Labeling useful isotopes

The method used for detection depends on the type of label used. Isotopic counting is employed for radioisotopes, colorimetry for enzyme assays, luminescence and fluorescence measurements can be achieved by means of photomultiplier tubes, while turbidimetry or nephelometry is used for particle enhanced assays. 

Dihydrogen (H2) is similarly protonated to by superacids, as was shown by studies using isotopic labeling. The structure of again involves 2e-3c bonding. 

Troost and Olavesen investigated the application of an internal standardization to the quantitative analysis of polynuclear aromatic hydrocarbons. The following results were obtained for the analysis of the analyte phenanthrene using isotopically labeled phenanthrene as an internal standard... 

There are many excellent examples of experiments using isotopic labeling in both organic chemistry and biochemistry. An interesting example is the case of lydroxylation of the amino acid phenylalanine which is carried out by the enzyme phenylalanine hydroxylase. 

The occurrence and extent of rearrangement of the 2-butyl cation have also been investigated by solvolysis studies using isotopic labeling. When 2-butyl tosylate is solvolyzed in acetic acid, C-2/C-3 rearrangement occurs only to the extent of 9% in the 2-butyl acetate which is isolated.Thus, under these conditions, most of the reaction proceeds by direct participation of the solvent. 

In the case of PtF Cl6 , it has even been possible to synthesize iso-topically labelled species using isotopically labelled HC1 (Figure 3.6). 

One of the possibilities is to study experimentally the coupled system as a whole, at a time when all the reactions concerned are taking place. On the basis of the data obtained it is possible to solve the system of differential equations (1) simultaneously and to determine numerical values of all the parameters unknown (constants). This approach can be refined in that the equations for the stoichiometrically simple reactions can be specified in view of the presumed mechanism and the elementary steps so that one obtains a very complex set of different reaction paths with many unidentifiable intermediates. A number of procedures have been suggested to solve such complicated systems. Some of them start from the assumption of steady-state rates of the individual steps and they were worked out also for stoichiometrically not simple reactions [see, e.g. (8, 9, 5a)]. A concise treatment of the properties of the systems of consecutive processes has been written by Noyes (10). The simplification of the treatment of some complex systems can be achieved by using isotopically labeled compounds (8, 11, 12, 12a, 12b). Even very complicated systems which involve non-... 

It is possible to carry out a chromatographic separation, collect all, or selected, fractions and then, after removal of the majority of the volatile solvent, transfer the analyte to the mass spectrometer by using the conventional inlet (probe) for solid analytes. The direct coupling of the two techniques is advantageous in many respects, including the speed of analysis, the convenience, particularly for the analysis of multi-component mixtures, the reduced possibility of sample loss, the ability to carry out accurate quantitation using isotopically labelled internal standards, and the ability to carry out certain tasks, such as the evaluation of peak purity, which would not otherwise be possible. 

Experiments were carried out using isotopically labelled methanol (97% 0) and ethanol (98% purchased from MSD Isotopes. Anhydrous isobutanol was purchased from Aldrich Chemical Co., Inc. and contained the natural abimdances of orygen isotopes, i.e. 99.8% and 0.2% O. Nafion-H was obtained fi om C. G. Processing, Inc. and Amberlyst resins were provided by Rohm and Haas. The 2SM-5 zeolite was provided by Mobil Research Development Corp. H-Mordenite, montmorillonite K-10, and silica-alumina 980 were obtained firom Norton, Aldrich, and Davison, respectively. y-AIumina was prepared from Catapal-B fi om Vista. 

FIGURE 3.4 Results from the May 2000 dose-response experiment conducted in situ within mesocosms installed at 4 sites in the Florida Everglades and using isotopically labeled Hg. Experimental conditions called for dosing at 0.5, 1.0, and 2.0 times the ambient loading rate... 

A related issue is whether the carbene, when it is involved, is in equilibrium with a ring-closed isomer, an oxirene.233 This aspect of the reaction has been probed using isotopic labeling. If a symmetrical oxirene is formed, the label should be distributed to both the carbonyl and a-carbon. A concerted reaction or a carbene intermediate that did not equilibrate with the oxirene should have label only in the carbonyl carbon. The extent to which the oxirene is formed depends on the stmcture of the diazo compound. For diazoacetaldehyde, photolysis leads to only 8% migration of label, which would correspond to formation of 16% of the product through the oxirene.234... 

By a combination of synthetic approaches, isotopic labeling, using tocopherols with 13C-labeling at C-5a and C-7a, EPR spectroscopy, and high-level DFT computations, it was shown that there is no radical formation at either C-5a or C-7a and that chromanol methide radical 10 does not occur in tocopherol.11 EPR failed to detect... 

In vitro labeling of proteins using isotope-coded affinity tags... 

The use of the in vivo labeling methods described above is limited by the fact that the sample must be grown in the presence of the labeling isotopes. In many cases, it is not feasible to perform in vivo metabolic labeling. For example, for human clinical samples it is not possible to perform in vivo labeling and yet it is highly desirable to obtain accurate quantitative information on protein expression levels within these samples. Therefore, robust methods are needed for quantitation of protein levels in the absence of in vivo labeling with isotopes. 

Kalkhof, S., Haehn, S., Ihling, C., Smyth, N., and Sinz, A. (2005b) Probing laminin self-interaction using isotope-labeled cross-linkers and ESI-FTICR mass spectrometry. Poster, Pierce Biotechnology web site. 

By hydrolysis under very mild alkaline conditions (with a boiling suspension of barium carbonate), ribonucleic acids have been shown to yield small quantities of cyclic phosphates as well as the normal nucleotides.96 These materials were identical electrophoretically with synthetic cyclic phosphates and were readily hydrolyzed to mixtures of 2- and 3-phosphates. Their formation in this way constitutes strong support for Brown and Todd s theory. The precise way in which the alkaline hydrolysis of the polynucleotide occurs has been studied using isotopically labeled water, and the results are in agreement202 with the scheme outlined above. 

Until recently, cell-free protein expression (also sometimes erroneously named in vitro protein expression) did not exhibit the productivity required for preparation of NMR samples, especially considering the high cost of using isotopically labeled starting material. Rather, it was exclusively used as an analytical tool that served to verify correct cloning or to study promotor sites. Because of the very low yields, detection of the expressed product usually required incorporation of a radioactive label (usually via 35S-methionine). 

With another immunophilin, FK binding protein (FKBP), experiments were performed using isotope editing of the [U-13C]-labeled inhibitor ascomycin (bound to unlabeled FKBP) [34], as well as by isotope filtering with unlabeled ascomycin derivatives (bound to labeled FKBP) [35],... 

Alternatively, it may be necessary to label a molecule by introducing an additional atom and in this case it has to be assumed that its presence does not alter the reactivity or metabolism of the molecule. Proteins, for instance, are often labelled with an isotope of iodine. It is very important that any labels used are firmly attached to the molecule otherwise invalid results will be obtained. 

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