Tritium substrate-labeling with

We have tried to detect the formation of a hydroperoxide of the substrate in the absence of the over-all reaction by using a substrate labelled with tritium in the p positions. 

Small isotope-effects can be detected by double-labeling techniques, in which the carbon skeleton is labeled with 14C, and the ratio of 14C to tritium is measured both in the substrate and the product. Care must be taken in the observation and interpretation of isotope effects determined from the hydrogen-isotope content of the product. Just as in non-enzymic reactions (see p. 154), discrimination against the substrate containing deuterium or tritium leads to an increase in the isotopic content of the substrate, and this decreases the apparent isotope-effect towards the end of the reaction. 

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... 

H]-(168) labelled with activated tritium with substrate dispersed on multipore filter crude yield — 23 mCi purified yield — 2 mCi specific activity 562 mCi mmol-1... 

Cerebroside sulphate labelled with tritium has been used as a substrate in an assay for cerebroside sulphatase activities using t.l.c. ... 

The reaction of UDP-glucuronate carboxylyase with TJDP- lucuronic acid, labeled in various portions with tritium, was investigated by Schutzbach (1968) in an approach to the study of the mechanism of the reaction. UDP-Glucuronic acid-3< reacted at the same rate as nonlabeled substrate or substrate uniformly labeled with C in the uronic acid moiety. When the H-Iabeled and [Pg.377]

Supported and unsupported forms of palladium have been used with tritiated water to label a wide variety of complex compounds, many of which do not survive the reductive conditions of catalyzed labeling with tritium gas. Generally, the recovery of substrate and the level of label incorporation are strongly dependent upon several reaction parameters, so that experimentation is usually required to optimize conditions for each new compound. 

Figure 3.22 Substrate labeled from tritiated water by alumina catalysis 3.3 Heterogeneous Catalysis in Solution with Tritium Gas...

The studies on the stereochemical course of 7a-hydroxylation are of note. Bergstrom et al. (1958) administered cholesterol-4-C, 7a-H and cholesterol-4-C, 7j3-H to bile fistula rats and isolated cholic acid. Ratios of to H were analyzed. From the substrate labeled at 7a only about 7% of the tritium was retained in the product isolated, while the 7j8 tritiated structure lost little or none of its radioactivity in transformation (Fig. 22). This experiment is similar to those carried out on steroid structures by Hayano et al. (1958, 1959) and Corey et al. (1958) which revealed that in the course of hydroxylations there is direct replacement of the hydrogen of the structural position involved with retention of configuration. 

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