Experiments with radioactive reagents

In discussing photoaffinity labeling experiments with radioactive reagents, I shall focus mainly on the labeling of protein receptors with small photola-bile ligands. The same principles may be applied to related experiments. The general approach to identifying a receptor in a mixture of proteins is first described. The mixture may be extremely complex, e.g. whole cells or a membrane preparation, or relatively simple, e.g. a purified multisubunit protein. Second, experiments to locate a label within a protein are described. This may be at the level of a low resolution peptide map or in favorable cases the identification of a labeled amino acid residue. 

Mohler et al., 1980), and the identification of the insulin receptor (Section 1.2.1). In all these examples specific labeling was achieved in extremely complex mixtures of polypeptides. 

Unbound radioactive photolysis products usually run close to the dye-front on electrophoresis. However, exceptions have been noted in which 

Bisson et al. (1980) labeled cytochrome oxidase with yeast cytochrome c to which an azido aryl group had been attached at Lys-13, and demonstrated that subunit II of the oxidase reacted specifically. In contrast, subunit III was attacked by a cytochrome c with an azido aryl group attached at Cys-102 (Moreland and Dockter, 1981). Lysine-13 and Cys-102 are on opposite sides of the cytochrome c molecule. 

In practice, clear cut results have been achieved, including a few cases in which a single amino acid is modified, but more or less intractable situations have also arisen. Some illustrative examples are now described. 

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Both forms, however, contained intact RNA and the full complement of capsid polypeptides (50) Further evidence that a substantial conformational alteration of entero- and rhinovirus capsids accompanies the D to C antigenicity change has been obtained by examining the accessibilities of individual capsid proteins in intact virions, A-particles and naturally-occurring empty capsids to lacto-peroxidase-catalyzed iodination or alkylation with acetic anhydride (5I> 52). The results of these experiments are summarized in Table lY. Since under the experimental conditions employed the radioactive reagents formed covalent bonds with tyrosine residues... 

In the presence of CH2-H4folate, FdUMP forms a specific, stable complex with thymidylate synthetase in which all components are covalently bound as depicted in Fig. 1. The affinity constant of this complex is suflSciently high that, with typical concentrations of components used in most experiments, the limiting reagent (FdUMP or enzyme) is completely bound. Using [ H]FdUMP of high specific activity, low levels of complexes present in solution may conveniently he assayed by retention on nitrocellulose filter membranes under conditions in which the free nucleotide is readily removed. The radioactivity remaining on the filter is determined to quantitate the complex. Other conventional methods (e.g., gel filtration, charcoal adsorption of free FdUMP, protein precipitation) may be used for this purpose, but are more tedious and apparently less eflicient. There are expectedly few proteins that will form isolable... 

Until recently, this has been one of the most useful of the isotope dilution methods because it allows the quantitative conversion of a small amount of an unlabelled compound to an isotopically labelled derivative. The unlabelled compound is reacted quantitatively with a radioactive agent of known specific activity and the radioactive derivative is isolated, purified and counted. The activity recorded indicates the amount of radioactive reagent it contains and since the stoichiometry of the reaction is known the amount of compound present can be calculated. There are two basic requirements. First, the compound to be analysed must be quantitatively converted in high and reproducible yield to the radioactive derivative second, there must be a good method of isolating and purifying the labelled derivative. The first experiments to use an isotope derivative technique focused on the determination of amino acids in a mixture [263-265]. The reagent used was I-pipsyl chloride (/ -iodobenzenesulphonyl chloride). 

Polybrominated Diphenyl Ethers. Three main metabolic peaks were identified in the feces from rats fed reagent-grade decaBDE for 8 days (El Dareer et al. 1987). Of the total decaBDE-derived radioactivity recovered in the feces, 1.5-27.9% constituted metabolites. Since absorption of decaBDE is minimal, El Dareer et al. (1987) suggested that metabolism apparently took place in the gastrointestinal tract. In parallel experiments in rats injected C-decaBDE intravenously, feces and gut contents contained 74% of the administered radioactivity 72 hours after the injection, and 63% of the excreted material was metabolites. In rats dosed intravenously with C-dccaBDE and with biliary cannulas, 7.17% of the dose appeared in the bile, and <1% of this amount was unchanged decaBDE. Only one metabolite was detected. The structure of the metabolites detected in these experiments was not defined. 

It is advised that you wear latex or vinyl exam gloves at all times in the laboratory. Even if a particular experiment does not require the use of hazardous chemicals, one can never be sure that those from a previous experiment have been properly disposed of. If volatile compounds are used, they should be stored under a fume hood at all times. If possible, students should work with these materials under the fume hood as well. The large amounts of materials that are often required for a laboratory group may soon fill the room with unpleasant and potentially hazardous vapors. This is particularily important if the reagent vapors are flammable (see Experiment 6) or radioactive (see Experiment 12). 

FIGURE 2 Footp rinting results of RNA polymerase binding to the lac promoter (see Fig. 26-5). In this experiment, the 5 end of the nontemplate strand was radioactively labeled. Lane C is a control in which the labeled DNA fragments were cleaved with a chemical reagent that produces a more uniform banding pattern. 

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