Enzyme activities, determination

FIGURE 5.6 Schematic representation of the immunosensor based on a Protein A-GEB biocomposite as a transducer, (a) Immobilization of RlgG on the surface via interaction with Protein A, (b) competitive immunoassay using anti-RIgG and biotinylated anti-RIgG, (c) enzyme labeling using HRP-streptavidin and (d) electrochemical enzyme activity determination. (Reprinted from [31] with permission from Elsevier.)... 

As the next enzymes of the heme pathway are active, uroporphyrinogen is converted to heptacarboxyporphyrinogen during the enzymatic reaction. This can be overcome if the incubation temperature is raised to 45°C, which inactivates uroporphyrinogen decarboxylase. Yet, 37°C is the standard temperature for enzyme activity determinations. Despite our neglect of the additionally formed porphyrins by uroporphyrinogen decarboxylase in the activity calculations, we found a highly reliable test performance. 

These methods for enzyme activity determination are easy and convenient for screening enzymes during purification procedures, especially for cellobiase and endoglucanase. Up to 72 of these types of samples can be run at the same time. For cellobiohydrolase it is more difficult, but it is still possible to run up to 24 samples at the same time. In this procedure, the samples in Corex tubes are continuously stirred while kept in a constant-temperature water bath. After incubation the tubes are removed, put on ice, and 5% TCA is added to stop the reaction. The solid substrate is subsequently removed by centrifugation. 

If the Km of alkaline phosphatase for compound I is 0.10 mM, what concentration of this substrate should be used for the enzyme activity determination ... 

Some commercially prepared sera are investigated similarly by the manufacturers, but others are submitted to a limited number of laboratories which have agreed to undertake such reference work in all cases it is desirable for the information accompanying commercially prepared sera to state the methods of analysis used by the reference laboratories, and this is essential for enzyme activity determinations. 

The results of enzymatic determinations of ceruloplasmin are often expressed in arbitrary units, and the values judged in the light of a series of results obtained in normal subjects by the same method. Expression of the enzyme activity in milligrams of ceruloplasmin per unit volume of serum is also possible. The relation between oxidase activity and the amount of ceruloplasmin in serum can be determined by measuring in parallel samples of sera both the oxidase activity and the change of optical density at 610 mix before and after the addition of ascorbic acid or cyanide. On the basis of the known absorbancy index, the ceruloplasmin concentration can be calculated (see Section 2.2.1) and the relation between it and the enzyme activity determined. Alternatively, purified human ceruloplasmin can be used for standardization of the enzymatic method. The ceruloplasmin content of the purified preparation can be determined colorimetrically or, in the case of a highly purified preparation, by nitrogen analysis. Predetermined increments of ceruloplasmin can then be added to aliquots of a selected serum. It is convenient to select a serum with relatively low ceruloplasmin level to start with. Serum of a patient with Wilson s disease, some of whom have no measurable amount of enzyme activity, would be ideal for the purpose however, Walshe (W5) has recently found an inhibitor in these sera. 

The measurement of enzyme activity plays a key role in clinical chemistry because increased enzyme activities in body fluids often indicate damages to the tissues and cells of certain organs. Enzyme activity determination is usually carried out by measuring the initial rate of the enzyme reaction of interest in the presence of a saturating substrate concentration. 

Horseradish peroxidase (HRP) is an archetypal heme peroxidase. It is a nonspecific enzyme used for studying the etfect of various substances on HRP-catalyzed electron-transfer reactions.1 2 For the enzyme activity determinations, many substrates and types of methods are used. One of these methods is lumino 1-dependent chemiluminescence (CL). The combination of the enzyme HRP/hydrogen peroxide (H202) system and a chemiluminescent method of detection allows for information to be obtained both about the result of the process and its course. 

The uses of GC to study enzymatic reactions in the amino acid metabolism have also been common. In such studies, measuring either a decreasing substrate concentration or an increase of the reaction product, GC can frequently offer greater sensitivity than other analytical methods. Alternatively, stereospecificity of some enzymatic reactions can be distinguished [505] if the resolution of optical isomers through GC is employed. Recent examples of the enzyme activity determinations are those concerning tryptophan pyrolase [506] and glutamic acid decarboxylase [507] in brain tissue. 

