Studies on the Preferential Specificity of Pancreatic Ribonuclease as Deduced from Partial Digests

Abstract

A two-dimensional "mapping" procedure (1) with paper electrophoresis and paper chromatography permits the separation and identification of the mono-, di-, tri-, and tetranucleotides obtained by digestion of tobacco mosaic virus ribonucleic acid with pancreatic ribonuclease (2).This procedure has also been used for the analysis of micrococcal nuclease digests of deoxyribonucleic acid (3) by the quantitative comparison of the major compounds found in incomplete as well as complete digests.Studies of this type afford a closer analysis of the hydrolytic activity of the nuclease than is possible if only the products of exhaustive digestion are examined.This report describes the investigation of partial pancreatic ribonuclease digests of ribonucleic acid from tobacco mosaic virus and from yeast.The results indicate that certain ribonuclease-sensitive bonds are cleaved much earlier in the course of digestion than others. EXPERIMENTAL PROCEDUREReagents and Materials-All spectrophotometric data were obtained in silica cells with a l-cm light path in a Beckman model DU spectrophotometer and are expressed as absorbancy (A).A Leeds and Northrup meter equipped with microelectrodes was used for the pH determinations.For paper chromatography with Whatman No. 3 MM paper, two solvents were used, Solvent A (isopropanol-concentrated NH*OH-Hz0 (7 : 1:2 by volume)) and Solvent B (40 g of (NHI)zS04 added to 100 ml of 0.1 M sodium phosphate, pH 7.0).For rechromatography, spots were located under ultraviolet light, cut out pointed at one end, and eluted with water or buffers as indicated.After chromatography with Solvent B, the separated spots were eluted in test tubes, and the ammonium sulfate removed by Norit A charcoal treatment (4) before rechromatography.Escherichia co& alkaline phosphatase (5)'