Formation from Trypsin and Modified Soybean Trypsin Inhibitor of a Complex Which upon Kinetic Control Dissociation Yields Trypsin and Virgin Inhibitor

It is shown that kinetic control dissociation of a complex prepared by mixing of equimolar quantities of trypsin and of modified (reactive site Arg (64)-Ile bond cleaved) soybean trypsin inhibitor (Kunitz) yields trypsin and predominantly (g95%) virgin (reactive site intact) inhibitor. If less than equimolar quantities of trypsin are used the fraction of inhibitor molecules in which the bond is resynthesized is directly proportional to the amount of trypsin used. A simple apparatus was constructed for monitoring the yield of virgin inhibitor obtained by kinetic control dissociation as a function of time after mixing of modified inhibitor and of trypsin. The reaction was found to be first order, a result consistent with the initial equilibrium formation of a loose complex of trypsin and modified inhibitor, L*, and its subsequent first order conversion to the stable trypsin inhibitor complex, C. The rate constant for this reaction was obtained and is ∼2 x 10-2 per sec at pH 4.00 and ∼2 per sec at pH 6. The rate of this reaction appears to be identical with the rate of change of ultraviolet absorbance at 260 nm observed in stopped flow studies.