Purification of ribonuclease T1 by affinity chromatography.

The purification procedure of ribonuclease T1 was greatly improved by introducing affinity chromatography with a new adsorbent, guanosine 5'-phosphate-aminohexyl-Sepharose 4B. 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.