Abstract

Abstract Ribonucleases (RNases) possess a variety of biological activities and, under certain conditions, are deleterious. Hence, design of selective inhibitors has been suggested as a strategy for treating RNase-related disorders. In the present study, isothermal titration calorimetry was used to measure ion effects on binding thermodynamics of the RNase A competitive inhibitor 2′-CMP as a representative system. The reaction cell (37°C) contained dialyzed RNase A (0.04–0.05 mM) in buffered solution (pH 5.5) of 50 mM Na + , K + , Ca 2+ , or Mg 2+ acetate, verified spectrophotometrically. Thirty-five sequential injections (4 μl each, 3 min apart) were made of 2′-CMP (1.2 mM) in ion-matching buffer. The data were corrected for heat of dilution. There was a 1:1 interaction in each case. The estimated parameters (±S.D.) were: K d = 4.84 ± 0.29 μM (Na + ); 5.62 ± 0.98 μM (K + ); 24.44 ± 6.96 μM (Ca 2+ ); 28.74 ± 0.43 μM (Mg 2+ ); Δ G o = −7.541 ± 0.037 kcal/mol (Na + ); −7.458 ± 1.03 kcal/mol (K + ); −6.574 ± 0.173 kcal/mol (Ca 2+ ); −6.442 ± 0.009 kcal/mol (Mg 2+ ); Δ H o = −22.357 ± 1.189 kcal/mol (Na + ); −21.917 ± 0.891 kcal/mol (K + ); −20.223 ± 1.503 kcal/mol (Ca 2+ ); −26.570 ± 1.579 kcal/mol (Mg 2+ ); and Δ S o = −0.048 ± 0.004 kcal/mol-K (Na + ); −0.047 ± 0.003 kcal/mol-K (K + ); −0.044 ± 0.005 kcal/mol-K (Ca 2+ ); −0.065 ± 0.005 kcal/mol-K (Mg 2+ ). Thus, all reactions were enthalpy-driven. Despite a 5-fold difference in K d between mono- and divalent ions, the ratio of ion hydration Δ G o to K d was constant. These data should be useful for molecular modeling and suggest that inhibitor activity will be a function of cellular conditions (normal or pathological).