Effect of water-soluble phospholipid polymers conjugated with papain on the enzymatic stability.
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.
Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, 113-8656, Tokyo, Japan.
Daisuke
D
Miyamoto
Junji
J
Watanabe
Kazuhiko
K
Ishihara
D001672:Biocompatible Materials / Q000138:chemical synthesis / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry; D008422:Materials Testing; D008689:Methacrylates / Q000737:chemistry*; D010206:Papain / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D011109:Polymethacrylic Acids; D011485:Protein Binding; D012995:Solubility; D014867:Water / Q000737:chemistry*
D001672:Biocompatible Materials; D004800:Enzymes, Immobilized; D008689:Methacrylates; D010743:Phospholipids; D011108:Polymers; D011109:Polymethacrylic Acids; C115766:poly(2-methacryloyloxyethyl-phosphorylcholine); D014867:Water; D010767:Phosphorylcholine; D010206:Papain