Purification of Ribonuclease T₁ by Affinity Chromatography

Abstract

The purification procedure of ribonuclease T₁ was greatly improved by introducing affinity chromatography with a new adsorbent, guanosine 5'-phosphate-aminohexyl-Sepharose 4 B. The enzyme was purified by only four steps with a high yield (68%) from Taka-Diastase powder. The purified enzyme preparation gave a single peak of protein with a small shoulder on DEAE-cellulose column chromatography. The peak fraction, amounting to approximately 90% of total proteins, was homogeneous ribonuclease T₁. Moreover the shoulder fraction was shown to contain another form of ribonuclease T₁ electrophoretically distinguishable from the original one. Comparison of the properties of the fraction containing almost equal amounts of both components with those of original ribonuclease T₁ shows that the other form of T₁ is identical with the original one in respect to amino acid composition and base specificity. We propose to designate this new form and original one as ribonuclease T₁ -B and T₁ -A, respectively.