Use of polynucleotide/polyacrylamide-gel electrophoresis as a sensitive technique for the detection and comparison of ribonuclease activities

Abstract

A technique is described in which the incorporation of a polynucleotide substrate into the matrix of a polyacrylamide gel allows the use of electrophoresis for the detection of polycationic ribonuclease activity rather than simply the presence of protein. Because use is made of the catalytic properties of ribonucleases, polynucleotide/polyacrylamide-gel electrophoresis is apparoximately 10(5) times more sensitive for the detection of these enzymes than conventional gel electrophoresis with the use of protein-staining dyes. Initial studies showed that the poor migration, in the gels, of highly charged polycationic ribonucleases in the presence of negatively charged synthetic polynucleotides could be overcome by high concentrations of spermine. The positively charged polyamine, by neutralizing the polyanionic polynucleotide, enabled these basic enzymes to migrate considerable distances in the gel. Electrophoresis of the RNAases under conditions of low pH, and incubation of the gel at neutral pH followed by staining for polynucleotide, resulted in coloured gels containing clear bands that define regions of enzyme activity. Alterations in spermine concentration or substrate identity caused changes in the positions of these bands, suggesting a dynamic interaction among the enzyme, polyamine and polynucleotide. Because of the advantages, in terms of selectivity and sensitivity of polynucleotide/polyacrylamide-gel electrophoresis, this technique was used to demonstrate the nuclease homogenity of three purified bovine muscle enzymes, and to compare these enzymes with each other, as well as with bovine pancreatic ribonuclease A.

Isoelectric focusing—polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities

“…In the latter instance, movement of all positively charged proteins through the network of negatively charged DNA in the slab gel is not possible, owing to the tight electrostatic binding between polypeptide and polynucleotide. As described previously (Karpetsky et al, 1980) this type of interaction may be overcome by titrating the negatively charged polynucleotide with an excess of a low-Mr polycation such as spermine, al-though some streaking is sometimes observed (Figs. 4b and 4c).…”

The ribonucleases of bovine skeletal muscle

“…Our purification, however, gave considerable increases in specific activity (more than 40000-fold for RNAases I and III). Furthermore, each RNAase is free of contamination by other RNAases, since each gave a single area of activity after gel electrophoresis or polynucleotide/polyacrylamide-gel electrophoresis [see the Experimental section and the following paper (Karpetsky et al, 1980)1 and a single symmetrical peak of activity on sucrose gradients (results not shown).…”

“…Although limited quantities of each RNAase, in terms of protein, were available after a lengthy purification procedure, the specific activities of the enzymes were high. In addition, results obtained by the use of polynucleotide/polyacrylamide-gel electrophoresis, a technique described in the following paper (Karpetsky et al, 1980), demonstrated the RNAase homogeneity of each of the purified enzymes.…”