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Labeling of proteins by reductive methylation using sodium cyano borohydride

Labeling of proteins by reductive methylation using sodium cyano borohydride

Journal of Biological Chemistry 254(11): 4359-4365

The free amino groups of proteins can be converted to the N,N-dimethyl derivatives by reaction with formaldehyde and a reducing agent such as NaBH4. The secondary and tertiary amines formed in the reaction retain the charge of the parent amines with only small differences in pK values. Substitution of the NaCNBH3 (which reduces Schiff bases but not aldehydes) for NaBH4 increases labeling efficiency 3-fold and eliminates the side reactions caused by NaBH4. NaCNBH3 is more stable to acidic conditions; reactions can be carried out at pH values as low as 6 rather than at pH 9 or above as required when NaBH4 is used. Nearly complete conversion of amino groups to the dimethyl derivatives occurs at molar ratios of formaldehyde to amine of 6 or greater; at equimolar ratios, as much as 70% of the added formaldehyde is covalently linked to protein. The specificity of the reaction was examined by several methods including studies with model compounds and 13C NMR spectroscopy of proteins 3H labeled with [13C]formaldehyde. The only groups labeled by this method were the .epsilon.-amino groups of lysyl residues and the .alpha.-NH2 terminus. No evidence was found for reduction of disulfides by NaCNBH3 or for cross-linking by formaldehyde. This procedure can be used to introduce methyl groups labeled with 13C, 14C or 3H into proteins. Labeling with 13C-enriched formaldehyde enables the study of perturbations in the chemical environment of lysyl residues by 13C NMR spectroscopy. With radioactive foramldehyde high specific activities with minimal change in protein structure can be attained.

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Accession: 005787042

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PMID: 571437

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