Determination of enzyme activity

Enzyme thermistors can be altered for measuring the activity of soluble enzymes. For this purpose, an inactive or empty Teflon column can be used as a reaction chamber. The sample and a buffer containing a suitable substrate in excess are passed through heat exchangers and thoroughly 

One alternative method for the determination of enzyme activities which is particularly effective at low enzyme concentrations involves enrichment with the enzyme by affinity binding (preferably of the reversible type) to an affinity column in the enzyme thermistor unit. The enzyme activity is determined by introducing a pulse of excess substrate. 

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. 

With sensors two distinct procedures are being used  

The product is indicated after a defined reaction period outside the measuring cell. 

The enzyme-catalyzed reaction is allowed to proceed in the measuring cell, the reaction rate being indicated by electronic differentiation of the current-time curve. 

The activity of cholinesterase in serum reflects the anabolic capacity of the liver. Values below normal (600-1400 U/l) indicate contact with cholinesterase inhibitors such as herbicides. 

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For many analytical methods there are no CRMs. It may also be that there is no primary standard, e.g. for determinations of enzyme activity, or that reliable methods for accurate determination do not exist. However, there is a requirement for RMs of some type. Samples which have previously been used within an FQA scheme may fulfil this purpose. When a large enough number of independent observations are made the mean is a good approximation to the true value (Sutton et... 

Wolfbeis O.S., Fiberoptic probe for kinetic determination of enzyme activities, Anal. 

S. Trajkovska, K. Tosheska, J.J. Aaron, F. Spirovski, and Z. Zdravkovski, Bioluminescence determination of enzyme activity of firefly luciferase in the presence of pesticides. Luminescence 20,192-196 (2005). 

B. H. Junker, J. Lonien, L. E. Heady, A. Rogers, and J. Schwender, Parallel determination of enzyme activities and in vivo fluxes in Brassica napus embryos grown on organic or inorganic nitrogen source. Phytochemistry 68, 2232 2242 (2007). 

Capsaicinoids are synthesized by the condensation of vanillylamine with a short chain branched fatty acyl CoA. A schematic of this pathway is presented in Fig. 8.4. Evidence to support this pathway includes radiotracer studies, determination of enzyme activities, and the abundance of intermediates as a function of fruit development [51, 52, 57-63], Differential expression approaches have been used to isolate cDNA forms of biosynthetic genes [64-66], As this approach worked to corroborate several steps on the pathway, Mazourek et al. [67] used Arabidopsis sequences to design primers to clone the missing steps from a cDNA library. They have expanded the schema to include the biosynthesis of the key precursors phenylalanine and leucine, valine and isoleucine. Prior to this study it was not clear how the vanillin was produced, and thus the identification of candidate transcripts on the lignin pathway for the conversion of coumarate to feruloyl-CoA and the subsequent conversion to vanillin provide key tools to further test this proposed pathway. 

Obviously, extrapolation procedures are impractical for routine determination of enzyme activities. When substrate saturation-curves conform to rectangular hyperbolas, reasonable concentrations of substrates should equal 10 to 20 times the respective Km values. As outlined above, application of this rule to assays of bilirubin UDP-glycosyltransferase activities is hampered by substrate inhibition and by occasional deviation from Michaelis-Menten kinetics. The best alternative in such cases may be to choose the concentrations at optimal enzyme activity. However, great care should be exercised in interpreting the results. When a bio-... 

Most authors use a mixture of the same composition as the test mixture for the incubation control, except for omission of UDP-sugar. As outlined above, complete suppression of endogenous synthesis is preferred by the present authors for determination of enzyme activities. 

A different approach to investigate active lignification during resistance reactions is provided by the determination of enzyme activities involved in lignin biosynthesis. Resistant plants are expected to be more strongly activated during or immediately preceding the resistance reaction compared to susceptible plants. Thus, phenylalanine ammonia-lyase (PAL) (43-45), cinnamic acid 4-hydroxylase (46), O-methyltransferases (44), and... 

Ghosal A, Hapangama N, Yuan Y, et al. Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes. Biopharm Drug Dispos 2003 24 375-384. 

In case of CDG-I IEF pattern, further investigations are initiated by the determination of PMM activity. PMM2 is the defective enzyme in the most widespread form of CDG known so far CDG-Ia (about 80% of all known CDG-cases). It is caused by mutations in the gene, which result in a severe decrease in PMM2 activity, catalysing the conversion of mannose-6-phosphate to mannose-1-phosphate in the cytosol (Fig. 4.5.1). Determination of enzyme activity may be carried out by incubation of protein extracts from patient-derived fibroblasts or leukocytes with [2-3H]mannose-... 

The [Eu(Tc)] assay for H202 can also be used for the determination of enzyme activities. This comprises all enzymes that produce (oxidases) or consume (catalase, peroxidases) H2O2. For example, the activity of glucose oxidase can be directly imaged after the addition of [Eu(Tc)] as indicator and glucose as substrate. Steady-state and time-resolved imaging schemes can be applied for the read-out of a microwell plate-based assay. The best results were obtained by the RLI method (Fig. 19) [115]. 

Alkaline Phosphatase. The first reported use of the automatic analyzer for determination of enzyme activity (M2) indicates that it is feasible for some determinations of this type. The schematic flow diagram for alkaline phosphatase is shown in Fig. 22. The method which has... 

Be familiar with the most useful enzyme determinations in the clinical biochemistry laboratory, understand the importance of timing in the determination of enzyme activity for diagnostic purposes, and know the utility of isozyme determinations. 

For the assay of enzymes with products and reagents that have no absorption, fluorescence or luminescence in the ultraviolet or visible region, developments in analytical infrared spectroscopy can be used. In particular, mid-Fourier transform infrared (mFTIR) spectroscopy has been successfully applied to the determination of enzyme activities and kinetics, e.g. of /i-fructosidasc, phosphoglucose isomerase and polyphenol oxidase [90]. The method could very well be a tool that may also be applied to a variety of other enzyme classes. The potential of high-throughput applications, however, has yet to be demonstrated. 

In direct enzyme assays, the initial rate can be determined readily and this is the quantity that should be used to specify enzyme activity. However, the situation is more complex in coupled reactions, since there is usually a lag phase as the intermediate product is made. For determination of enzyme activity, it is essential that the rate of the ancillary reaction is not a limiting factor, which means that the activity of the coupled enzyme should be in sufficient excess (> 10-fold) over that of the enzyme of interest. 

Determination of enzyme activity level is important in many applications in clinical medicine, in food science and nutrition, in agricultural chemistry, in biochemistry, and in physiology. Because of the large number of these applications we are highly selective in the following discussion. 

But an important point is to realize that (o) comparisons at the molecular level cannot be established between species, since the same enzyme formed by various species may have different specific activities and require different cofactors. (6) Very large errors can be made, and actually have been made in the past, by using poor techniques for the determination of enzyme activity. At the present time, directly active enzymes such as amylase (19, 20), lipase (16), and ribonuclease (21) are measured with a reasonable degree of accuracy. Good techniques and specific substrates are also available for chymotrypsin, trypsin, carbox3 ptidases A and B. But the special problem here is that proteolytic enzymes must be activated and that the activation step must be carefully controlled in complex mixtures (Figs. 5 and 6). 

Moore BD, Stevenson L, Watt A, Flitsch S, Turner NJ, Cassidy C, Graham D (2004) Rapid and ultra-sensitive determination of enzyme activities using surface-enhanced resonance Raman scattering. Nat Biotechnol 22 1133-1138... 

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