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The influence of bond chemistry on immobilized enzyme systems for ex vivo use
Author(s) -
Comfort Ann R.,
Mullon Claudy J.P.,
Langer Robert
Publication year - 1988
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260320419
Subject(s) - chemistry , agarose , cyanogen bromide , immobilized enzyme , sepharose , glutaraldehyde , covalent bond , bromide , chromatography , affinity chromatography , enzyme assay , enzyme , hydroxylamine , nuclear chemistry , organic chemistry , biochemistry , peptide sequence , gene
The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50–80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25–30%) with tresyl chloride activated agarose and oxirane activated acrylic. The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in portein leaching was reached after a few hours of incubatttion and the transient leaching period was well represented byu a logarithimic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme‐linked immunnosorbant assay (ELISA), no immune responce was detected. The transient leaching profile was shortenend by washingthe enzyme‐support conjugate with 1 M hydroxylamine, pH8.5 intermolecular cross‐linking with glutaraldehyde also improves the enzyme‐support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.