Stabilization of enzymes by multipoint covalent attachment to agarose-aldehyde gels. Borohydride reduction of trypsin-agarose derivatives

The process of borohydride reduction of one-point-attached and multipoint-attached trypsin (amine)-agarose (aldehyde) derivatives has been studied. We have tested the effect of different variables that control the intensity of this reduction process on the activity and stability of the resulting derivatives. In this way, we have been able to establish the optimal conditions for this process: 1 mg ml−1 borohydride, pH 10.0, 25°C, 30 min of reaction time, and presence of benzamidine (competitive inhibitor of trypsin). In these conditions, the reduction of Schiff's bases formed between the enzyme and the support is very intense, the remaining aldehyde groups on the support are reduced completely, and the deleterious effects of borohydride on trypsin structure are negligible. We have also studied these deleterious effects of borohydride when reductions were performed in more drastic experimental conditions. A broad range of experimental conditions seems to be useful to test the reduction of a broad spectrum of multipoint-attached enzyme (amine)-support (aldehyde) derivatives.