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+, Ca2+, or Mg2+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: Kd = 4.84 ± 0.29 μM (Na+); 5.62 ± 0.98 μM (K+); 24.44 ± 6.96 μM (Ca2+); 28.74 ± 0.43 μM (Mg2+); ΔGo = −7.541 ± 0.037 kcal/mol (Na+); −7.458 ± 1.03 kcal/mol (K+); −6.574 ± 0.173 kcal/mol (Ca2+); −6.442 ± 0.009 kcal/mol (Mg2+); ΔHo = −22.357 ± 1.189 kcal/mol (Na+); −21.917 ± 0.891 kcal/mol (K+); −20.223 ± 1.503 kcal/mol (Ca2+); −26.570 ± 1.579 kcal/mol (Mg2+); and ΔSo = −0.048 ± 0.004 kcal/mol-K (Na+); −0.047 ± 0.003 kcal/mol-K (K+); −0.044 ± 0.005 kcal/mol-K (Ca2+); −0.065 ± 0.005 kcal/mol-K (Mg2+). Thus, all reactions were enthalpy-driven. Despite a 5-fold difference inKd between mono- and divalent ions, the ratio of ion hydration ΔGo toKd 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).
Footnotes
- Abbreviations:
- RNase
- ribonuclease
- ang
- angiogenic
- ΔhydGo
- free energy change of hydration
- ITC
- isothermal titration (micro)calorimetry
- npt
- nonpancreatic-type
- pt
- pancreatic-type
- RI
- 50-kDa protein ribonuclease inhibitor
- Received November 29, 2001.
- Accepted February 12, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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