Binding quantitative

The most common methods used to determine protein concentration are the dye-binding procedure using Coomassie brilliant blue, and the bicinchonic-acid-based procedure. Various dyes are known to bind quantitatively to proteins, resulting in an alteration of the characteristic absorption spectrum of the dye. Coomassie brilliant blue G-250, for example, becomes protonated when dissolved in phosphoric acid, and has an absorbance maximum at 450 nm. Binding of the dye to a protein (via ionic interactions) results in a shift in the dye s absorbance spectrum, with a new major peak (at 595 nm) being observed. Quantification of proteins in this case can thus be undertaken by measuring absorbance at 595 nm. The method is sensitive, easy and rapid to undertake. Also, it exhibits little quantitative variation between different proteins. 

At the cellular level cisplatin causes a block in the cell cycle at the point where DNA repair occurs, the S/G2 interface. The stages of the cell cycle can be monitored using flow cytometry75. In this technique cells are fixed and stained with propidium iodide, a DNA fluorochrome, which binds quantitatively to DNA. The degree of fluorescence is therefore directly proportional to the DNA content of the cell, which in turn is an indicator of the position of the cells in the cell cycle. The ovarian cell line, SK-OV-3, was incubated with [Au(acetato)2(damp)] for 4 hours at concentrations of either 30 pM or 100 pM and cell cycle analysis determined from DNA content. Using this technique it was apparent that the gold(III) complex [Au(OAc)2(damp)], unlike cisplatin, did not exert a cell cycle specific effect69. 

MAO inhibitors with a propargylamine moiety inhibit the enzyme in a mechanism-based fashion. They first bind competitively to MAO and then are oxidized by the FAD moiety of the enzyme to active intermediates which bind quantitatively to C-5 of the FAD isoalloxazine ring (Scheme 17) (for review see Gerlach and coworkers ). Since this deactivation reaction is irreversible and quantitative, chlorgyline and selegiline have been used to map and quantitate MAO A and MAO B activities in the tissues of various animal species Most of the quantitation of MAO A and B in human brains was done... 

The results of adsorption experiments may be used to evaluate binding constants provided that Rj and, sometimes, known. We consider to be the most uncertain of the parameters as it may vary with species, tissue, and preparation. Furthermore, direct measurements are rarely attempted. They would require an estimation of membrane area from a known quantity of vesicles together with the measured uptake of a solute assumed to bind quantitatively all the negative charges. Instead, be estimated indirectly using a fluorescent... 

The sol should bind quantitatively and irreversibly to the day gallery surfaces in order to drive the intercalation reaction and predude desorption of the sol in the presence of a strong electrolyte. 

N.m.r. and Raman difference spectroscopy have shown that methylmercury(ii) ions bind quantitatively to inosine at pH 8 by displacement of the proton at N-l. o The binding of Mn + ions to inosine 5 -triphosphate has been studied by... 

Besides yielding qualitative information, these biologically and pharmaceutically motivated applications of SMD can also yield quantitative information about the binding potential of the ligand-receptor complex. A first advance in the reconstruction of the thermodynamic potential from SMD data by discounting irreversible work was made by Balsera et al. (1997) as outlined in Sect. Reconstruction of the potential of mean force below. 

Poso A, R Juvonen and J Gynther 1995. Comparative Molecular Field Analysis of Compounds wii CYP2A5 Binding Affinity. Quantitative Structure-Activity Relationships 14 507-511. 

In order to interpret the data in Table 5.1 in a quantitative fashion, we analysed the kinetics in terms of the pseudophase model (Figure 5.2). For the limiting cases of essentially complete binding of the dienophile to the micelle (5.If in SDS and 5.1g in CTAB solution) the following expression can be derived (see Appendix 5.2) ... 

Quantitative Structure—Activity Relationships. Many quantitative stmcture—activity relationship (QSAR) studies of progestins have appeared in the Hterature and an extensive review of this work is available (174). QSAR studies attempt to correlate electronic, steric, and/or hydrophobic properties to progestational activity or receptor binding affinity. A review focusing on the problems associated with QSAR of steroids has been pubUshed (175). 

Direct quantitation of receptor concentrations and dmg—receptor interactions is possible by a variety of techniques, including fluorescence, nmr, and radioligand binding. The last is particularly versatile and has been appHed both to sophisticated receptor quantitation and to dmg screening and discovery protocols (50,51). The use of high specific activity, frequendy pH]- or p lj-labeled, dmgs bound to cmde or purified cellular materials, to whole cells, or to tissue shces, permits the determination not only of dmg—receptor saturation curves, but also of the receptor number, dmg affinity, and association and dissociation kinetics either direcdy or by competition. Complete theoretical and experimental details are available (50,51). 

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). 

Using any of the carrier proteins available in highly purified form, eg, TBG or TBPA, a convenient and accurate quantitative determination of and is possible by displacement of radioiodinated or T. This procedure enables their quick determination at low concentrations even in the presence of coundess other substances that occur in body duids (31). In a similar fashion, intact cell nuclei or solubilized proteins from rat fiver cell nuclei, which display high affinities for thyroid hormones, especially T, have been used to establish relative binding affinities of many thyromimetic compounds (7). 

Pharmacokinetic studies are designed to measure quantitatively the rate of uptake and metaboHsm of a material and determine the absorbed dose to determine the distribution of absorbed material and its metaboHtes among body fluids and tissues, and their rate of accumulation and efflux from the tissues and body fluids to determine the routes and relative rates of excretion of test material and metaboHtes and to determine the potential for binding to macromolecular and ceUular stmctures. 

The situation is different if the substrate is a prochiral or meso compound. Since these molecules have a center or plane of symmetry the binding of pro-S or pro-R forms is equivalent. The chirahty appears only as a result of the transformation. Hence, at least theoretically, the compound can be converted to one enantiomer quantitatively. 

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