[Show abstract][Hide abstract]ABSTRACT: Papain is widely used in several laboratories as a reference enzyme and, as such, needs to be highly pure. A one-step purification of the enzyme is described here starting from spray-dried papaya latex. After protection of the essential SH group with a monomethoxypolyethylene glycol derivative synthesised in our laboratory, the mixture of papaya enzymes is submitted to fractionation on S-Sepharose Fast Flow. This procedure leads to the isolation of the S-pegylthiopapain conjugate devoid of any contamination nor by the enzyme's irreversibly oxidized form nor by one of the other proteinases present in the original mixture. Thereafter, S-pegylthiopapain may be quite easily converted into its fully active form.