THIOL PEGYLATION FACILITATES PURIFICATION OF CHYMOPAPAIN LEADING DIFFRACTION STUDIES AT 1.4 (A)OVER-CIRCLE RESOLUTION

Thiol pegylation of a protein may profoundly affect its chromatographi c behavior on ion-exchange supports as a result of charge shielding ef fects induced by the presence of the polyethylene glycol (PEG) chain(s ) at the surface of the polypeptide. When PEG chain(s) is(are) covalen tly bound via disulfide bonds, thiol pegylation is reversible and may be used in the context of purifying enzymes such as chymopapain, the d ithiol proteinase from papaya latex, investigated here. Reaction of ch ymopapain with a dithiopyridyl poly(ethylene glycol) (PEG) reagent, po ssessing an extended spacer arm, followed by cation-exchange chromatog raphy on S-Sepharose Fast Flow, afforded for the first time an homogen eous preparation of the native form of this proteinase. This constitut ed the key for obtaining highly diffracting crystals for chymopapain ( as the protected S,S'-dimethylthio derivative) exhibiting diffraction spots visible up to a resolution of 1.4 Angstrom.