Determination, of enzymes

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... 

Characterization of inhibition modality, and from this quantitative determination of enzyme-inhibitor dissociation constants, constitutes the only rational, quantitative means of assessing relative compound affinity for a target enzyme. 

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). 

Methods utilizing the determination of enzymically released methanol, either after distillation,59,64 or directly in the reaction mixture,109 just like the method of manometric determination of the carbon dioxide released from hydrogen carbonate buffer,55 have not found... 

Determination of enzyme 02 consumption Measures true enzyme activity good discrimination heterozygotes, normals and galactosemics Warburg apparatus (S12)... 

Dixon, M. (1952) The determination of enzyme inhibition constants. Biochemical Journal, 55, 170-171. 

Lineweaver, H. and Burk, D. (1934) The determination of enzyme dissociation constants. Journal of the American Chemical Society, 56, 658-666. 

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). 

Remote double labelling techniques have been used successfully in the determination of enzyme KIEs (Kiick, 1991). A variant of this technique was applied to a nonenzymatic reaction by Matsson and co-workers (Axelsson et al., 1990). They determined the primary carbon and secondary deuterium KIEs for the SN2 reaction between methyl iodide and hydroxide ion in 50% dioxane-water at 25°C. The a-carbon KIE was determined by the UC method (Axelsson et al. 1987,1991). In this method, a mixture of substrate molecules labelled with UC (tm = 20.4 min) and 14C is used. The reactants and products... 

Many reports about activation volume determinations of enzyme reactions do not state the experimental conditions clearly. In such cases it is difficult to interpret the AVJs. As AVJs nearly always are dependent upon substrate concentration, it is also difficult to interpret effects in vivo from in vitro experiments when substrate concentration is not specified. With only one more determination of AVJ, e.g., one at high and one at low substrate concentration (with respect to Km), much more information can be obtained than from just a single determination. 

Tanner, J J., R.M. Hecht, and K.L. Krause. 1996. Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceralde-hyde-3-phosphate dehydrogenase at 25 Angstroms Resolution. Biochemistry 35 2597-2609. 

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. 

Saranpuetti C et ai. Determination of enzymes from Colletotrichum sp. AHU9748 essential for lepidimoide production from okra polysaccharide, J Biosci Bioeng 102 452—456, 2006. 

Selected entries from Methods in Enzymology [vol, page(s)] Computer programs, 240, 312 infrared S-H stretch bands for hemoglobin A, 232, 159-160 determination of enzyme kinetic parameter, 240, 314-319 kinetics program, in finite element analysis of hemoglobin-CO reaction, 232, 523-524, 538-558 nonlinear least-squares method, 240, 3-5, 10 to oxygen equilibrium curve, 232, 559, 563 parameter estimation with Jacobians, 240, 187-191. 

Data analysis flow chart, 240, 314-315 data point number requirements, 240, 314 determination of enzyme kinetic parameters multisubstrate, 240, 316-319 single substrate, 240, 314-316 enzyme mechanism testing, 240, 322 evaluation of binding processes, 240, 319321 file transfer protocol site, 240, 312 instructions for use, 240, 312-313. 

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... 

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. 

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. 

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