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 degrees 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 microl 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 microM (Na(+)); 5.62 +/- 0.98 microM (K(+)); 24.44 +/- 6.96 microM (Ca(2+)); 28.74 +/- 0.43 microM (Mg(2+)); DeltaG(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+)); DeltaH(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 DeltaS(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 DeltaG(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).