Carbon-13 nuclear magnetic resonance spectroscopy of [2-13C]carboxymethylcytochrome c

Horse heart cytochrome c has been carboxymethylated under various reaction conditions using [2-13C]bromoacetate. Direct analysis of reaction products using 13C nuclear magnetic resonance spectroscopy shows that the protein can be much more extensively modified than has previously been assumed. The proximity of one carboxymethylmethionine residue to the paramagnetic center of the ferric protein allows it to be distinguished from a more constant carboxymethylmethionine residue on the basis of the chemical shift of its labeled methylene group. Refolding of cytochrome c after alkylation at low pH apparently gives a different configuration of modified methionine residues within the protein compared to that produced by alkylation at neutral pH in the presence of cyanide.