ENZYMATIC-ACTIVITY OF CHYMOTRYPSIN AND ITS POLY(ETHYLENE GLYCOL) CONJUGATES TOWARD LOW AND HIGH-MOLECULAR-WEIGHT SUBSTRATES
ENZYMATIC-ACTIVITY OF CHYMOTRYPSIN AND ITS POLY(ETHYLENE GLYCOL) CONJUGATES TOWARD LOW AND HIGH-MOLECULAR-WEIGHT SUBSTRATES
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DOI：
10.1021/bc00022a007
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发表时间：
1993-07-01
影响因子：
4.7
通讯作者：
KOPECEK, J
中科院分区：
文献类型：
--
作者：
CHIU, HC;ZALIPSKY, S;KOPECEK, J
以甲氧基丁二酰亚胺碳酸酯(SC-PEG)为氨基修饰剂，制备了天然糜蛋白酶及其聚乙二醇偶联物，考察了其对低分子底物和高分子底物的活性。以三肽和四肽对硝基苯胺为底物，分别作为N(α)-苄氧基甲酰基衍生物、N-(2-羟丙基)甲基丙烯酰胺(HPMA)共聚物或聚乙二醇酯(HPMA)的N-末端，以及蛋白质白蛋白和偶氮白蛋白作为底物。每个凝乳酶分子平均含有14个聚乙二醇链的广泛修饰的胰凝乳蛋白酶(PEG14CHT)和含有10个聚乙二醇链的适度修饰的凝乳酶(PEG10CHT)都以与天然酶相似的速率降解低分子对硝基苯胺，而在PEG10CHT的情况下，降解速率高于天然酶。合成的高分子量底物和偶氮白蛋白也被聚乙二醇改性的酶降解。然而，聚乙二醇酶的这种酶反应速率比天然的要低。与偶氮白蛋白相比，天然白蛋白可以抵抗两种PEGCHTs的降解，但很容易被天然的糜蛋白酶消化。所获得的结果表明，聚乙二醇酶的底物大小依赖的专一性不能仅仅用空间位阻来解释。聚乙二醇酶对蛋白质底物活性的降低与聚乙二醇酶将蛋白质从周围排除的能力以及蛋白质展开对降解敏感性的影响是一致的。
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.