Enzymatic activity of chymotrypsin and its poly(ethylene glycol) conjugates toward low and high molecular weight substrates.

Native chymotrypsin and its polyethylene glycol (PEG) conjugates, obtained using the succinimidyl carbonate of methoxy-PEG (SC-PEG) as the amino group modifying reagent, were tested for their activity toward several low and high molecular weight substrates. Tripeptide and tetrapeptide p-nitroanilides either as N alpha-benzyloxycarbonyl derivatives or attached via their N-terminals to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers or to PEG, as well as the proteins albumin and azoalbumin, were used as substrates. Extensively modified chymotrypsin bearing on average of 14 PEG chains per chymotrypsin molecule (PEG14CHT) and a moderately modified one containing 10 PEG chains (PEG10CHT) both degraded the low molecular weight p-nitroanilides at rates comparable to, and in the case of PEG10CHT, greater than, the rates exhibited by the native enzyme. Synthetic high molecular weight substrates and azoalbumin were also degraded by the PEG-modified enzymes. However, rates of such enzymatic reactions were lower for the PEG-enzymes than for the native one. Native albumin, as compared to azoalbumin, resisted degradation by both PEGCHTs yet was readily digested by the native chymotrypsin. The results obtained indicate that substrate-size-dependent specificity of PEG-modified enzymes cannot be explained solely by steric hindrance considerations. The decreased activity of PEG-enzymes toward protein substrates is consistent with the well-documented ability of PEG to exclude proteins from its surroundings and with the influence of protein unfolding on the susceptibility to degradation.