Accelerator mass spectrometry for assaying irreversible covalent modification of an enzyme by acetoacetic ester

Protein modification (sometimes known as crosslinking) often requires two or more steps to affix a small molecule irreversibly. Two-step reductive alkylation of the enzyme rabbit muscle aldolase with ethyl 3-C-14-acetoacetate and sodium cyanoborohydride attaches less radioactivity than with cyanoborohydride omitted. The C-14 level incorporated into aldolase corresponds to only about 15-30 modified protein molecules per million. Accelerator mass spectrometry (AMS) provides the only technique currently available for investigating the shorter chains from CNBr-cleavage of modified aldolase. Examination of individual fragments reveals that reductive alkylation of the active site lysine in the presence of cyanoborohydride (+BH3CN) is negligible when compared with the extent of covalent modification in the absence of cyanoborohydride (-BH3CN). Labeling by ethyl acetoacetate cannot result from simple acetoacetylation, because dialysis with hydroxylamine does not wash it out. The amount of C-14 incorporated from ethyl 3-C-14-acetoacetate without cyanoborohydride is roughly proportional to the number of tyrosine residues in each CNBr-fragment, and we surmise that ethyl acetoacetate attaches irreversibly via a reaction specific to that amino acid. Cyanoborohydride inhibits this reaction, but appears to diminish the susceptibility of the active site tyrosine (which is close to a lysine in the tertiary structure of aldolase) less than other tyrosine residues. (Int J Mass Spectrom 179/180 (1998) 185-193) (C) 1998 Elsevier Science B.V.