Simplified method for purification of ribonuclease H from calf thymus

Abstract An improved purification procedure for the isolation of ribonuclease H(hybrid nuclease; RNA-DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) from the thymus of 4-to 6-months-old calves yields two highly active forms of the enzyme, designated as ribonuclease H1 and H2. Their isoelectric points are 5.0 +/- 0.05 and 5.25 +/- 0.05, respectively; their molecular weight, estimated from gel filtration, is in both cases 64,000 +/- 2000. On sodium dodecyl sulfate gel electrophoresis, two principal bands were identified, with molecular weights of 32,000 and 21,000. The nature of the nucleotides at the 3'-OH terminals, produced initially by the enzymic hydrolysis of hybridized RNA, was examined and shown to be a function of the divalent metal ion employed as activator.