Résumé : Thiol pegylation of a protein may profoundly affect its chromatographic behavior on ion-exchange supports as a result of charge shielding effects induced by the presence of the polyethylene glycol (PEG) chain(s) at the surface of the polypeptide. When PEG chain(s) is(are) covalently bound via disulfide bonds, thiol pegylation is reversible and may be used in the context of purifying enzymes such as chymopapain, the dithiol proteinase from papaya latex, investigated here. Reaction of chymopapain with a dithiopyridyl poly(ethylene glycol) (PEG) reagent, possessing an extended spacer arm, followed by cation-exchange chromatography on S-Sepharose Fast Flow, afforded for the first time an homogeneous preparation of the native form of this proteinase. This constituted the key for obtaining highly diffracting crystals for chymopapain (as the protected S,S'-dimethytthio derivative) exhibiting diffraction spots visible up to a resolution of 1.4 Å.