Tight-binding inhibition of angiogenin and ribonuclease A by placental ribonuclease inhibitor

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Abstract
The dissociation rate constant of the angiogenin-placental ribonuclease inhibitor complex was determined by measuring the release of free angiogenin from the comples in the presence of scavenger for free placental ribonuclease inhibitor (PRI). In 0.1 M NaCl, pH 6, 25 .degree.C, this value is 1.3 .times. 10-7 s-1 (t1/2 .simeq. 60 days). The Ki value for the binding of PRI to angiogenin, calculated from the association and dissociation rate constants, is 7.1 .times. 10-16 M. The corresponding values for the interaction of RNase A with PRI, determined by similar means, are both considerably higher: the dissociation rate constant is 1.5 .times. 10-5 s-1 (t1/2 = 13 h), and the Ki value is 4.4 .times. 10-14 M. Thus, PRI binds about 60 times more tightly to angiogenin than to RNase A. The effect of increasing sodium chloride concentration on the binding of PRI to RNase A was explored by Henderson plots. The Ki value increases to 39 pM in 0.5 M NaCl and to 950 pM in 1 M NaCl, suggesting the importance of ionic interactions. The mode of inhibition of RNase A by PRI was determined by examining the effect of a competitive inhibitor of RNase A, cytidine 2''-phosphate, on the association rate of PRI with RNase A. Increasing concentrations of cytidine 2''-phosphate decrease the association rate in manner consistent with a competitive mode of inhibition.

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