Tritium labeling experiments

Binding assays for the saxitoxins were conducted with homogenized rabbit brain and saxitoxin exchange-labelled with tritium at C-11 (92, 93). If the various saxitoxins were available with suitably intense radiolabels, then the equilibrium dissociation constant, K, could be measured directly for each. Since only saxitoxin is currently available with the necessary label, the binding experiments instead measure the ability of a compound to compete with radiolabelled saxitoxin for the binding site. The value obtained, Kj, corresponds to the uilibrium dissociation constant, K, that would be observed for the compound if it were measured directly. Affinity is defined for this assay as the reciprocal of Kj. The affinities of several of the saxitoxins (94) are summarized in Figure 11, expressed relative to saxitoxin and plotted on a logarithmic scale. 

Reduction of aldonolactones and their derivatives with isotopically modified reducing agents leads to sugars labeled at the anomeric center. Glycosides substituted with deuterium or labeled with tritium are widely employed for kinetic isotope-effect measurements, mechanistic studies, isotope-tracing experiments, and so on. 

The most frequently used combination of radioisotopes in double labeling experiments is tritium and 14C. Ideally the ratio of the activities in the corresponding channels (Fig. 7.16) should to be high enough to insure the carbon counts in the tritium channel are negligible compared to tritium counts. Usually this is not achieved and a correction must be applied. 

An ingenious experiment showed that this cyclization is not as simple as it seems. If the starting material is labelled with tritium (radioactive 3H) next to the ring, the product shows exactly 50% of the label where it is expected and 50% where it is not. 

Before one tries to construct a steric picture of the events that occur at the active site of the lyase, it is necessary to discuss the history of the re-isolated substrate, which also became labelled with tritium during the above experiments. In order to locate the tritium in these 2-aminopropanols, they were dissolved in deuterium oxide and incubated with HLADH and diaphorase [33]. Although NMR monitoring... 

The postexchange mixture in the tritium-labelling experiment has not been subjected to oxidation, and if the tritiated (saturated) B-ring product had been formed, it probably would not have undergone reoxidation and its bromoacetylated derivative has been separated from 148 in the course of its HPLC purification. 

Energetically, Reaction 2 would be far simpler for the enzyme to carry out. To study these two proposals, an experiment was set up in which the enzyme reaction was conducted in tritiated water (30). One could distinguish between the two reactions on the basis that Reaction 2 would incorporate tritium into the final product, pyruvate, during the addition reaction. In this case, Reaction 2 would yield tritiated pyruvate. When this experiment was conducted and the pyruvate was isolated from the tritiated water on a Dowex-l-Cl column, it was found that pyruvate was labeled with tritium. Unfortunately, however, when the boiled enzyme was incubated with pyruvate and tritium, the pyruvate was also tritiated. Apparently then at pH 8.5, pyruvate picked up tritium by enolization. 

The investigation of isotopic separations in systems with cyclic polyethers has been carried out up to now for the elements lithium, calcium and sodium, in particular. Among these elements, the enrichment of Li is of essential importance for the production of tritium and that of the heavy calcium isotopes for medical labeling experiments. An enrichment aspect does not exist for the monoisotopic element sodium. Investigations with the radioactive nuclides Na and Na are obviously of interest for fundamental investigations because these isotopes can be easily and precisely measured by their y-activity. Except for uranium, most of the investigations on other chemical exchange systems with metal ions are also based on measurements with lithium and calcium, respectively. 

In the first experiment, tritium-labeled M-3-G was added to an ongoing fermentation of Cabernet Sauvignon grapes after the third day of the fermentation. This coincided with the maximum level of M-3-G in the must. Figure 1. After 24 hours, a new analysis of both malvidin-3-glucose by chromatography and the concentration of radioactivity by liquid scintillation counting showed decreases. However, the radioactivity had dropped almost by half, while the M-3-G level had decreased only by about 13%. 

To determine whether the -p mechanism was operating in the Hofmann degradation, several labelling experiments were carried out by Weygand, Daniel, and Simon 156>. They examined the decomposition of 2-tritioethyltrimethyl ammoniumhydroxide at 150 °C and the reaction of the bromide salt with phenyllithium at room temperature. Since only a small amount of tritium was found in the trimethylamine when the hydroxide was decomposed, they concluded that the a -(3 path was not important. However, 54% of the original activity was present in the trimethylamine when the bromide salt was treated with phenyllithium,... 

