Development and characterization of a polymer gel with an immobilized enzyme to measure L-glutamate.
Glutamate dehydrogenase (GDH) is used in an enzyme electrode to measure L-glutamate. GDH is covalently immobilized in a hydrophilic, permeable, and semirigid gel produced by the copolymerization of polyacrylamide and N-acryloxysuccinimide. Experimental conditions necessary to retain GDH in the gel with high efficiency and minimum denaturation are optimized. The abilities of enzymatic cofactors and coenzymes, NADH, NAD, ATP, ADP, GTP, and ZnCl2, to protect the enzyme during immobilization are explored. Under optimum experimental procedures an enzyme-containing gel is produced that is reproducible and long lasting in its functional behavior. The gel responds to the presence of L-glutamate with high velocity, the delay being less than 500 ms; high specificity, being 1000-fold more responsive to L-glutamate than D-glutamate, D- or L-aspartate, and N-acetylhistidine; and high sensitivity, a concentration of about 3 microM can be measured.
D000180:Acrylic Resins; D000818:Animals; D002021:Buffers; D002417:Cattle; D004566:Electrodes; D004800:Enzymes, Immobilized; D005782:Gels; D005969:Glutamate Dehydrogenase; D005971:Glutamates / Q000032:analysis*; D018698:Glutamic Acid; D066298:In Vitro Techniques; D007700:Kinetics; D000099166:Polyacrylamides
D000180:Acrylic Resins; D002021:Buffers; D004800:Enzymes, Immobilized; D005782:Gels; D005969:Glutamate Dehydrogenase; D005971:Glutamates; D018698:Glutamic Acid; D000099166:Polyacrylamides