A NOVEL FLUOROGENIC SUBSTRATE FOR RIBONUCLEASES - SYNTHESIS AND ENZYMATIC CHARACTERIZATION

The synthesis and enzymatic characterization of DUPAAA, a novel fluoro genic substrate for RNases of the pancreatic type is described. It con sists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a flu orophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, hav e been attached by means of phosphodiester linkages. Due to intramolec ular quenching the intact substrate displayed very little fluorescence . Cleavage of the phosphodiester bond at the 3'-side of the uridylyl r esidue by RNase caused a 60-fold increase in fluorescence. This allowe d the continuous and highly sensitive monitoring of enzyme activity. T he substrate was turned over efficiently by RNases of the pancreatic t ype, but no cleavage was observed with the microbial RNase T-1. Compar ed to the dinucleotide substrate UpA, the specificity constant with RN ase A, RNase PL3 and RNase U-s increased 6-, 18-, and 29-fold, respect ively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in th e binding of elongated substrates. Studies on the interactions of RNas e inhibitor with RNase A using DUPAAA as a reporter substrate showed t hat it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.