Relative Importance of Electrostatic Forces in the Interaction of Ribonuclease and Ribonucleic Acid

Recent studies on the inhibition of pancreatic ribonuclease with polyanions (1-12) indicated that competing electrostatic interaction forces predominantly determine whether the cationic enzyme interacts with the polyanionic inhibitor or whether it interacts with its high or low molecular weight anionic substrate (ribonucleic acid or nucleotide cyclic 2’) 3’-phosphate). Similar polyanionic inhibition of many other cationic enzymes has been observed (cf. for example, (8, 13)). Current studies on ribonuclease suggest that one or two histidine residues are present in the active center of this enzyme (14, 15) and that at least one of these residues must be protonated in order to bind the low molecular weight substrate (16, 17). Models of tertiary structure of ribonucleaser (18) postulate a binding region with a striking concentration of accessible cationic groups. These observations bring up the questions, to what extent are long range electrostatic forces important in the first stage of the enzyme reaction, that is, in the interaction between enzyme and its substrate (9)) and can this be studied by inhibition with polyions? We investigated the inhibition of bovine pancreatic ribonuclease activity on ribonucleic acid by using the polyanionic inhibitor, polyglucose sulfate, at different pH values and at different salt concentrations. We chose a pH range in which only the degree of ionization of ribonuclease (RNase) changed and both the polyglucose sulfate and the ribonucleic acid (RNA) were in a fully ionized form. As the pH was raised, the net positive charge of the RNase decreased, and together with this the electrostatic attraction forces between RNase and RNA and between RNase and polyglucose sulfate decreased to an equal extent. Similarly, the electrostatic attractive forces decreased at any given pH value as the size of the ion atmosphere around the polyions increased with increased salt concentration. Thus, if other than electrostatic forces participate in causing preferential interaction between RNase and RNA rather than between RNase and polyglucose sulfate, these specific forces should be revealed at higher pH values and at higher salt concentrations. This is expected to be manifested by polyglucose sulfate becoming a less potent inhibitor at high pH values and increased salt concentrations, and more polyglucose sulfate than RNA will be needed to achieve 50% inhibition of RNase activity. Previous qualitative observations have shown lower potency of the polyanionic inhibitors of RNase at high pH values (1, 2, 11, 12, 19).