Radiolabeled substrates

It is appropriate at this stage to evaluate procedures that will be used to assess the effectiveness of bioremediation, which have been discussed in Chapter 13. These may include (a) use of radiolabeled substrates (although these will not generally be permitted in field operations) and the application of C-labeled substrates, (b) evaluation of the occurrence of metabolites, and (c) evaluation of markers such as specific enzymes. 

Support for this hypothesis on iso-LAS is given through the findings of Nielsen et al. [100] in a degradation study carried out with radiolabelled substrate, in which it was reported that most of the iso-LAS isomers underwent ultimate biodegradation (79-90%), although some of their carbon (10-20%) was released as water-soluble intermediates. Furthermore, Kolbener et al. [90] could identify structures... 

In vitro techniques for studying DDI potential are based on the metabolism of known marker substrates. Two assay types are typically used to study DDIs the turnover of drug-like probes monitored by LC-MS/MS methods or the use of spectrophotometer (plate reader) based methods. As each technique has unique advantages and shortcomings, assay use has not been standardized across the industry. Although techniques based on the turnover of radiolabeled substrates have also been developed [94—97], these methods are used infrequently and will not be discussed further. 

Several things may be done if the researcher has difficulty in detecting an enzyme activity of interest in a homogenate, or elsewhere. A more sensitive assay technique may be used, if one is available. The concentration of enzyme may be increased, as the rate of product formation is directly proportional to [E]. The incubation time for enzyme with substrate can be increased, although the caveats discussed in O Section 3.3.2 must be borne in mind. The reaction volume may also be increased, while maintaining concentrations of reactants constant this approach is particularly useful if product is separated and detected by chromatography, or if a column is used to separate radiolabeled substrate from product, because the increased amount of product formed in unit time will result in enhanced signal size. 

When utilizing radiolabeled substrates, it is especially important to search for impurities resulting from radiolysis. This is particularly true if the stabihzing agent, often supplied by the vendor, has been removed during any laboratory procedure. It should be recalled that a radioactive compound can decompose chemically as well as via radioactive decay. In many cases, the rate of chemical decomposition for a radioactive compound is greater than the nonradioactive counterpart. This increased decomposition, which can result in a progressive increase in the apparent specific activity, may be a result of the... 

A enzyme kinetic technique, introduced by Britton and co-workers "", that permits one to measure the equilibrium distribution of enzyme-bound substrate(s), inter-mediate(s), and product(s). In this procedure, radiolabeled substrate or product is initially permitted to react with enzyme for sufficient time to equilibrate. At thermodynamic equilibrium, all the different enzyme-bound species will be present at concentrations reflecting their stability relative to each other. One can then add a large excess of unlabeled substrate. Under this condition, any unbound or newly released radiolabel will mix with the large unlabeled pool of substrate or product, where it will undergo substantial reduction in its radiospecific activity. This dilution effectively reduces or eliminates any significant recycling of released radiolabel. One can then... 

Dobbins DC, Pfaender FK. 1988. Methodology for assessing respiration andcellular incorporation of radiolabeled substrates by soil microbialcommunities. Microb Ecol 15 257-273. 

The concentration of the free drug (cu) in the ultrafiltrates or the concentration of bound fraction (Cb) in the retentate can be quantified by LC-MS/MS by means of a calibration curve. If radiolabeled substrate has been applied, liquid scintillation counting can be employed. 

Samples obtained are analyzed by chromatographic means, e.g. LC-MS/MS, HLPC using UV detection or radiodetection, if radiolabeled substrates are applied. Metabolic stability as final result is given as percent remaining after incubation by dividing peak area of parent compound in the sample by the area of the time 0 sample and multiplication by 100. Also evaluation of parent and metabolites detected is possible for generat-... 

Vesicular uptake studies are performed using the rapid filtration technique as reported by Chu et al. 1997. The transport medium contained the radiolabeled substrate (e.g. 3H-ethinylestradiol-3-0-sulfate or -3 -O-glucuronide, 3 H-estradiol-17(>-l)-glucuronide,... 

More direct approach to the problem is based on measuring rapid presteady state kinetics with the use rapid chemical quench and stop-flow techniques (Johnson, 1995 Fierke and Hammes, 1995). These techniques allow monitoring individual rates of binding, conversion and dissociation of substrate. The most effective variant of such an approach is based on using a single turn over kinetics in which enzyme is taken in excess over radiolabeled substrate. 

This hydroxylase carries out the 1-hydroxylation of the compound 25-hydroxyvitamin D3 (25-OH-D3) to form the product 1,25-dihydroxy-vitamin D3 [l,25-(OH)2-D3]. The HPLC assay developed replaces those using radiolabeled substrates. 

The separation of the substrate and the product was accomplished by reversed-phase HPLC on an ODS column. The column was eluted isocratically with a mobile phase of methanol-water (33 67 v/v). The compounds were detected at 254 nm. The separations obtained are shown in Figure 9.121. Radiolabeled substrates were also used, and the eluent was assayed for radioactivity on fractions collected during the elution. 

The direct radioisotope tracer method, in which the accumulation of radiolabeled product from added radiolabeled substrate over time yields a rate estimate, is not very practical for measuring rates of nitrification in the environment. Capone et al. (1990) were able to quantify nitrification rates using but the isotope is so shortlived (10 min half-life) that its use is usually impractical. 

Figure 2b shows the results of a rapid-quench single-turnover experiment performed with EPSP synthase with enzyme in excess over the radiolabeled substrate, PEP. The data show the transient formation and decay of the tetrahedral intermediate, which led to its subsequent isolation and stmcture determination. 

The fact that the isotherm data for L-PA were better fitted to the Freundlich isotherm at lower concentrations agrees with previous studies on other template systems, where the isotherm data were often best fitted to tri-Langmuir models [25]. Competitive assays using radiolabelled substrates allowed the identification of a class of sites with a very low surface coverage ca. 1 nmol/g) and high binding constants (up to 1 x 10 /M) (Table 5.3). 

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