A study on the glutaraldehyde activation of hydrophilic gels for immobilized enzymes

Abstract

One of the techniques for fixing enzymes is to link enzymes chemically to a water-insoluble support through the reaction of spacers. Because a variety of well-designed supports have become available, this method is better suited for obtaining stable immobilized enzymes than other method. Particularly, size-exclusion chromatography gels, which are spherical microparticulates and have pressure durability, are interesting supports because immobilized enzymes can be prepared in packed columns to use under high pressure. In the present study, glutaraldehyde, one of the most frequently used spacers,'-5 was used to activate size-exclusion chromatography gels, G6000PW (rigid gel) and Toyopearl (semirigid gel) (Toyo Soda, Tokyo, Japan), synthesized from glycidylmethacrylate as a monomer. These gels have been reported to have little interaction with proteins.6 EXPERIMENTAL MaterialsBovine serum alubumin (BSA) and pronase were purchased from Sigma Chemical Co. (St. Louis, MO) and Kaken Kagaku (Tokyo, Japan), respectively. G6000PW and Toyopearl size-exclusion chromatography gels used as matrices of fixation of these proteins were obtained from Toyo Soda (Tokyo, Japan). Glutaraldehyde (25%) and all other chemicals were purchased from Nakarai Chemicals (Kyoto, Japan). Pure glutaraldehyde was prepared by saturating commercial glutaraldehyde solution with NaCl, performing an extraction with ether, and distilling the extract at 71°C in vacuo (10 mmHg). An aqueous pure glutaraldehyde solution had a single absorbance at 280 nm. Water was purified by a Milli R/Q water purifier (Millipore, Bedford, MA). Glutaraldehyde Activation of SupportsG6000PW and Toyopearl were modified as follows. Both of the gels were tosylated in pyridine by the conven-* To whom all correspondence should be addressed. tional method. These gels were washed with water, dilute sulfuric acid, and sodium carbonate, and then were allowed to swell in acetonitrile. The tosylated G6000PW and Toyopearl were aminated with hexamethylenediamine, namely G6000PW-HMDA and TP-HMDA. The overall reaction is represented by R-OH (gel) + p yridine Subsequently, the aminated samples were activated with glutaraldehyde. Assay for Fixed PronaseThe unit of pronase activity was determined by the casein-Folin color m e t h~d .~ One power unit (PU), used in this study, was defined to cause the formation of one micromole tyrosine per minute by one milliliter wet immobilized pronase. RESULTS AND DISCUSSIONUsing G6000PW-HMDA (0.5 mL, wet) and commercial glutaraldehyde (0.25%, 20 mL), the variation of the absorbance (235 nm) of the reaction mixture during the activation with glutaraldehyde was measured at various times. The results obtained are shown in Figure 1. As is seen in Figure 1, the absorbance decreased as a function of time, reached the minimum value, and then increased. The reaction time to reach the minimum absorbance was almost the same (ca. 2 h) for both gels. In order to explore the reason for this unique change, pure glutaraldehyde was used instead. When the pure glutaraldehyd...

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