A New Affinity Adsorbent for Guanyloribonuclease Guanylyl-(2′-5′)-Guanosine Coupled to Aminohexyl-Sepharose

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Abstract
Guanylyl-(2′-5′)-guanosine binds to RNase T1 in 1: 1 stoichiometry with a dissociation constant of 0.22 mM at pH 5.0 and 25°C. This nucleotidc, coupled to aminohexyl-Sepharose 4B, is able to serve as an affinity adsorbent for guanyloribonuclease [EC 3.1.4.8]. The strength of interaction between the adsorbent and various guanyloribonucleases at pH 5.0 was found to decrease in the following order: RNase N1> RNase F1> RNase T1> RNase St. The bound enzymes can be released from the adsorbent either by increase of ionic strength or by increasing the pH from 5.0 to 7.5. The interaction between RNase T1 and the adsorbent is weakened by the presence of a low concentration of 2′-, 3′-, or 5′-GMP, which are competitive inhibitors of the enzyme. RNase F1 was purified to homogeneity by use of this affinity adsorbent.