30b] Ribonuclease T2 from Taka-Diastase

Abstract

This chapter discusses the preparation of T2 from Taka-Diastase. The amount of RNase T2 contained in Taka-Diastase is variable. It appears from DEAE-cellulose column chromatography of water extracts of Taka-Diastase (pH 7.5 activity: pH 4.5 activity = 2.3) that less than 20% of the total activity at pH 4.5 in crude extracts is responsible for RNase T2-A and T2-B. RNase T2 thus obtained is purified about 1000-fold and is colorless and homogeneous on chromatography, electrophoresis, and sedimentation analysis. About 80% of the RNase T2 activity is recovered in samples stored frozen for several months at neutrality; repeated freezing and thawing of enzyme solutions leads to loss of activity. About 15% inactivation is observed on lyophilization at pH 6.0. RNase T2 is inhibited by heavy metal ions, especially Cu++, but only slightly by Mg++ and Ca++. Most purified RNase T2 preparations are not activated by EDTA as consistently as are partially purified enzyme preparations. RNase T2 splits preferentially the internucleotide bonds between the 3'-adenylic acid group and the 5'-hydroxyl group of adjacent nucleotides in RNA, with the intermediate formation of adenosine 2', 3'-cyclic phosphate, and consequently the secondary phosphate ester bond of other nucleotides in RNA via the nucleoside 2', 3'-cyclic phosphate.