Increased activity and stability of poly(ethylene glycol)-modified trypsin.

The reaction of trypsin with activated monomethoxypoly(ethylene glycol) with various molecular masses led to the development of a series of poly(ethylene glycol)-modified trypsins (PEG-trypsins). On determining the catalytic properties of PEG-trypsin using N-benzoyl-L-arginine p-nitroanilide as a substrate, a three- to fourfold increase in the maximal velocity of hydrolysis was found to occur, whatever the size of the PEG moiety used. PEG-trypsin with higher molecular mass moieties showed lower Michaelis constant values. The activation of trypsin was neither reversed by nucleophiles such as hydroxylamine, nor prevented when modification was carried out in the presence of benzamidine or in the presence of the polypeptidic soybean trypsin inhibitor. Chemical modification of about 80% of the free amino groups with PEG chains significantly improved the resistance to heat and detergents. This might result from the formation of a highly hydrogen-bonded structure around the enzyme.