Chemical fixation of chymotrypsin to water-insoluble crosslinked dextran (Sephadex) and solubilization of the enzyme derivatives by means of dextranase
✍ Scribed by Rolf Axén; Per-Åke Myrin; Jan-Christer Janson
- Book ID
- 102762306
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 1970
- Tongue
- English
- Weight
- 801 KB
- Volume
- 9
- Category
- Article
- ISSN
- 0006-3525
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✦ Synopsis
Abstract
Catalytically active chymotrypsin derivatives can be synthesized from cyanogen bromide‐activated Sephadex G200. In most cases the apparent catalytic activity of the covalently fixed enzyme appears to be considerably decreased in comparison to the activity of the free enzyme. However, by proper choice of the reaction conditions for the activation, enzyme conjugates with high activity, even toward a high molecular substrate, can be synthesized. These latter derivatives may be of practical value for the digestion of proteins. Crosslinked dextran as carrier was chosen because of the possbility, of digesting it enzymatically by dextranase. Sephadex G200, if activated at or below pH 10.3, will combine with chymotrypsin to yield digestable products. Changes of apparent kinetic properties of the fixed enzyme can accordingly be studied during the degradation process. On the solubilization of the insoluble conjugate, a total recovery of activity of the fixed enzyme can be obtained in cases the carrier has been activated by a sufficiently mild procedure. The high apparent Michaelis constant K~m~ of insoluble chymotrypsin–Sephadex toward N‐acetyl‐L‐tyrosine ethyl ester shifts back on solubilization to the value of free chymotrypsin. We therefore propose that the decreased activity of an insoluble chymotrypsin–Sephadex is due to diffusional effects shown by the gel matrix toward the substrate. Similarly observed shifts in optimum pH are explained by accumulation of hydrogen ions in the gel. The organic chemical reaction used for coupling the enzyme to the polymer can therefore be performed without decreasing the inherent catalytic activity of the enzyme. The route described for fixing chymotrypsin to Sephadex followed by solubilization of the products may be useful as a synthetic method for binding proteins, peptides, and other amino group‐containing substances to soluble carriers, e.g., for the modification of pharmaceuticals.