Isotope competition

The protocol described in Section 7.1.2 involves isotopic competition, but with the different isotopomers held in separate containers. Equations 7.10 to 7.13 apply equally well to a type of competition experiment known in biochemistry as the perturbation method for determining KIE s of reversible enzyme catalyzed reactions. The perturbation method differs from simultaneous non-competitive measurements in several important ways. One begins by mixing equilibrium concentrations of substrate and product but with one component (substrate or product) at a different isotopic composition than the other. Thus, the mixture is in chemical, but not isotopic equilibrium. At this stage no enzyme is present and the interconversion is... 

A. Chemical vs. Isotopic Competitive Methods Two types of competitive methods can and have been used. They are the chemical competitive and the isotopic fractionation techniques. In the chemical competitive method, the isotopic compounds A or A compete with a chemically different species, B, for reaction with C. The method is, therefore, not applicable to unimolecolar reactions and requires samples of A and A of appreciable isotopic enrichment. Furthermore, the species B must react with C at a rate of similar order of magnitude s A or A do. Consider for simplicity reactions first order in each of the reactants... 

The isotopic competitive method is one of wide versatility and simplicity, especially when the isotopic pair have an abundance ratio of the order of 10 or less. For many of the stable isotopes e.g. C13, N15, O18, such studies have been carried out with material of natural abundance and many difficult synthetic problems are eliminated. However, in the use of this method, care must be exercised to eliminate effects due to isotopic exchange between reactants, intermediates, and products. In a number of cases the problem of isotopic homogeneity arises. We shall discuss the latter in connection with the problem of intramolecular isotope effects. 

We shall consider, first, the kinetics of a system undergoing an isotopic competitive intermolecular reaction, first order in the concentration of the isotopic species and of arbitrary order with respect to other reactants. Later we shall show that the results are applicable to intermolecular isotopic studies of any higher order... 

Application of the Isotopic Competitive Method to Systems at the Tracer Concentration Level... 

Intramolecular Isotope Effects — A Special Case of the Isotopic Competitive Method... 

If a compound A BA decomposes to form A B and A, then the decomposition of ABA into AB and A or into A B and A will show an intramolecular isotope effect or an intramolecular isotopic competition. The general solution to the kinetics of such a system involving the species A BA, ABA, and ABA all reacting at different rates to give the products A B, AB, A, and A is rather unwieldy. We shall present several particular solutions to the kinetics of the system. 

The isotopic competitive method, whether the analyses are made mass spectrometrically or by radioactivity measurements, is very sensitive to impurities. In the use of radioactive isotopes, the sample to be counted must be decontaminated of other chemical species containing the same radioactive nuclide and it must be chemically pure to avoid dilution. Both of these effects must be reduced to the order of 0.1 per cent, which is a very exacting restriction. In the mass spectrometric method one must avoid impurities which give ion peaks (mjq), either from the parent or from a fragment, at the same mfe as the product analyzed. A scan of the mass spectrum usually enables one to detect such impurities and provides the basis for further purification processes. The latter must all be isotopically nonfractionating. 

In either the radioactive or the mass spectrometric determination only the relative isotope ratios need be determined for the isotopic competitive method, except for the correction factors of the form (l + Ra0)/(l + Rxf). 

Fig. 4. The quantity of meteorite-derived sulfur, Sm, in the samples illustrated in Fig. 3, as a function of nitrogen content. Values of Sm were calculated from observed sulfur contents using a model based on mixing of indigenous and meteoritic sulfur followed by isotopically competitive loss of sulfur from the lunar surface, see text. Nitrogen content is employed here as a surrogate for soil maturity, see for example, Kerridge (1993). The clear correlation of Sm with maturity constitutes evidence for addition of meteoritic sulfur to the lunar regolith. From Kerridge et al (1975).

Nothing published since 1975 calls into question our model invoking influx of meteoritic sulfur and isotopically competitive loss of part of the resulting sulfur population. Curiously, a paper appeared shortly afterwards purporting to show, without benefit of isotopic data, that the lunar regolith contained meteoritic sulfur, but without the isotopic evidence for the substantial amount of missing sulfur, it represented an exercise in futility. Our model remains the best available description of the behavior of sulfur on the lunar surface, with the caveat that it is still... 

It is often more advantageous to use comparative methods, that is, to work with the mixture of the two compounds in the experiments, which ensures identical experimental conditions for the parallel reactions. The isotope competition method does not require pure isotopic compounds, it can be used even with compounds of natural isotopic composition, and thus it can be applied to the determination of C, N, and 0 isotope effects. However, the isotopic composition must be measured with high accuracy, in the case of stable isotopes usually by mass spectrometry, in the case of radioisotopes by measuring the change in the specific activity. In order to obtain the kinetic isotope effect, one needs to determine the isotopic composition of the test compound, first at the start of the reaction then again after the reaction has taken place to a known extent. The isotope effect can also be obtained from the isotopic analysis of the reaction products. In the latter case, the method of the evaluation of k /k from the experimental data can be found, for example, in the book of Vertes and Kiss (1987). 

Vogel discovered an alternate pathway of ornithine biosynthesis from glutamic acid involving AT-acetylglutamate 114, 116). He has obtained evidence that this is the major pathway for ornithine formation in E. colt and Neurospora 127). The pathway shown in Fig. 5 is of minor importance for ornithine biosynthesis but is the major one for proline. These conclusions have been substantiated by isotope competition experiments 128). The steps in the acetylglutamate pathway is shown in Fig. 6. 

Dougall, D. K., and Fulton, M. M., 1967, Biosynthesis of protein amino acids in plant tissue culture IV. Isotope competition experiments using glucose-U-C and potential intermediates. Plant Physiol., 42 941. 

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