DETERMINATION OF BASE SPECIFICITY OF MULTIPLE RIBONUCLEASES FROM CRUDE SAMPLES

Ribonucleases are widely found in the tissues of living organisms, but the functions of individual ribonucleases are not clear. To facilitat e characterization of individual ribonucleases, I have developed a rap id method to separate and identify each ribonuclease from a crude samp le by gel electrophoresis instead of by time-consuming purification st eps. The ribonucleases in a crude sample are first separated by RNA-ca st SDS-polyacrylamide gel electrophoresis and then eluted from the gel after ethidium bromide staining. To determine the base specificity of each ribonuclease, a 5' labelled oligonucleotide with known sequence is added to the enzyme eluate and the digested products are analyzed b y denaturing gel electrophoresis. The base specificity of bovine pancr eatic ribonuclease (RNase A), bullfrog oocyte-specific ribonuclease (R C-RNase), human serum ribonucleases and sweet potato leaf ribonuclease s were determined by this method. Other properties of individual ribon ucleases, e.g. substrate preference, may also be determined from crude samples by this method without further purification steps.