Radioactive isotopes are commonly used for competitive KIE measurements in a double-label experiment, yielding kn/kj or ko/kj ratios on kcat/ M- This technique typically utilizes tracer-level radioactivity in the position of interest (primary or secondary) to monitor the transfer of radioactivity from reactant to product, and requires a remote label (e.g. C) in order to measure the conversion of unlabelled substrate to product. As an example, [ring- C(U)]benzyl alcohol and [l- H]benzyl alcohol (Scheme 10.2) can be used to simultaneously measure the primary and a-secondary kn/kj effects in the reaction catalyzed by alcohol dehydrogenase (ADH), as the tracer tritium is incorporated randomly into primary and a-secondary positions [6, 10]. 

Reticuline labeled with tritium was used in experiments to show 29) that thebaine (and consequently also codeine and morphine) arises from (— )-reticuline, which is in agreement with the known absolute configuration of these morphinane bases. Its intermediate is salutaridinol-I (not its epimer II) where the absolute configuration of the hydroxyl group was determined by isolation of L-glyceric acid (after degradation). 

Labeling. Incorporation of tritium on exposure of a nonirradiated protein to HST or TI has been termed /3-labeling in contrast to y-labeling which refers to the tritium introduced into the protein as a consequence of radical formation by y-radiolysis. Earlier investigations (30, 37) showed that in the standard (y + / )-labeling experiment, where the time of exposure to HST is 4 hours, /3-labeling accounted for 10% or... 

Normalized Specific Activities Among Native Proteins. Figure 9 shows a comparison of tritium distributions for native proteins irradiated to about 6 Mrads (except for myoglobin which was irradiated to 23 Mrads). Each bar represents the average normalized specific activity of five separate labeling experiments for ribonuclease and of two for each of the other proteins. The tritium distributions have many similarities. The activities of proline and methionine are generally high. Lysine and histidine are heavily labeled in most proteins, while threonine and serine... 

The acid-catalysed conversion into phenols is achieved in the same way. Deuterium and tritium labeling experiments have established that this rearrangement involves a 1,2-hydride shift from carbon to oxygen (the NIH shift, named after the National Institute of Health, Bethesda,USA, where it was discovered) ... 

Radioisotopic tracer techniques were applied to study the coadsorption of n-octadecane and stearic acid on a metal surface immersed in a n-octadecane solution of stearic acid. Dual labeling was employed for determining the surface concentrations of both n-octadecane and stearic acid. n-Octadecane was labeled with tritium and stearic acid with carbon-14. The results of half-hour adsorption experiments provide direct proof of coadsorption of polar and nonpolar materials on iron, copper, silver, and platinum surfaces. The films produced on silver and copper by 19-hour adsorption consisted of approximately one molecular layer of stearic acid and two molecular layers of octadecane. A new model is proposed to describe the structure of this thick coadsorbed film. 

Very recently, Abeles et al. 12) reported the results of tritium labeling experiments of enzyme-bound coenzyme by H-labeled propanediol which they feel contradict our mechanism. Their data may be readily interpreted in terms of our mechanism. In our view, the tritiation of the coenzyme occiurs as the result of the stereospeciflc removal of the ion... 

The incorporation C. autumnale) of 0-methylandrocymbine (CXVII R = CH3, tritium label in the OCH3 at carbon 2) and CXVIII (i C at carbon 6) into colchicine (15 and 10% incorporation, respectively) with no randomization of label indicated the general correctness of the proposed scheme. Multiple labeling experiments with synthetic CXVIII have confirmed many details of the biosynthesis. CXIX was incorporated into colchicine with no change in the ratio, proving that... 

Surface ionization studies of Reaction 1 necessarily involved the detection of the much heavier product, KBr, which was constrained by the conservation of momentum to scatter close to the center of mass (c.m.). In this case, the small recoil velocity of the product KBr from the c.m. is masked by the spread of the c.m. velocity vectors arising from the distribution of parent beam velocities. Thus, it becomes diflBcult to extract any information about the motion of the KBr relative to the c.m., which would provide the desired description of the mechanism and energetics of the reaction (9). High velocity resolution experiments have since been done by Bernstein and coworkers (10,11), but the studies of Reactions 2 and 3 by Martin and Kinsey (8) were the first to provide some information on the dynamics of these reactions from a measurement of product distributions. In these experiments tritium labeling allowed radioisotope... 

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