Tritium labeled proteins

For direct measurements of the protein adsorption at an interface the method of radioactive indicators was elaborated using tritium labeled proteins [153]. The method allows to measure the adsorption of proteins in a wide range of concentration down to very low values of 10 -10 M. Labeled proteins are obtained by means of tritium thermal activation. This operation replaces hydrogen atoms in C-H bonds by tritium, which also partially penetrates into labile positions of 0-H, N-H and S-H bonds. Protein is purified from labile tritium... 

Hormone receptors for steroids were discovered in the early 1960s, when the technology to radioactively mark steroids became available. By obtaining tritium-labeled estradiol, Jensen could show the existence of an intracellular protein component that bound specifically to this hormone and that was called the estradiol receptor (ER). 

Therefore, those hydrogen atoms that are present in stable H-bonds will resist exchange with the solvent. H-bonds lying on the surface of many proteins readily undergo exchange. In addition to deuterium, one can also use tritium-labeled water to follow the kinetics of protein hydrogen exchange. 

Residue in Malt. Treatment. Samples of steeped barley (Kindred Variety, Northem-B grade, low protein 44.8% moisture) were treated with tritium-labeled... 

Our binding assays also identified M. brassicae proteins of the OBP-Type 2 class. Several of these proteins from both male and female antennal extracts were observed to bind the four tritium-labeled analogs of Zll-16 Ac, 16 Ac,... 

The tissue distribution of CSPs was examined by amplifying CSP cDNAs from different parts of the M. brassicae body, including legs (unpublished) and female pheromonal glands (Jacquin-Joly et al., 2001), as well as antennae and proboscis. Moreover, proteins extracted from these tissues appeared to bind tritium-labeled pheromone components. Figure 17.4A shows binding of Zll-... 

Traditional methods of C-terminal end determination55 were hydrazine degradation, carboxypeptidase digestion, and tritium labeling. Unfortunately, no chemical methods analyzing amino acid sequence sequentially from the C-terminal end is available with reliability similar to that of Edman degradation. Carboxypeptidase digests a protein and... 

The only other known study on the binding of A9-tetrahydrocannabinol to plasma proteins was performed with electrophoretic techniques on tritium-labeled material and human plasma (14). The compound was 90 -95% associated with lipoproteins. 

Ultrafiltration has been used to determine the protein bound fraction of many drags, such as methadone (Wilkins et al. 1997), phenylacetate and phenylbu-tyrate (Boudoulas et al. 1996), etoposide (Robieux et al. 1997), doxorubicin and vincristine (Mayer and St-Onge 1995), disopyramide (Echize et al. 1995), and ketamine and its active metabolites (Hijazi and Boulieu 2002). Schumacher et al. (2000) have shown the applicability for the determination of erythro-cyte/plasma distribution. The method of UF has been applied in the measurement of free unaltered thyroxin or after displacement by salicylate as well after displacement by heparin in healthy people and in patients with non-thyroidal somatic illness (Faber et al. 1993). The protein binding of tritium labeled, antidiabetic repaglinide and its displacement by warfarin, furosemide, tolbutamide, diazepam, glibenclamide and nicardipine were determined by ultrafiltration (Plumetal. 2000). 

Proteins are relatively easy to label by exposure to tritium gas. This method was first described by Hembree et al. According to their procedure, the protein is situated in a thin porous membrane filter of polymer material (cellulose ester) 0.5 pm thick. Protein solution in the membrane filter is then exposed to H2 using microwave discharge. Isotopic exchange (H -> T) occurs. The labeled protein is then purified by chromatography. 

Another tritium labeling method is the reductive methylation 128,129) dehyde is allowed to combine with amino groups of a protein and is then reduced using sodium borohydride. The details will be given later in a paragraph concerned with labeling. 

C-Carbon is now utilized more often in accordance with the development of new labeling techniques which involve so-call i natural labeling, i.e. replaces by isotopic exchange in molecules of the proteins to be labeled. The application of is more convenient than tritium labeling. Radiocarbon may be measure by various gas flow counters and semiconductor detectors, but liquid scintillation counting is still preferred. 

In some eases one of these two counterjarts is additionally labeled with a radionuclide, chromophore, fluorophore, enzyme, etc. to facilitate the detection and to make the method more sensitive. Leucoagglutinin labeled with tritium and protein A labeled with FITC can serve as examples. 

Figure 6-20. Polio virus-induced proteins from the cytoplasm of infected HeLa cells. Proteins were separated by SDS acrylamide gel electrophoresis and the gels fractionated using an autogeldivider. (O) Distribution of tritium-containing proteins from cells infected and then labeled under conditions where only virus-specified proteins are made. ( ) Distribution of the label of C labeled purified virions. [From Summers et al., Proc. Natl. Acad. Sci. US, 54 505 (1965).]...

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

Proof that a lysine residue has been modified can be readily obtained because pyridoxyl derivatives of lysine possess characteristic white-blue fluorescence (Ronchi et al. 1969). In addition, they have a distinctive absorption maximum at 325 nm with of 9710 cm (Fisher et al. 1963). Finally, a radiochemical label can be introduced by reducing the pyridoxal-5-phosphate protein complex with tritium-labelled sodium borohydride. The peptide containing the derivatized lysine can therefore be detected either by fluorimetry, spectrophotometry or radiochemical techniques following routine procedures of proteolytic digestion and fractionation. Acid hydrolysis in 6 N HCl for 24 hr of peptides containing pyridoxal-5-phosphate lysine yields pyridoxyl-lysine since phosphate esters are readily hydrolyzed under these conditions. Pyridoxyl-lysine is eluted between lysine and histidine from a 55 cm column of Beckman 50 resin with 0.15 M citrate buffer pH 5.28. 

Retinyl esters 102a-c (1 mCirnl ) stored under argon at —60°C in toluene containing 40 /ig of 2-t-butyl-4-methoxyphenol and 4 pL of pyridine are quite stable. After 1 year about 60% decomposition was noted, due to radiolysis in the case of 102c. Retinoic acid 103 under similar conditions is also radiochemically stable, but after 4 months the material has to be repurified. Specific activities of tritium-labelled retinoids in the 10-40 Cimmol range have been found necessary in view of the discovery and use of cellular retinoid binding proteins. ... 

Separated proteins are detected by use of Coomassie dyes, silver staining, radiography (exposure of photographic film to emissions of isotopicaUy labeled polypeptides), or fluorographic analysis (x-ray film exposed to tritium-labeled polypeptides in the presence of a scintillator). The latter two methods represent the greatest analytical sensitivity, because they are 100 to 1000 times more sensitive than the Coomassie dyes. 

When the plasma protein binding properties of triamcinolone were studied it was found that tritium-labeled triamcinolone was present in the unbound state to a much greater extent than hydro- 0... 

Exactly how organostannanes exert their toxicity is complex and not well understood. It has been known since 1958 that organostannanes disrupt oxidative phosphorylation in mitochondria and this action was subsequently demonstrated to arise from inhibition of ATP synthases. A tritium labelling study indicated that tri-n-butyltin chloride interacts with a specific ion channel sub-unit in ATP synthase. A second mitochondrial protein, stannin (Snn) has also been identified as a triorganotin receptor responsible for tissue-specific toxicity. ... 

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