Studies on the State of Tryptophan Residue in Ribonuclease T<sub>1</sub> and Carboxymenty<sup>1</sup> Ribonuclease T<sub>1</sub>

Abstract

1) In order to investigate the state of the tryptophan residue in RNase T1 [EC 2. 7. 7. 26] and carboxymethyl RNase T1 (CMRNase T1), fluorescence emission spectra of these enzymes were measured in aqueous solutions and at various concentrations of urea and guanidine-HCl, using 295 mμ excitation light. The maxima of emission spectra of RNase T1 and CMRNase T1 were at 320 mμ which is different from 350 mμ, the maximum wavelength for N-acetyl-tryptophan. The maxima of these enzymes in 8 M urea and 7 M guanidine-HCl were, however, located at about 350 mμ. From these results, it was deduced that the tryptophan residue is buried in these protein molecules. 2) The tryptophan residue in RNase T1 or CMRNase T1 was resistant to the oxidation by NBS and to the modification by 2-hydroxy-5-nitrobenzyl bromide, while it was accessible to such chemical modifications at higher concentrations of urea or guanidine-HCl. The conversion to the accessible states took place in parallel with the shift of the fluorescence band. 3) Quenching of RNase T1 fluorescence caused by addition of various nucleotides was studied and the dissociation constants of RNase T1nucleotide complexes were determined. 4) Possible roles of the tryptophan residue in RNase T1 were discussed.