Preparation and properties of trypsin-digested ribonuclease T1 split at the single arginyl peptide bond.

Abstract

Ribonuclease T1 [EC 3.1.4.8] was selectively hydrolyzed by digestion with trypsin at the peptide bond of the single arginine residue at position 77. Selective hydrolysis was achieved by blocking the xi-amino group of Lys-41 with 2-methoxy-5-nitrotropone and subsequent digestion with trypsin in the presence of 2M urea. The trypsin-digested ribonuclease T1 was composed of two polypeptide chains containing 77 and 27 residues, though the two chains were covalently linked by a disulfide bond between Cys-6 and Cys-103. The modified enzyme lost enzymatic activity toward RNA and the ability to bind to 3'-GMP. The circular dichroic spectrum of the modified protein suggested that its conformation was extensively destroyed. It is concluded from the present results that the continuity of the peptide chain at the arginine residue is extremely important for maintaining the active conformation of the enzyme protein and for the enzymatic function of ribonuclease T1.