Enzymatic method with polyphenol oxidase for the determination of cysteine and N-acetylcysteine

Thiols, such as cysteine and N-acetylcysteine, are included in many pharmaceutical products for their mucolytic properties. The method described here uses mushroom polyphenol oxidase (PPO) to determine two thiols and consists of measuring the lag period in the formation of the product generated as PPO acts on o-diphenol in the presence of a thiol. In the experimental conditions, o-quinone is formed enzymatically and then reacts stoichiometrically with the thiol, originating the corresponding thiol-diphenol adduct, which does not absorb visible light. Once the thiol has been used up, the o-quinone can be observed in the medium. It must be borne in mind that the inhibition of PPO is practically null at low concentrations of thiol, and the only effect observed is the formation of the thiol-diphenol adduct. In the following, an exact kinetic method capable of rapidly and accurately assaying thiols with PPO and o-diphenol is optimized and is shown to be a straightforward way of calculating thiol concentration. The method has been successfully applied to the determination of cysteine in model solutions and of N-acetylcysteine in pharmaceutical products.