In vitro inhibition of alcohol dehydrogenase by acrylamide: interaction with enzyme-sh groups

Acrylamide, a reactive electrophile, caused a concentration-dependent inhibition of alcohol dehydrogenase (ADH) activity (purified) which was reversed by prior addition of glutathione. Reaction of thiol groups of this enzyme with or/Aophthalaldehyde (OPT), a fluorescent reagent, exhibited characteristic fluorescence maxima at 330 nm excitation and 420 nm emission. Addition of acrylamide to the enzyme resulted in a concentration-dependent (acrylamide and protein) quenching of fluorescence of thiol groups when compared with fluorescence quenching caused by N-ethylmaleimide (NEM), a known thiol ligand. The results demonstrate that acrylamide-induced inhibition of purified ADH activity is mediated through its specific interaction with -SH groups in the enzyme molecule.