Abstract
An improved, simple and efficient method for preparation of monomethoxypolyethylene glycol (PEG) activated with p-nitrophenylchloroformate (PNP-PEG) and its use as a potent modifier of protein under mild conditions are described. Modification of bovine serum albumin with PNP-PEG was compared with that done with PEG activated with N, N′-carbonyldiimidazole or cyanuric chloride. The reaction of PEG, activated with either p-nitrophenylchloroformate or cyanuric chloride, with bovine serum albumin at 4°C reached a plateau within 1h, whereas protein modification using PEG activated with N, N′-carbonyldiimidazole was rather 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 the free amino acid groups were modified at 4°C for 2h, and the modified enzyme still had 33% residual enzyme activity. Immunochemical studies showed that the highly modified enzyme (67% modification with 18% residual enzyme activity) had lost its immunogenicity and had become much less sensitive to protease digestion.
