Identification of functional arginine residues in ribonuclease A and lysozyme.

A specific color reaction has been developed for the detection of N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine-containing peptides. The reaction is based on the fact that hydroxylamine converts the blocking group to cyclohexanedione dioxime, which forms a red nickel complex. N-7, N-8-(1,2-dihydroxycyclohex-1,2-ylene)-L-arginine-containing peptides can also be detected by diagonal electrophoresis from the change of electrophoretic mobility of these peptides on interaction of the blocking group with borate. Since the modified arginine residues are resistant to tryptic cleavate, changes in tryptic peptide patterns can also be utilized to identify the presence of modified arginine residues. A combination of these approaches was used to identify the arginine residues modified by cyclohexanedione treatment. Bovine panctreatic RNase A loses approximately 90% of its activity on cyclohexanedione treatment with the modification of 2 to 3 arginine residues. Arginine-39 reacts most rapidly and its modification contributes most to inactivation of the enzyme. Arginine-85 also reacts rapidly with cyclohexanedione. Arginine-10 reacts slowly and no reaction was observed with arginine-33. Removal of the blocking groups by hydroxylamine treatment resulted in complete recovery of enzyme activity in samples where arginine-39 and arginine-85 had been modified, whereas 80% of activity was regained from samples where arginine-10 had also been modified. With egg white lysozyme, all 11 arginine residues react with cyclohexanedione, resulting in partial inactivation of the enzyme. The fully modified enzyme retains 35% of its activity. Since arginine residues are important for electrostatic interaction between the enzyme and the negatively charges cell surface, even the modified, basic residues can provide the necessary positive charges. In the presence of borate, activity is almost completely abolished, since the modified arginine-borate complex has a reduced net positive charge. Upon removal of the blocking groups by hydroxylamine, even the fully modified lysozyme regains complete activity. With the exception of the most reactive arginine (residue 5), modification of all other arginine residues contributes equally to inactivation of the enzyme. The possible reason for the importance of arginine-5 in maintaining activity is discussed. Advantages of the present method for the selective reversible modification of arginine residues of proteins and for the identification of reactive arginine residues are evaluated.