An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. I. Purification and properties of two forms of RNase U2.

Abstract

An affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, was prepared by the periodate oxidation of AMp followed by coupling and condensation with amino-hexyl-Sepharose 4B. RNase U2, a purine-specific RNase, was specifically bound to this adsorbent at pH 4.5 and eluted critically at pH 5.9 in the presence of 1 M NaCl, corresponding to the pH dependence of the binding of 2'-AMP to RNase U2. By using this affinity chromatography as a main tool, a simplified and effective purification method for RNase U2 was established with a high yield of 58%. Another form of RNase U2 with low specific activity, named RNase U2-B, was eluted at a slightly higher pH from this adsorbent. RNase U2-B was indistinguishable from the original enzyme (RNase U2-A) in base specificity, affinity for ApA, molecular weight and amino acid composition, but was clearly different in specific activity, molecular activity for ApA, isoelectric point and conformation of molecule. This affinity adsorbent is also effective for the detection or isolation of small amounts of base-specific RNases in crude cell extract.