[Modification of RNA ligase histidine residues by diethylpyrocarbonate]

Abstract

The practicality of Tris-HCl buffer for modification of histidine residues by diethylpyrocarbonate (DEPC) was studied using a model protein-hexokinase. It was found that modification was selective at pH 7.5. Conditions for modification of one histidine residue in the protein molecule were specified. In 30 mM Tris-HCl buffer pH 7.5, 10-min interaction of RNA-ligase with 0.3 mM DEPC was accompanied by modification of one histidine residue, as a result of which the ability to form a covalent AMP-RNA-ligase complex decreased 3 times. Modification of two histidine residues of RNA-ligase resulted in a complete loss of the enzyme activity. At increasing DEPC concentration modification affected all of the seven histidine residues of RNA-ligase. The kinetic parameters (Km and V) for the native and modified enzymes were determined and compared.