Protein-type fluorescence

Lymphocyte A lymphocyte is a particular type of white blood cell that is involved in many of an organism s immune responses. Subpopulations of lymphocytes with microscopically identical anatomy can be distinguished because their surface membranes contain different arrays of proteins. The staining of these proteins with fluorescently tagged monoclonal antibodies allows the subpopulations to be enumerated by flow cytometry. 

Usually, when studying protein-protein interaction, fluorescence variation of the probe can indicate the type of the interaction. For example, one can find out whether the interaction between the two proteins is cooperative or not, etc... Two examples to illustrate this, we used 2,p-toluidinylnaphthalene-6-sulfonate (TNS) as a fluorescent probe to study the interaction between cytochrome c and cytochrome hj core and to follow the binding of cytochrome b2 core on flavodehydrogenase. The three proteins are extracted from the yeast Hansenula anomala (see chapter 1, paragraph 6Q. 

The diagnostics of protein complexes is an intricate problem if a molecule contains more than one absorption/fluorescent center (Permyakov, 1992). The problem becomes much more complex if, in addition, the protein specimen (ensemble of molecules) is a mixture of several chemically nonidentical types of molecules (subensembles) which cannot be separated, i.e. their partial concentrations are unknown. The second situation is typical for the special kind of proteins, namely, fluorescent proteins (FPs) (Piatkevich et al., 2010). The solutions of FPs are usually mixtures of several typ>es of molecules, which are chemically different and have their own set of photophysical properties (Verkhusha et al., 2004). In this case for unambiguous interpretation of experimental data it is necessary to make simultaneous measurements of a large number of parameters, i.e. to simultaneously apply (or, better, synthesize) several spectroscopic methods. 

Hydrophobic probes have been widely used to monitor changes in the exposure of aliphatic and aromatic residues of food proteins (Nakai and Li-Chan, 1988). Two of the more popular probes are of the anionic type, l-anilinonaphthalene-8-sulfonic acid (ANS ) and c/5-parinaric acid (CPA). Table 2 shows the hydrophobicity of food proteins monitored by these two probes (Li-Chan, 1991). These probes have high quantum yields of fluorescence in nonpolar environment, and are therefore useful to monitor the accessible or surface hydrophobicity of proteins. However, fluorescence of the ANS probe is influenced by solvents which favor the rigid planar configuration, including aqueous MgCl2 so-... 

Two types of fluorescent proteins have been isolated from luminous bacteria and studied in detail. The first of them are the blue fluorescent lumazine proteins (LumPs) containing lumazine as their chromophores, which were isolated from P. phosphoreum and P. fischeri (Gast and Lee, 1978 Koda and Lee, 1979 O Kane et al.y 1985). The second are the yellow fluorescent proteins (YFPs) containing a chromophore of FMN or riboflavin, isolated from P. fischeri strain Y-l (Daubner et al., 1987 Macheroux et ai, 1987 ... 

Gonczy This is very difficult to quantitate. As you have seen from the immunofluorescence images, there are many microtubules on either side in wild-type moreover, anaphase B takes places within a couple of minutes. Therefore, it will be difficult to uncover potential transient changes in microtubule numbers using fixed specimens. However, we have generated a green fluorescent protein... 

Alexa488 bound to IFABP monitored by steady-state fluorescence was fitted to a two-state reversible unfolding model. This modified protein is slightly less stable (midpoint of 4.5 M compared to 4.7 M for wild-type IFABP). 

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