Abstract
The covalent attachment of activated polyethylene glycol2 (PEG2) of 10, 000 daltons to nonessential groups on a serine proteinase II (SepII) from Aspergillus sojae produced two modified preparations (PEG2-SepII-S and PEG2-SepII-L). The molecular weights of PEG2-SepII-S and PEG2-SepII-L were about 170, 000 and 280, 000, respectively. The PEG2-SepII-S lost about 80% of its antigenicity, while the PEG2-SepII-L completely lost its antigenicity. In comparison of kinetic parameters with SepII there was less than 40% variation in Km, but the values of Kcat towards succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine 4-methylcoumaryl-7-amide (Suc-LLVY-MCA) or succinyl-Lalanyl-L-alanyl-L-valyl-L-alanine p-nitroanilide (Suc-AAVA-pNA) decreased to about 70% less than that of SepII. The modified preparations have about 20 % activity towards fibrin hydrolysis and a low affinity for a protein proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), with a molecular weight of 23, 000, while the preparations have high affinity for a low molecular weight microbial inhibitor, chymostatin. The stoichiometry of the reaction of α2-macroglobulin (α2M) with PEG2-SepII-S showed that PEG2-SepII-L bound to α2M in a molar ratio of 1:1. No appreciable differences were observed in the pH stabilities of the modified enzymes and the native one at pH 3.6, while the modified enzymes were more stable than that of the native one at pH 11.5. The two modified preparations were labile at 50°C, but the native enzyme was completely stable at 50°C.
