SYNTHESIS OF RIBONUCLEASE LABELED WITH C-13 ON METHIONINE-29

The Cε{lunate} methyl group of methionine-29 of RNAase was enriched with 13C. The synthesis involved the reaction of RNAase with 13CH3I at pH 4. S-Methylmethionine-29 RNAase was recovered in 80% yield. This sulfonium derivative was subsequently demethylated with 0.1 M mercaptoethanol at pH 8.5, 25°C for 4 days. These conditions allowed the demethylation reaction to successfully compete with the reaction of the thiol with the four disulfide bridges in RNAase. After dialysis, concentration and chromatography, native RNAase with approx. 50% of its Met29 methyl groups enriched in 13C was recovered as was unreversed S-Methylmethionine-29 RNAase. Both proteins showed full enzymatic activity toward cytidine 2′:3′-cyclic monophosphate. 13C-methyl signals from enriched RNAase and the sulfonium derivative were observed at 13.8 and 26.7 ppm from TMS respectively. Preliminary denaturation studies with the methylated protein suggest that 13C enrichment of methionine methyl groups in RNAase will be a useful technique for following the unfolding transition at these sites of the protein. © 1979.