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 ( >95%) virgin (reactive site intact) inhibitor. I f 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 10V2 per set at pH 4.00 and -2 per set 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. inhibitor with trypsin as a discriminating criterion between the two forms of the inhibitor. This criterion, while correct, is quite cumbersome to use and does not permit an accurate determination of the virgin-modified inhibitor distribution. The application of disc gel electrophoresis (5, 6) as a method for measuring the fractions of virgin and of modified inhibitor in a sample (2) made the problem tractable again.