Alternative reducing agents for reductive methylation of amino groups in proteins

Dimethylamine borane and trimethylamine borane were compared with sodium cyanoborohydride as reducing agents for the reductive methylation of amino groups in proteins. Dimethylamine borane performed nearly as well as cyanoborohydride; either reducing agent (15 mM) with 20 mM formaldehyde gave extensive methylation of turkey ovomucoid (5 mg/ml) in 2 h at 22 degrees. Trimethylamine borane gave equivalent levels of methylation only with a higher concentration of formaldehyde, suggesting that it is an even milder reducing agent than the other two under these conditions. With all three reducing agents, the pH optimum for methylation covered a range of pH 6-9. Methylations should be performed at the lowest possible pH to avoid side reactions of formaldehyde with the protein. Possible effects of these reducing agents on the disulfide bonds of proteins were studied. No reduction of disulfides of turkey ovomucoid was observed using each of the three reducing agents under conditions for methylation.