Cysteine 66 in human butyrylcholinesterase is not free, but is blocked by a variety of sulfhydryl reactive molecules.

The 340 kDa butyrylcholinesterase (BChE) tetramer in human plasma is best known for its protective role against the toxic effects of organophosphorus pesticides and nerve agents. Each subunit is stabilized by 3 internal disulfide bridges plus one disulfide bridge between 2 subunits. These disulfide bridges account for 7 of the 8 cysteines known to be in BChE, leaving 1 cysteine that appears to be free. A free cysteine could disrupt structure and function by disulfide interchange. However, mass spectrometry analysis of purified human plasma BChE shows that the potentially free Cys66 is not free. Instead, it is covalently modified by sulfinic acid or persulfide, sulfonic acid, nitrosothiol, and thiomethyl. The absence of a dominant adduct suggests adducts are formed adventitiously from electrophiles in plasma or liver. The Cys66 adducts explain why Cys66 is unreactive with iodoacetic acid and mercury under physiologic conditions. Adducts are released from Cys66 by treatment with 10 mM dithiothreitol at 100˚C, allowing carbamidomethylation of Cys66. The adducts on Cys66 serve to protect BChE from disulfide interchange.