EVALUATION OF A NEW REAGENT FOR COVALENT ATTACHMENT OF POLYETHYLENE-GLYCOL TO PROTEINS

Methoxypolyethylene glycol of molecular weight 5000 was converted to a reactive succinimidyl carbonate form (SC‐PEG). The usefulness of this new polymeric reagent for the covalent attachment of polyethylene glycol to proteins was evaluated. SC‐PEG was found to be sufficiently reactive to produce extensively modified proteins under mild conditions within 30 min, showing the highest reactivity around pH 9.3. The commonly used succinimidyl succinate derivative of methoxypolyethylene glycol (SS‐PEG) served as a reference standard to which the new reagent was compared. The stability of the polymer‐protein linkages, studied on a series of PEG‐modified bovine serum albumins, provided the single most important difference between the two activated polymers. Urethane‐linked PEG‐proteins obtained through the use of SC‐PEG showed considerably higher chemical stability than SS‐PEG‐derived conjugates. The measured rate constants of aminolysis (using N alpha‐acetyllysine) and hydrolysis showed that SC‐PEG is slightly less reactive yet more selective of the two reagents. Hydrolysis of the active groups on SC‐PEG was on average twofold slower than that on SS‐PEG. The differences in the rates of aminolysis were even smaller than those in hydrolysis. PEG‐trypsin conjugates produced by both activated polymers showed similar properties: they had no proteolytic activity, well‐preserved esterolytic activity, and enhanced activity toward p‐nitroanilide substrates. Michaelis‐Menten constants of the modified enzymes were determined using N alpha‐benzyloxycarbonyl‐L‐arginine p‐nitroanilide. These measurements indicated that the attachment of PEG to trypsin caused an increase in both the rate of turnover of the substrate and its affinity toward the modified enzymes. Through a series of experiments involving the appropriate polymeric and low‐molecular‐weight model compounds, it was demonstrated that these increases in amidolytic activity were unrelated to tyrosyl residues acylation by either one of the activated polymers. 1992 The Swiss Political Science Review