Reagent with enzyme

The enzymes hydrogenase, nitrogenase, and formate dehydrogenase can be used to equilibrate reducing reagents with H2O/H2, N2NH3, and CO2/HCOOH, respectively.(53) In no case do the enzymes involve expensive noble metals as catalysts. 

Notes. When using biotin-labeled secondary antibodies instead of enzyme-labeled antibodies, you have first to detect biotin with enzyme-labeled (strept) avidin and proceed further with the Substrate Step (9). Do not add normal serum, non-fat dried milk, culture media or other potential sources of biotin to (strept)avidin-containing reagents. This may result in reduced sensitivity. Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate. 

Enzyme membranes can be prepared by adsorbing the enzyme on the surface of a suitable native or synthetic membrane, or, in the case of membranes with large pores, by impregnating the whole membrane with enzyme. The resulting enzyme membrane can be stabilized by covalently cross-linking the adsorbed protein with a suitable bifunctional reagent (8 ). 

The biocatalytic reduction of carboxylic acids to their respective aldehydes or alcohols is a relatively new biocatalytic process with the potential to replace conventional chemical processes that use toxic metal catalysts and noxious reagents. An enzyme known as carboxylic acid reductase (Car) from Nocardia sp. NRRL 5646 was cloned into Escherichia coli BL21(DE3). This E. coli based biocatalyst grows faster, expresses Car, and produces fewer side products than Nocardia. Although the enzyme itself can be used in small-scale reactions, whole E. coli cells containing Car and the natural cofactors ATP and NADPH, are easily used to reduce a wide range of carboxylic acids, conceivably at any scale. The biocatalytic reduction of vanillic acid to the commercially valuable product vanillin is used to illustrate the ease and efficiency of the recombinant Car E. coli reduction system." A comprehensive overview is given in Reference 6, and experimental details below are taken primarily from Reference 7. 

Reagents for Enzyme-Immunoassay. Tris buffer contained 0.05 M Tris (hydroxymethyl) aminomethane, pH 7.5 0.05, with 0.1% human serum albumin and 0.01% sodium azide. The H O -methanol fixative contained 0.3% in absolute methanol prepared just before use... 

Antibodies can be combined with enzymes and color reagents or radioactive antigens to produce quantitative testing for drugs. The ELISA or Enzyme-Linked ImmunoSorbant Assay uses antibodies generated against the Ag to be tested for covalently linked to an enzyme which can catalyze a color change reaction such as the NADH to NAD conversion (Xmax at 340 nm). When the Ag-Ab complex is formed the enzyme is activated and the color can be detected. 

A typical assay with the casein or hemoglobin substrates (Basic Protocol 1 or Alternate Protocol) takes 2 hr following reagent and enzyme preparation. Assays employing the azo-substrates (Basic Protocol 2) are considerably shorter, 1 hr, si nee the color development step is not necessary. 

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