Simultaneous non-competitive measurements

A partial resolution to some of the problems with the non-competitive technique is to carry out the reactions of the separated isotopomers at the same time, and under the same conditions, but in different containers (say in a common thermostat). In this fashion one can directly compare isotopic differences as the reactions progress. For example, if the concentration of product or substrate can be followed spectropho-tometrically, one might use a two-beam instrument with the two samples placed next to each other. The photometric signal, then, is proportional to the difference in the absorption, A, of light and heavy species, and therefore to the difference in their concentrations, (provided the experiment is carried out in a region where the Lambert-Beer law is valid, and the molar extension coefficients are equal for both isotopomers), see Fig. 7.1. 

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Fig. 7.1 Spectrophotometric simultaneous non-competitive measurement of KIE. In this technique the reaction mixture containing the reference isotopomer is placed in the reference cell and the mixture with the other isotopomer in the sample cell (at identical concentrations). The two cells are placed in a common thermostat (www.chemguide.co.uk)...

Table 7.1 Isotopic differences in reaction progress as a function of fi, (simultaneous non-competitive measurement of isotope effects)...

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... 

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