin inhibitor (Kunitz) is doubleheaded
Abstract: Further evidence is presented for the formation of a ternary complex between α-chymotrypsin (EC 3.4.21.1) and soybean trypsin inhibitor as well as between α-chymotrypsin and a preformed complex of soybean trypsin inhibitor and trypsin (EC 3.4.21.1). This is well in agreement with our earlier sedimentation equilibrium studies. We report on different elution patterns of the ternary forms as compared to the inhibitor trypsin complex and the individual components in gel filtration studies. We also demonstrate the decrease of a given chymotryptic activity on a substrate if the solution is mixed with another one containing the preformed stoichiometric inhibitor-trypsin complex. A fourth piece of evidence for the formation of a chymotrypsin-inhibitor-trypsin complex is the appearance of a difference spectrum in a absorbance, when chymotrypsin is mixed with the inhibitor-trypsin complex. Inhibition studies with purified inhibitor show that one molecule of inhibitor binds two molecules of α-chymotrypsin, with dissociation constants K1 about 1 μM and K2 about 300 nM at pH B. The site with weaker affinity for chymotrypsin is specifically blocked by stoichiometric amounts of trypsin. Purification of commercially available preparations of soybean trypsin inhibitor (Kunitz) (‘inhibitor’) to apparent homogeneity using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and first-order association kinetics with β-trypsin, is achieved by a combination of gel filtration and ion-exchange chromatography. The kinetics of the interaction of chymotrypsin with inhibitor or with inhibitor-trypsin complex were measured in a stopped-flow photometer by following the displacement of proflavine from the active site of chymotrypsin. A complete reaction scheme is presented with all rates and equilibrium constants as well as their pH-dependence.
Publication Year: 1981
Publication Date: 1981-01-01
Language: en
Type: article
Indexed In: ['crossref', 'pubmed']
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Cited By Count: 25
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