A SIMPLE AND EFFICIENT METHOD FOR PREPARATION OF MONOMETHOXYPOLYETHYLENE GLYCOL ACTIVATED WITH P-NITROPHENYLCHLOROFORMATE AND ITS APPLICATION TO MODIFICATION OF L-ASPARAGINASE

An improved, simple and efficient method for preparation of monomethox ypolyethylene glycol (PEG) activated with p-nitrophenylchloroformate ( PNP-PEG) and its use as a potent modifier of protein under mild condit ions are described. Modification of bovine serum albumin with PNP-PEG was compared with that done with PEG activated with N,N'-carbonyldiimi dazole or cyanuric chloride. The reaction of PEG, activated with eithe r p-nitrophenylchloroformate or cyanuric chloride, with bovine serum a lbumin at 4 degrees C reached a plateau within 1 h, whereas protein mo dification using PEG activated,with N,N'-carbonyldiimidazole was rathe r slow and gave a low yield. The remaining activity of L-asparaginase modified with PNP-PEG was much higher than that of the enzyme modified to the same degree with PEG activated with cyanuric chloride. At a 20 molar excess of PNP-PEG having a molecular weight of 5,000, 55% of th e free amino acid groups were modified at 4 degrees C for 2 h, and the modified enzyme still had 33% residual enzyme activity. Immunochemica l studies showed that the highly modified enzyme (67% modification wit h 18% residual enzyme activity) had lost its immunogenicity and had be come much less sensitive to protease digestion.