Protein dye systems

Future Trends and Scope on Induced Circular Dichroism in Protein-Dye Systems... 

Almost all HCS assays will require at least two fluorescent dyes. One is often to identify which objects are cells or nuclei and a second is to look for specific subcellular expression or localization of a protein or morphological change. A third dye can be used for additional information, such as cell type, co-localization of a second protein, etc. Systems that simultaneously measure two or more wavelengths are advantageous not only for speed but also to enable live cell assays and the measurement of kinetic response to the addition of a reagent. Environmental control is sometimes required for these live-cell assays. Most higher throughput applications are done with fixed cells for convenience. 

Gringorten J L, Witt D P, Milne R E, et al. (1990). An in vitro system for testing Bacillus thuringiensis toxins The lawn assay. J. Invertebr. Pathol. 56 237-242. Bradford M (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 248-254. 

Over the years, spectrophotometry (UV-VIS) has been used to study such physicochemical phenomena as heats of formation of molecular addition compounds and complexes in solution, determination of empirical formulas, formation constants of complexes in solution, hydration equilibria of carbonyl compounds, association constants of weak acids and bases in organic solvents, tautomeric equilibria involving acid base systems, protein-dye interactions, chlorophyll-protein complexes, vitamin A aldehyde-protein complex, association of cyanine-dyes, determination of reaction rates, determination of labile intermediates, and dissociation constants of acids and bases. 

ACID DYES Commercial acid dyes contain one or more sulfonate groups, thereby providing solubility in aqueous media. These dyes are apphed in the presence of organic or mineral acids (pH 2—6). Such acids protonate any available cationic sites on the fiber, thereby making possible bonding between the fiber and the anionic dye molecule. Wool, an animal fiber, is an amphoteric coUoid, possessing both basic and acidic properties because of the amino and carboxylic groups of the protein stmcture. In order to dye such a system, coulombic interactions between the dye molecule and the fiber must take place ie, H2N" -wool-COO + H2N" -wool-COOH. The term acid dye is appHed to those that are capable of such interactions. Acid dyes... 

Fluorescent materials are very important in medical research. Dyes such as fluorescein (21) can be attached to protein molecules, and the protein can be traced in a biological system by exciting the fluorescein and looking for its emissions. The use of a fluorescent material allows the detection of much smaller concentrations than would otherwise be possible. Because fluorescent materials can be activated by radioactivity, they are also used in scintillation counters to measure radiation (see Box 17.2). 

Ioffe VM, Gorbenko GP, Tatarets AL, Patsenker LD, Terpechnig EA (2006) Examining protein-lipid interactions in model systems with a new squarylium fluorescent dye. J Fluoresc 16 547-554... 

In addition to the described above methods, there are computational QM-MM (quantum mechanics-classic mechanics) methods in progress of development. They allow prediction and understanding of solvatochromism and fluorescence characteristics of dyes that are situated in various molecular structures changing electrical properties on nanoscale. Their electronic transitions and according microscopic structures are calculated using QM coupled to the point charges with Coulombic potentials. It is very important that in typical QM-MM simulations, no dielectric constant is involved Orientational dielectric effects come naturally from reorientation and translation of the elements of the system on the pathway of attaining the equilibrium. Dynamics of such complex systems as proteins embedded in natural environment may be revealed with femtosecond time resolution. In more detail, this topic is analyzed in this volume [76]. 

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