Fluorometric assay for ADP-ribosylarginine cleavage enzymes

A continuous fluorometric assay for enzyme activities which remove ADP-ribose linked to proteins at arginine was developed. The substrate analog, N alpha-dansyl-N omega-(1,N6-etheno-ADP-ribosyl)arginine methyl ester, was used to assay the catalytic activities of dinitrogenase reductase activating glycohydrolase from Rhodospirillum rubrum and nucleotide pyrophosphatase from Crotalus adamanteus. The assay is based on the increase in fluorescent emission by ethenoadenine accompanying the enzyme-catalyzed hydrolysis of the substrate. The assay has been used to detect activities of 10 fmol substrate cleaved per minute. The substrate anomerizes to give a 40:60 equilibrium of alpha:beta ribosylguanidinium anomers, allowing the determination of enzyme stereospecificity. The substrate was used to determine the kinetic parameters and products of the N-glycohydrolase and the pyrophosphatase.