Carbon 13 Nuclear Magnetic Resonance Spectroscopy of Myoglobins Carboxymethylated with Enriched [2-13C]Bromoacetate

Cyanoferrimyoglobins of harbor seal and sperm whale were carboxymethylated with enriched [2-13C] bromoacetate. The enriched adducts were readily observed by 13C nuclear magnetic resonance. Resonances were identified by comparison with enriched adducts to appropriate small molecules, and T1 values were determined under various conditions. In addition to the expected adducts to the NH2-terminal residues and to histidine and lysine, an alkali-labile glycolate ester product was observed. Denatured forms were obtained by carboxymethylation in 8 m urea which led to complete conversion of all histidine residues to the dicarboxymethyl form. By carrying out carboxymethylation in stages it was possible to limit the labeling to either the normally external or the normally internal set of histidine residues. Treatment under denaturing conditions led to carboxymethylation of both methionine residues. The T1 values place some limits on the interpretation of the degrees of mobility of the adduct carbon nuclei.