Fluorescence binding measurements

Although intended for the biochemistry lab, this experiment provides analytical students with a practical characterization analysis. Of particular interest is the use of Job s method to determine the number of TNS (2-p-toludinylnaphthalene-6-sulfonate) binding sites on calmodulin, fluorescence is measured at 475 nm using an excitation wavelength of 330 nm. 

Fluorescence lifetime measurements can increase the analytical specificity when analyzing mixtures (1-4) and can indicate changes in chemical binding of the fluorophores under various environmental conditions (5). 

A single tyrosine is in the C-terminal portion of the transcription factor 1 (TF1), a type II procaryotic DNA binding protein encoded by Bacillus subtilis phage SPOl. Time-resolved fluorescence decay measurements yielded... 

Figure B3.6.11 The binding of retinol to p-lactoglobutin that has been denatured by exposure to high pressure. The sample contaiining 270 pM p-lactoglobulin was pressurized to 400 MPa for 15 min. After release of pressure, retinol in ethanol was added and fluorescence was measured as a function of time. Parameters final protein concentration 8.5 pM in 20 mM phosphate buffer . ex = 330 nm k9m = 470 nm. Circles, native p-lactoglobulin triangles, pressurized p-lactoglobulin. Reprinted from Ikeuchi et al. (2001) with permission from the American Chemical Society.

Binding of the components in a neutral aqueous solution was confirmed by potentiometric titrations. The feasibility of electron transfer between the components was predicted by cyclic voltammetry and an efficient outer-sphere fast electron transfer was foreseen. Fluorescence spectroscopy measurements showed that the formation of a defined donor-acceptor complex worked even in water at neutral pH. Electron transfer as the quenching mechanism was proved by laser flash photolysis. 

Cabaniss, S.E. and Shuman, M.S. (1988) Fluorescence quenching measurements of copper-fulvic acid binding. Anal. Chem., 60, 2418-2421. 

Recent detection methods for glycan array include fluorescent assay, SPR, MALDI-TOF mass spectrometry, and nanoparticle assay. Fluorescence-based measurement is the prevalent principle for detecting binding to glycan microarrays. Rhodamine [9],... 

Figure 7-14. Example of a fluorescence titration. Intrinsic protein fluorescence is measured in a titration of the E. coli single-strand DNA binding protein to poly-dT. A mixture of protein (at 0.25pM) and DNA was titrated into protein solution (also at 0.25 pM) so that the concen-...

In this paper, the results on solution and Interfaclal properties of a cationic celluloslcs polymer with hydrophobic groups are presented. Interaction of such polymers with added surfactants can be even more complex than that of "unmodified" polymers. In the past we have reported the results of Interactions of unmodified cationic polymer with various surfactants Investigated using such techniques as surface tension, preclpltatlon-redlssolutlon, viscosity, solubilization, fluorescence, electroklnetlc measurements, SANS,etc.(15-17). Briefly, these results showed that as the concentration of the surfactant Is Increased at constant polymer level significant binding of the surfactant to the polymer occurred leading to marked Increases In the surface activity and viscosity. These systems were able to solubilize water Insoluble materials at surfactant concentrations well below the CMC of polymer-free surfactant solutions. Excess surfactant beyond that required to form stoichiometric complex was found to solubilize this Insoluble complex and Information on the structure of these solubilized systems has been presented. 

There are three tyrosine residues in Fre, Tyr 35, Tyr 72, and Tyr 116, close to the flavin-binding pocket (Fig. 1.2). Fluorescence lifetime measurements of the wild-type and mutant Fre/flavin complexes showed that electron transfer from Tyr 35 to the excited FAD isoalloxazine is responsible for the fluorescence quenching [6]. The average positions of the bound FAD and the three tyrosine residues of the protein are shown in the figure. To study the fluctuations in the... 

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