Enzyme colorimetric method

A wide variety of enzymes have been used in conjunction with electrochemical techniques. The only requirement is that an electroactive product is formed during the reaction, either from the substrate or as a cofactor (i.e. NADH). In most cases, the electroactive products detected have been oxygen, hydrogen peroxide, NADH, or ferri/ferrocyanide. Some workers have used the dye intermediates used in classical colorimetric methods because these dyes are typically also electroactive. Although an electroactive product must be formed, it does not necessarily have to arise directly from the enzyme reaction of interest. Several cases of coupling enzyme reactions to produce an electroactive product have been described. The ability to use several coupled enzyme reactions extends the possible use of electrochemical techniques to essentially any enzyme system. 

The same reaction was recently proposed to detect creatine kinase (CK), an enzyme of high clinical significance in relation to the investigation of skeletal muscle disease and the diagnosis of myocardial infarct or cerebrovascular accidents. As ATP is a reaction product obtained from the reaction of ADP with creatine phosphate catalyzed by CK, this enzyme can be indirectly measured by the CL intensity read from the subsequent reaction of ATP with luciferin. Using the technique of electrophoretically mediated microanalysis (EMMA), it is possible to detect the enzyme using nanoliter volumes of biological sample with an improved speed and simplicity with respect to a conventional colorimetric method [100],... 

Many automated instruments measure enzyme activity using fixed time colorimetric methods. Some, however, can be classed as reaction rate analysers, e.g. the centrifugal analysers, and these instruments determine the reaction rate from either the initial slope of the reaction curve or from repeat measurements at fixed intervals. In both methods the slope of the line is taken to represent the activity of the enzyme. 

Some enzymes with improved single amino acid specificity are commercially available. An example is phenylalanine dehydrogenase (EC 1.4.1.1), derived from bacterial sources, which acts on phenylalanine with the simultaneous conversion of NAD to NADH. Quantitation of the phenylalanine is based on determining the amount of NADH produced using standard procedures. In the direct methods, the absorbance at 340 nm is measured, whereas in the colorimetric methods, the reaction is coupled to an electron acceptor... 

Colorimetric methods were developed as a nonradioactive alternate method of detection. Enzymes such as HRP or AP, which are usually conjugated to the secondary antibody, can convert substrates to colored precipitates that accumulate on the blot and generate a colored signal. Such substrates include 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for AP and 3,3 -diaminobenzidine... 

Some colored compounds have also been linked to peptides or proteins for the detection and measurement within visible spectra ranges. Colorimetric methods are relatively nonspecific but they can be made specific by linking them to enzymatic or immunologic reactions (see Immunoassay section). In these cases, enzymes produce colored products from colorless substrates, rather than radionuclides, are used to label particular antigens and antibodies (Winder and Gent 1971). 

Several techniques have been developed for the determination of purine and pyrimidine derivatives in food sample and in particular for hypoxanthine quantification biosensors (220-223) and electrochemical methods making use of immobilized enzyme electrode (224 -227), electrochemical enzymatic-based HA methods (228,229), enzyme reaction with fluorimetric detection (230), radioimmunoassay (231), colorimetric methods (232), capillary electrophoresis (233), and TLC (234). Many HPLC methods have also been developed and are reported in Table 4 (235-247) the most recent ones are described next. 

In addition to analyzing compounds, enzyme sensor has been used to determine the freshness of meats. Xanthine oxidase has been used to determine the levels of xanthine and hypoxanthine that are accumulated from purine degradation during muscle aging so as to monitor fish freshness for a long time. Traditional methods including the automated colorimetric method (54) were time consuming. Jahn et al (55) developed a dipstick test by... 

Protein phosphatase inhibition is usually detected by colorimetric methods, but the development of a biosensor requires the search of other transduction techniques. Electrochemistry has been widely used in biosensors because of the simplicity, easy to use, portability, disposability and cost-effectiveness of the devices. As protein phosphatase is not an oxidoreductase enzyme, our work has been devoted to the investigation of novel enzymatic substrates, electrochemically active only after their dephosphorylation by the protein phosphatase. Nevertheless, colorimetric assays have been used for the optimisation of several experimental parameters. 

In order to demonstrate the viability of the approach, protein phosphatase inhibition was first performed with the enzyme in solution and detected by colorimetric methods. Two microcystin variants, microcystin-LR and microcystin-RR, were used. Both enzymes were inhibited by these toxins, although to a different extent. The 50% inhibition coefficients (IC50) towards microcystin-LR were 0.50 and 1.40 pgL 1 (concentrations in the microtitre well) for the Upstate and the GTP enzymes, respectively. Hence, the Upstate enzyme was more sensitive. The IC50 towards microcystin-RR were 0.95 and 2.15 pgL-1 for the Upstate and the GTP enzymes, respectively. As expected, microcystin-LR was demonstrated to be a more potent inhibitor. 

Acetylcholinesterase inhibition was first performed with the enzyme in solution and detected by colorimetric methods. As the inhibition is irreversible and stoichiometric, it is possible to calculate the toxin concentration by the percentage of the enzyme that has not been inhibited and remains active. The use of several enzyme concentrations and the... 

When the amperometric biosensor was used, results correlated with those obtained by colorimetric methods. The two sensitive enzymes allowed the detection of 0.5 nM anatoxin-a(s) and the limits of detection obtained with the two insensitive mutants were 16- and 50-fold higher than those obtained with the sensitive ones. 

A colorimetric method for determining mz/o-inositol has been described.104 The color is developed with the phosphomolybdotungstic acid reagent of Folin and Denis, after oxidation of the inositol with bromine. Enzymic methods, based on the inositol dehydrogenases of Acetobacter suboxydans44 and Aerobacter aerogenes,106 have also been used. These methods could be employed with any cyclitol readily attacked by the respective enzyme systems (see pp. 144 and 147). 

The purification of many enzymes has been achieved by use of gel-filtration chromatography, ion-exchange chromatography, or other chromatographic procedures. Eluates from such columns may be analyzed for carbohydrate and protein content, as well as for enzymic activity. Such analyses have revealed that, for glycoenzymes, the enzymic activity, the carbohydrate content, and the protein content are distributed identically in the eluates from the columns. Results of this type of experiment for ribonuclease B are shown in Fig. 1. In these experiments,8 ribonuclease B and a hydrolyzate of the enzyme were subjected to gel filtration on Sephadex, and the eluates were analyzed for protein and for carbohydrate by appropriate colorimetric methods. A control experiment containing ribonuclease A and D-man-nose was treated similarly. The data in Fig. 1 show that the carbohydrate component in the unhydrolyzed sample of ribonuclease B... 

Neutral lactase (p-Galactosidase) is an enzyme produced on an industrial scale, which catalyzes the hydrolysis of lactose. Industry utilizes this enzyme in the production of low-lactose dairy products, whey treatment, and fermented lactic products. The goal of this study is to demonstrate that a new colorimetric method to measure the enzymatic activity is adequate for use as a standard method. 

Lespinas F, Dupuy G, Revol F, Aubry C. Enzyme urea assay A new colorimetric method based on hydrogen peroxide measurement. Clin Chem 1989 35 654-8. 

The oxypurines can be determined by their ultraviolet absorption, changes in absorption following reaction with enzymes, colorimetric reactions, or combinations of these methods. The assay of large quantities of reasonably pure oxypurines is quite simple practical problems, however, arise in the determination of small quantities in the presence of unknown impurities. No completely satisfactory methods for the determination of these compounds have been developed. One of the difiBculties in interpreting some of the results in the literature is to decide whether the cerebrations of the investigators exceed the limitations of their methods. 

---