Position-specific labeling

The application of substrates isotopically labeled in specific positions makes it possible to follow the fate of individual atoms during the microbial degradation of xenobiotics. Under optimal conditions, both the kinetics of the degradation, and the formation of metabolites may be followed— ideally when samples of the labeled metabolites are available. Many of the classical studies on the microbial metabolism of carbohydrates, carboxylic acids, and amino acids used radioactive... 

The comparative biochemistry of glycolysis has been studied very effectively using D-glucose labeled in specific positions with C14. The distribution of C14 in the key intermediates and end products, compared with those... 

Isotopic labels (and especially enriched materials) have proven crucial in the investigation of the mechanisms of homogeneously catalyzed reactions [130]. Further, isotope effects on the rate or the equilibrium constant of a reaction can be diagnostic, and structural information can be provided by isotope-induced changes in the chemical shifts of neighbouring nuclei, and/or alterations in the coupling pattern of the detected spectra. The isotope- and position-specific information inherent to NMR techniques are ideally suited for the analysis of isotope effects in catalysis [131]. 

In their approach of applying transition matrices for the description of the carbon transfer in metabolic networks, Wiechert et al. [16] defined the labeling pattern of a molecule via cumulated isotopomer (cumomer) fractions, whereby the isotopomers belonging to the same cumomer fraction share labelings at specific carbon positions. For a Cj compound, three 1-cumomer fractions... 

A specific variant of El MS is isotope ratio (IR) MS [46]. It is based on electron impact ionization with maximized ionization probability. IR MS is limited to the analysis of gases of high volatility and low reactivity such as CO2, N2 or SO2. The analytes of interest thus have to be transformed into one of these gases before introduction into the IR MS. Information on the position of C labelings in the analyte can be only obtained, if all carbons are isolated position specific and subsequently combusted. In this context Corso and Brenna [47] showed position specific analysis by IR MS for methylpalmitate through pyrolytic fragmentation. IR MS exhibits an extremely high precision of 0.00001 % for the isotope ratio measurement and is optimal to quantify low label enrichments [48]. This is especially important for in vivo studies with ani-... 

One must be aware of the possible occurrence of certain problems in isotope dilution analysis. One of these is incomplete isotopic exchange, in which the active and inactive atoms do not mix. This lack of exchange can be due to differing physical and chemical states of tracer and inactive materials. Steps must be taken to ensure complete exchange. One must also be sure that the labeled position in any compound is relatively inert. If the atom in question is very labile, one can get a reduction in specific activity without any dilution having taken place. To compare specific activities, all samples must be counted under identical conditions with proper corrections for self-absorption in samples of varying mass. 

For the label of specificity, criterion (2), we must appeal to a more limited area of study, to spectroscopic and diffraction data. The most definitive data are, no doubt, those which indicate atom positions in the molecular aggregate. Thus, x-ray diffraction, neutron diffraction, and certain nuclear resonance studies of solids can provide more or less direct evidence that there are H atoms which occupy positions of close approach (hence bonding distance) to two other atoms. Electron diffraction spectra can yield the same information for gaseous species. More easily obtained, however, are IR and Raman spectra, which reveal specific involvement of H atoms by peculiarities in their vibrationeil degrees of freedom in the molecular aggregate. Finally, high resolution proton magnetic resonance studies provide a sensitive index of the electronic environment of the H atoms. 

The source of the atoms which comprise the PQC cofactors was determined from isotopic labeling studies in which cell cultures were fed tyrosine that was labeled at specific positions with and NMR analysis of the PQQ isolated from those cultures indicated that PQCI was formed from a tyrosine and a glutamate, with all carbon and nitrogen atoms of the two amino acids incorporated into Remarkably, it was subsequently... 

The subject of phospholipase assays has been concisely reviewed [1-5]. A standard approach employs phospholipids with radioisotopes incorporated into specific positions in the molecule. By the appropriate choice of labeling, the specificity of the enzymes can readily be established and as little as a few picomoles of product can be detected. The use of isotopes has been helpful in the measurement of phospholipase activity using the membranes of whole cells or isolated subcellular fractions previously labeled with radioactive phospholipid precursors. 

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