Miyamoto Daisuke, Watanabe Junji, Ishihara Kazuhiko
Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, 113-8656, Tokyo, Japan.
Biomaterials. 2004 Jan;25(1):71-6. doi: 10.1016/s0142-9612(03)00474-5.
To maintain enzymatic activity during long-term storage by conjugation with water-soluble 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers (PMPC-COOH) having various molecular weights with a carboxylic group on the terminal, such compounds were synthesized as a polymer modifier using a photoinduced living radical polymerization technique. A poly(ethylene oxide) with a carboxyl group (PEO-COOH) was used as the control. The PMPC-COOHs were reacted with the amino groups of the enzyme, papain, via amide bonds. With an increase in the molecular weight in the range between 5 and 20K of the PMPC-COOH, the modification degree and alpha-helix content of the conjugated papain slightly decreased, but the remaining enzymatic activity did not depend on the molecular weight of the PMPC-COOH. However, when a much higher molecular weight PMPC-COOH (40K) was conjugated with a reduction in the modification degree, alpha-helix content was higher compared with the other PMPC-conjugated papain. Modification with PEO-COOH showed little reduction of the alpha-helix content of papain. The time dependence of the remaining enzymatic activity of the polymer-conjugated papains was evaluated during storage at 40 degrees C. The native papain diminished activity within one week. PEO-conjugated papain had decreased activity with time, but after one week it had half its initial level. The same tendency was observed when papain was modified with PMPC-COOHs 5 and 40K, that is, the enzymatic activity did not decrease even when they were stored for 4 weeks. We concluded that the PMPC chain could stabilize the enzyme by control of the molecular weight of the PMPC and modification degree to the enzyme.
为了通过与具有不同分子量且末端带有羧基的水溶性2-甲基丙烯酰氧基乙基磷酰胆碱（MPC）聚合物（PMPC-COOH）共轭来在长期储存期间保持酶活性，使用光引发活性自由基聚合技术合成了此类化合物作为聚合物改性剂。将带有羧基的聚环氧乙烷（PEO-COOH）用作对照。PMPC-COOH通过酰胺键与木瓜蛋白酶的氨基反应。随着PMPC-COOH分子量在5至20K范围内增加，共轭木瓜蛋白酶的修饰度和α-螺旋含量略有下降，但剩余酶活性并不取决于PMPC-COOH的分子量。然而，当更高分子量的PMPC-COOH（40K）共轭且修饰度降低时，与其他PMPC共轭木瓜蛋白酶相比，α-螺旋含量更高。用PEO-COOH进行修饰显示木瓜蛋白酶的α-螺旋含量几乎没有降低。在40℃储存期间评估了聚合物共轭木瓜蛋白酶剩余酶活性的时间依赖性。天然木瓜蛋白酶在一周内活性降低。PEO共轭木瓜蛋白酶的活性随时间降低，但一周后其活性为初始水平的一半。当木瓜蛋白酶用5K和40K的PMPC-COOH修饰时观察到相同趋势，即即使储存4周它们的酶活性也没有降低。我们得出结论，PMPC链可以通过控制PMPC的分子量和对酶的修饰度来稳定酶。
