Fluorescent compounds, protein

Isolation of F and P. The first attempt to isolate and purify the substances responsible for the light emission of M. norvegica was made by Shimomura and Johnson (1967). They isolated two substances, a protein (P) and a fluorescent compound (F), which produce a blue light... 

Fig. 3.2.2 Influence of pH on the initial light intensity of euphausiid luminescence when the fluorescent compound F and protein P are mixed in 25 mM sodium phosphate buffers of various pH values, each containing 1M NaCl, at near 0°C. Both F and P were obtained from Meganyctiphanes norvegica. From Shimomura and Johnson, 1967, with permission from the American Chemical Society.

The fluorescent compound F, a luciferin, emits blue light (Amax 476 nm Fig. 3.2.4) in the presence of molecular oxygen and the protein P, a luciferase. In the luminescence reaction, F is changed into an oxidized form (structure 8, Fig. 3.2.6). The luminescence reaction is highly sensitive to pH, with a narrow optimal range around pH 7.8 (Fig. 3.2.2) the optimum salt concentration is 0.15 M for NaCl... 

However, engineering of fluorescent marker proteins to determine subcellular protein localizations and associations in planta can be quite challenging since plant cells contain a number of autofluorescent compounds (e.g., lignin, chlorophyll, phenols, etc.,) whose emission spectra interfere with that of the most commonly used green or red fluorophores and their spectral variants... 

Amino acids accelerate and proteins retard the rate of Cu(II)-catalyzed oxidation of di-2-pyridyl ketone hydrazone (173) yielding fluorescent compounds. This has been applied for the analysis of amino acids and proteins378. 

Often, treatment of samples with fluorescence labeling agent reacts with primary and secondary amines to give a fluorescent compound. This is especially important for detecting amino acids in protein hydrolyzates. Fluorescence detectors may also be integrated into a high performance liquid chromatographic (HPLC) system. 

The most obvious fluorescent compound in milk is riboflavin, which absorbs strongly at 440-500 nm and emits fluorescent radiation with a maximum at 530 nm. Riboflavin in whey is measured easily by fluorescence (Amer. Assoc. Vitamin Chemists 1951). Proteins also fluoresce because of their content of aromatic amino acids. Part of the ultraviolet radiation absorbed at 280 nm is emitted at longer wavelengths as fluorescent radiation. A prominent maximum near 340 nm is attributable to tryptophan residues in the protein. Use of fluorescence for quantitation of milk proteins was proposed by Konev and Kozunin (1961), and the technique has been modified and evaluated by several groups (Bakalor 1965 Fox et al. 1963 Koops and Wijnand 1961 Porter 1965). It seems to be somewhat less accurate than desired because of difficulties in disaggregating the caseinate particles and in standardizing instruments. It also involves a basic uncertainty due to natural variations in the proportions of individual proteins which differ in tryptophan content. 

Fluorescence spectroscopy offers several inherent advantages for the characterization of molecular interactions and reactions. Firstly, it is 100-1000 times more sensitive than other spectrophotometric techniques. Secondly, fluorescent compounds are extremely sensitive to their environment. For example, vitamin A that is buried in the hydrophobic interior of a fat globule has fluorescent properties different from molecules that are in an aqueous solution. This environmental sensitivity enables characterization of viscosity changes such as those attributable to the thermal modifications of triglyceride structure, as well as the interactions of vitamin A with proteins. Third, most fluorescence methods are relatively rapid (less than 1 s with a Charge Coupled Device detector). One particularly advantageous property of fluorescence is that one can actually see it since it involves the emission of photons. The technique is suitable for at-line and on/in-line process control. 

Imaging assays (DiscoveRx PathHunter, Bioimage) Can use recombinant green fluorescent or yellow fluorescent fusion proteins to measure kinase-stimulated signalling events such as protein translocation to nucleus or membrane to cytosolic translocations Biological assay can present kinase in native form examines functional consequences of test compound Difficult to develop lower throughput Nickischer (2006) Traskjr (2006)... 

III) and 2-am no-3-methyl-9H-pyrido[2,3-b]indole (IV) in a pyro-lyzate of soy protein. These fluorescent compounds could be protein-bound in roasted foods. Detailed experiments on the interaction between food proteins and such compounds of toxicological importance are being carried out in Japan. 

Interference by the fluorescence of proteins, compounds, or nonspecific adsorption of the tracer on proteins. 

H -tetramethylbenzidine in anionic-cationic mixed micelles has been studied in detail by ESR . The photochemistry of the semi-oxidised forms of eosin Y and rose bengal have been investigated in colloidal solutions. Relevant to the fluorescence of proteins is a study of fluorescence quenching of indolic compounds by amino-acids in SOS, CTAB, and CTAC micelles O Rate constants for proton transfer of several hydroxyaromatic compounds have been measured in a variety of surfactant solutions. Photoprotolytic dissociation does not require exit of the reactant molecules from the micelles. Micellar solutions can be used to improve the fluorescence determination of 2-naphthol by inhibiting proton transfer or proton inducing reactions z2. jpe decay of the radical pair composed of diphenylphosphonyl and 2,4,6-trimethyl benzoyl radicals in SDS is affected by magnetic... 

In addition to the photo-switchable cyanine dyes, other photo-activatable dyes such as caged fluorescent compounds [5] and photo-chromic rhod-amine [22], as well as photo-switchable fluorescent proteins, such as PA-GFP [23], Kaede [24] and EosFP [25], KikGR [26], Dronpa [27,28] and rsFastLime [29], and photo-switchable CFP2 [30] can also be used for STORM/PALM/ FPALM imaging. 

A serum or urine sample contains many compounds that fluoresce. Thus the sample matrix is a potential source of unwanted background fluorescence and must be examined when new methods are developed. The most serious contributors to unwanted fluorescence are proteins and bilirubin. However, because protein excitation maxima are in the spectral region of 260 to 290 nm, their contribution to overall background fluorescence is minor when excitation occurs above 300 nm. 

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