J. Biochem, 1977, Vol. 81, No. 4 1031-1039
© 1977 Japanese Biochemical Society
research-article |
Circular Dichroism Studies on the N-Bromosuccinimide Oxidation of Ribonuclease from Aspergillus saitoi
Department of Microbiology, Hoshi College of Pharmacy Ebra, Shinagawa-ku, Tokyo 142
The NBS oxidation of RNase M, which is base non-specific, was studied mainly by circular dichroism spectroscopy. It was found that tryptophan residues of RNase M were resistant to NBS at pH's from 2.5 to 6.0, but were oxidized at pH 2.0. The CD spectrum of the native RNase M gave sharp negative bands at 298 nm (band A) and 289 nm (band B) and a very complex broad band around 280–265 nm.
On increasing addition of NBS, band A decreased first, then band B. The decrease in band A seemed to parallel the decrease of enzymatic activity. The wavelengths of bands A and B indicated that these bands were due to tryptophan residues in RNase M. The amino acid composition of the NBS-oxidized RNase M indicated that, in the early stage of NBS oxidation, only tryptophan residues were lost and little decrease of tyrosine residues was observed.
In 8 M urea, RNase M was resistant to NBS at pH's between 4.5–6.0. However, at pH 4.0, tryptophan residues of RNase M were susceptible to NBS. The mode of oxidation of RNase M was different from that in an aqueous solution, that is, band A and B decreased in parallel at the early stage of oxidation.
The changes of UV and CD spectra of RNase M in 8 M urea were measured as a function of pH. A marked transition of the spectra was observed at pH 4.0. A similar experiment in aqueous solution gave a marked transition of the CD difference spectrum at pH 2.0. These results suggest that in the presence and absence of 8 M urea, a conformational change in RNase M induced by decrease in pH rendered tryptophan residues in RNase M susceptible to NBS.
In the early stage of NBS oxidation, the gross structure of the enzyme was very similar to that of the native enzyme as regards the CD spectrum at 230–210 nm, but the conformations around tyrosine residues were affected.