The early observation that ferric iron could not be used as substrate by ferrochela-tase [3], led to the development of an assay for enzyme activity determination based on the use of DTT (dithiothreitol) to maintain the iron ions in the reduced +2 form. This requirement for DTT in the enzymatic assay was assumed to be an essential feature, and accordingly, either DTT or some other reducing agent was always included in the buffers used in ferrochelatase purification as well as in the activity assay [9, 49]. However, as Porra et al. [50] and Punekar and Gokhale [51] pointed... 

Automated wet analytical reactions can be performed in autoanalyzer systems to monitor intracellular compounds [167]. Ahlmann et al. reported the development of an on-line system for analysis of intracellular penicillin G amidase produced by genetically modified bacteria. The time delay between sampling and photometric detection was 30 min. The whole system included automated sampling, cell disruption, and enzyme activity determination [167]. 

However, the main goal was to study the behavior and performance of a novel bioartificial material (obtained with an innovative method involving prefreezing and inversion steps) as a suitable matrix for the entrapment of proteins or enzymes in a stable manner. The tests performed (determination of enzyme activity, determination of and V ax kinetic parameters, repeatability test) confirmed both that the enzyme immobilised in the bioartificial polymeric matrix maintained its catalytic activity unchanged and that the catalytic reaction rate was comparable with that of the free a-amylase reaction (used as control). 

Analytical procedures available for enzyme activity determinations are many and usually several alternatives exist. A proper selection should be based on sensibility, reproducibility, flexibility, simplicity and availability. Spectrophotometfy can be considered as a method that fulfils most, if not all, such criteria. It is based on the absorption of light of a certain wavelength as described by the Beer-Lambert law ... 

Several other analytical procedures are available for enzyme activity determination. Fluorescence, this is the ability of certain molecules to absorb light at a certain wavelength and emit it at another, is a property than can be used for enzymatic analysis. NADH, but also FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) have this property that can be used for those enzyme requiring that molecules as coenzymes (Eschenbrenner et al. 1995). This method shares some of the good properties of spectrophotometry and can also be integrated into an HPLC system, but it is less flexible and the equipment not so common in a standard research laboratory. 

The enzyme activity was determined from the culture extract of SSF samples S g (dry weight) fermented substrate was extracted with 100 ml wata containing 0.1 % Tween-80 by shaking for 1 h at room temperature (25°C). At the end of the extraction, the suspension was centrifuged (3000 rpm, 10 min) and the supernatants were stmed at 4°C until use for enzyme activity determinations. 

Chemical cytometry is an analytical technique in which a single cell is isolated and lysed and its contents are separated and detected. The separation step permits quantitative information of multiple analytes to be readily determined. Multianalyte detection is important to measure enzyme activity, determine how biochemical species covary, and elucidate regulatory pathways at the level of individual cells. 

This should not be interpreted as a call for the immediate rejection of high relative molecular mass substrates for enzyme activity determination. In all likelihood, two types of standards (low and high relative molecular masses) may be widely used in the near future. However, it is likely that the low relative... 

Enzyme activity determination Extracellular signal-regulated protein kinase Mixing fluorescently labeled peptide, protein, inhibitor, electrophoretic separation of phosphorylated product and nonphosphorylated substrate Fluorescence... 

Fig. 2. Activity of subtilisin DY determined with benzyloxycarbonyl-L-alanyl-L-alany1-1-leucine p-nitroanilide in dependence on the amount of the enzyme in mg/ml (A) and the time course of enzyme activity determination in min (B).

Benzyloxycarbonyl-L-alanyl-L-alanyl-L-leucine p-nitroanilide is a useful substrate for determination of the activity of subtilisin (Lyublinskaya et al., 1974 1977). Figure 2 (A) shows the activity of subtilisin DY determined with this substrate as a function of the amount of the enzyme and Figure 2 (B) shows subtilisin DY activity in dependence on the time of enzyme activity determination. 

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