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Evidence That the Two Binding Sites for Trypsin on Chicken Ovoinhibitor Are Not Equivalent

DISSOCIATION OF THE COMPLEXES WITH PORCINE TRYPSIN

From the ¹ From the Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Berkeley, California 94710

Complexes between chicken ovoinhibitor (OI) and porcine trypsin were allowed to dissociate in the presence of the active site titrant, p-nitrophenyl p'-guanidinobenzoate, which reacts extremely rapidly with free active trypsin (T). The rate of the slower, "postburst" reaction with titrant, which is a measure of the rate of dissociation of the complexes, showed a distinct break not observed previously with bovine trypsin (Zahnley, J. C., and Davis, J. G. (1970) Biochemistry 9, 1428–1433). The OI-trypsin complexes dissociate according to the scheme

OI-T₂ OI-T OI + T + T

With porcine trypsin, rate constants for dissociation of the OI-T₂ and OI-T complexes were 7.5 x 10^-2 and 4 to 8 x 10^-4 s^-1, respectively. The OI-T₂ complex obtained with porcine trypsin dissociated 6 to 8 times faster than that obtained with bovine trypsin, perhaps reflecting structural differences between the two trypsins. The greater ratio of dissociation rate constants, k_d, with porcine trypsin (> 100, against 9 for bovine trypsin) shows clearly that the two binding sites for trypsin on the single polypeptide chain of chicken ovoinhibitor are not equivalent. This appears to be due to intrinsic differences between the two sites, rather than to changes induced by the binding of the first trypsin molecule. An association rate constant, k_a, of 8 x 10⁴ m^-1 s^-1 for formation of the OI-bovine trypsin (1:1) complex was calculated from the inhibition constant (K_i) and the k_d. This value of k_a is lower than those previously reported for other naturally occurring trypsin inhibitors.

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