Immobilized enzymes: The coupling of biologically active proteins to ethylene-maleic anhydride copolymers of different anhydride content
✍ Scribed by Leon Goldstein
- Publisher
- Elsevier Science
- Year
- 1972
- Tongue
- English
- Weight
- 401 KB
- Volume
- 50
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Immobilized derivatives of biologically active proteins in which the protein is covalently bound to a water-insoluble polymeric carrier have found many applications as easily removable specific reagents and, in column form, for continuous processes (l-4). A commercially available polymeric acylating reagent, a 1: 1 copolymer of ethylene and maleic anhydride, EMA, has been widely used for the preparation of highly active water-insoluble polyanionic derivatives of enzymes, protein inhibitors, and antibodies (l-7). EMA reacts mainly with the a-amino groups of the lysyl residues on a protein (5,6), Experience has shown that both the binding of protein and the recovery of biological activity in the insoluble derivatives varied within rather wide limits with different commercial EMA samples, presumably due to differences in the anhydride content of the samples (5,6,8). This communication describes a method for the controlled hydrolysis of ethylene-maleic anhydride copolymers, to obtain EMA samples of well-defined anhydride content. The binding of several enzymes to EMA samples containing 30 to 85% anhydride was investigated. The conditions for optimal recovery of enzymic activity and of immobilized protein were determined.
MATER.IALS AND METHODS
Commercial samples of EMA (average molecular weight 20,000, Monsanto Company, St. Louis, Missouri) were dried by heating over phosphorus pentoxide at 110" in vacua for a few days until an anhydride content of no less than 85% was obtained. Water-saturated ether was prepared from redistilled sodium-treated ether (9) to eliminate alcohol and other impurities. Acetyl-n-tyrosine ethyl ester and benzoyl-n-arginine ethyl ester were obtained from Miles Laboratories, Elkhart, Indiana. All other reagents were of the best grade available commercially.
Controlled hydrolys& of EMA. Oven-dried EMA of anhydride content 40