Specific activity, radiolabeled

Applications in agrochemicals [42, 43], pharmaceuticals [44,45], and positron emission tomography (PET) [46, 47, 48 49] have resulted in the resuscitation of the Wallach reaction The Wallach technique provides high-specific-activity F-radiolabeled aromatic fluoride for PET studies, in contrast to the low-specific-ac-tivity product by the Balz-Schiemann route... 

The second criterion for mechanism-based enzyme inactivation concerns the stoichiometry of enzyme modification. If mechanism-based inactivation of an enzyme is the result of specific covalent modification of an essential active site amino acid residue, then one radiolabeled inactivator molecule should be incorporated per active site. The stoichiometry of specific radiolabeling should also correlate with the extent of inactivation. In multimeric enzymes which display negative cooperativity, it is possible to observe complete inactivation following substoichiometric modification of the enzyme (Johnston et al., 1979). 

ACC synthase is inactivated during the catalytic reaction ([54], see Section 5.2.3). Inactivation probably occurs by covalent binding of vinylglycine produced from AdoMet to the enzyme [55,56]. Thus, when AdoMet radiolabelled at its aminobutyryl moiety is used as substrate, the ACC synthase protein can be specifically radiolabelled, although enzyme activity is lost. When this [ C]AdoMet labelling method was applied, a 50 kDa... 

Concentration (mCi/ml) = (activity purified radiolabeled protein (cpm/ ml)/specific activity (cpm/mmol)) specific activity (mCi/mmol). 

The results presented above indicate that exogenous hormone does not necessarily equilibrate effectively with all endogenous pools so that a true turnover rate is obtained. In view of these problems, the turnover rates obtained by measuring the rate of dilution of specific activity of radiolabelled hormone should ideally be compared with those obtained by an independent method. 

The introduction of tritium into molecules is most commonly achieved by reductive methods, including catalytic reduction by tritium gas, PH2], of olefins, catalytic reductive replacement of halogen (Cl, Br, or I) by H2, and metal pH] hydride reduction of carbonyl compounds, eg, ketones (qv) and some esters, to tritium-labeled alcohols (5). The use of tritium-labeled building blocks, eg, pH] methyl iodide and pH]-acetic anhydride, is an alternative route to the preparation of high specific activity, tritium-labeled compounds. The use of these techniques for the synthesis of radiolabeled receptor ligands, ie, dmgs and dmg analogues, has been described ia detail ia the Hterature (6,7). 

Other methods of sensitive detection of radiotracers have been developed more recently. Eourier transform nmr can be used to detect (nuclear spin 1/2), which has an efficiency of detection - 20% greater than that of H. This technique is useful for ascertaining the position and distribution of tritium in the labeled compound (14). Eield-desorption mass spectrometry (fdms) and other mass spectral techniques can be appHed to detection of nanogram quantities of radiolabeled tracers, and are weU suited for determining the specific activity of these compounds (15). 

Radiotracers have also been used extensively for the quantitative rnicrodeterrnination of blood semm levels of hormones (qv), proteins, neurotransmitters, and other physiologically important compounds. Radioimmunoassay, which involves the competition of a known quantity of radiolabeled tracer, usually I or H, with the unknown quantity of semm component for binding to a specific antibody that has been raised against the component to be deterrnined, is used in the rnicro deterrnination of physiologically active materials in biological samples (see Immunoassay). 

An important consideration for all radiopharmaceuticals and especially radiolabeled biologically active molecules is specific activity. There are two types of specific activity radionucHdic and biological. RadionucHdic specific activity refers to the ratio of the number of atoms of a particular radioisotope to the total number of atoms of the element. For Tc, the radionuchdic specific activity is the number of Tc atoms to the total number of Tc and Tc atoms. Because all isotopes of an element ate chemically identical, a low specific activity may lead to a low yield in the synthesis of a radiopharmaceutical if a significant proportion of the reagents is consumed by the undesited isotopes. 

Muelder and Shadoff (3) prepared C-2,3,7,8-Cl4-DBpD (0.9 mCi/ mmole) by chlorination of C-2,7-dichlorodibenzo-p-dioxin made from potassium C-2,4-dichlorophenate. The preparation of tritium-labeled 2,3,7,8-Cl4-DBpD is justified because the radiolabeled intermediates are less expensive and more accessible and because a higher specific activity is potentially attainable. Here, we consider the optimal conditions for the reaction sequence designed to obtain products of high chemical and radiochemical purity shown at the top of p. 8. 

