CHROMATOGRAPHIC BEHAVIOR OF MPEG-PAPAYA PROTEINASES CONJUGATES EXAMINED ON ION-EXCHANGE AND HYDROPHOBIC GEL MEDIA

The four cysteine proteinases, papain, chymopapain, caricain, and endo proteinase Gly-C were isolated and purified as the catalytically compe tent species from the commercially available latex of the tropical tre e Carica papaya L. Their free thiol function (cysteine-25), which is e ssential for activity, was protected in the form of a mixed disulfide containing a 5 kDa polyethylene glycol (PEG) chain. The second (noness ential) free thiol function (cysteine-117) of chymopapain was blocked similarly. Caricain was also derivatized through acylation of its amin o functions by PEG chains (average: 15 moles of PEG per mole of enzyme ). The chromatographic behavior of these conjugates was examined on io n-exchange and hydrophobic gels and compared to the chromatographic be havior of the unpegylated proteinases. The results indicated that char ge-shielding effects by PEG chain(s), surrounding the different protei nases, plays(play) a key role in the course of separation of pegylated and unpegylated species by ion-exchange chromatography. Similarly, PE G chain(s) is(are) able to mask hydrophobic regions on the surface of the proteinases. However, the affinity showed by PEG itself for the hy drophobic ligands immobilized on the matrix is the preponderant factor determining the behavior of the PEG-proteinases conjugates on Fractog el TSKButyl-650.