One limitation of this method is that the specific activity of the radiolabel is progressively diluted as the radiolabelled transmitter is released from neurons and replaced by that derived from unlabelled substrate. This method also assumes that there is no compartmentalisation of the terminal stores, yet there is ample evidence that newly synthesised acetylcholine and monoamines are preferentially released. An alternative approach is to monitor the rate at which the store of neurotransmitter is depleted after inhibition of its synthesis (Fig. 4.1). However, the rate of release of some neurotransmitters (e.g. 5-HT) is partly governed by their rate of synthesis and blocking synthesis blunts release. 

High-specific activity D-P- H] panthothenic acid (Figure 10.8) was prepared from commercially available p-[3-3H]alanine using Escherichia coli strain DVl, which converted 85 to 90% of the input p-[3-3H]alanine to extracellular D-[3- H]panthoth-enate under appropriate growth conditions. The radiolabeled vitamin was purified... 

One of the first practical applications for these fluorescent labelled heparins was to examine the heparin binding behavior of different proteins and peptides under study in our laboratories. To this end we used a modification of the dot-blot assay described by Hirose and colleagues (13). F-D labelled heparin (-1 fluorescein/heparin) was radiolabelled with 125Iodine using iodobeads, to a specific activity of approximately 0.5 x 106 cpm/pg. Solutions of proteins with known heparin-binding capacities were dotted on nitrocellulose paper. A series of replicates... 

Antibody. Rat monoclonal antibody 34A was purified from nu/nu mouse ascites fluid as described (79). The 34A was radiolabeled with 125I using IDO-GEN (Pierce, Rockford, IL) method to a specific activity of 2 to 4 x 105 cpm/pg, and conjugated with NGPE as previously described (7). 

One of the first decisions to be made when designing an experiment is the method of detection to be used with a particular solute. If radiolabeled material is available, a simple method of analysis is to count the radiolabel appearing in the receiver compartment as a function of time. While convenient, this can be a dangerous practice. Depending upon the type of radioisotope, its position in the molecule, and its specific activity, radiolabeled compounds can be subject to a variety of chemical and solution-catalyzed degradation pathways. If the stock solution contains a significant amount of radioactive impurities or generates them as a result of solution instability, then the possibility for preferential transport of... 

A very simple technique is to use a radiolabeled ligand (usually a well-known substrate) of the specific transporter of interest. A recent suggestion for functional quantitation of the apparent affinities (fQ values) to P-gp using Caco-2 cells and the substrate taxol has been published [143], The method can be described simply as (1) determination of b —> a transport of3 H -taxol in normal, untreated Caco-2 cells and (2) determination of b —> a transport of 3H-taxol in the presence of verapamil (0.2 mM). The difference between these two components represents the active transport via P-gp. The two concentrations of the test compound are chosen as approximately 0.25 x K, and 4.0 x K, and for the inhibition of taxol transport, and in the study of Gao et al. [143], 16 pM and 250 pM of the test compound were used... 

These factors make 125I the iodine label of choice for radiolabeling biological molecules. Its commercial availability from a number of suppliers at relatively low cost further adds to its popularity. Even though it has lower specific activity than 131I, iodine-125 still provides much greater sensitivity than 14C, 32P, 35S, or 3H in labeling biomolecules. In fact, the use of a radioactive iodine label can create probes that have 150-fold more sensitivity than tritiated molecules and as much as 35,000 times the detectability of 14C-labeled molecules (Bolton and Hunter, 1986). 

The following procedure describes the iodination process for the Bolton-Hunter reagent and its subsequent use for the radiolabeling of protein molecules. Modification of other macromolecules can be done using the same general method. For particular labeling applications, optimization of the level of iodine incorporation may have to be done to obtain the best specific radioactivity with retention of biological activity. 

Feinberg, A.P., and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6-13. 

Kobayashi, H., Sato, N., Saga, T., Nakamoto, Y., Ishimori, T., Toyama, S., Togashi, K., Konishi, J., and Brechbiel, M.W. (2000) Monoclonal antibody-dendrimer conjugates enable radiolabeling of antibody with markedly high specific activity with minimal loss of immunoreactivity. Eur. J. Nucl. Med. 27, 1334-1339. 

The synthesis of the title compound, 214, the active-site-directed photoaffinity radiolabel for androgen-binding proteins ( ABP ), has been accomplished180,181 by treatment of excess 17a-[( )-2-tributyltin(IV)ethenyl]androsta-4,6-dien-17 -ol-3-one, 215, with sodium iodide-125 of specific activity 27 Cimmol-1 in a sodium acetate-AcOH buffered solution and a solution of 30% H2O2 in glacial AcOH (equation 77). 

---