Protein fluorescence quenching

A direct demonstration of binding of alcohol to LADH in the absence of coenzyme has been made (253) by utilizing the spectroscopic changes and protein fluorescence quenching that occur when the chromophoric substrate hydroxymethyl ferrocene binds. It was not definitively established, however, that the alcohol binds in the substrate binding pocket. 

Phase 3. The steady-state production of NADH. NADH fluorescence remains enhanced and protein fluorescence quenched. Protons are liberated at the same rate as NADH is produced. 

Lehrer SS (1971) Solute perturbation of protein fluorescence—quenching of tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 10 3254-3263... 

Figure 5.4 The reaction of pig heart lactate dehydrogenase with lactate and NAD. The four panels show (a) two phases of NADH formation (enzyme bound and free), (/>) NADH fluorescence, which is predominated by the enzyme-NADH complex after pyruvate dissociation, (c) protein fluorescence quenching monitoring concentration of all enzyme-NADH complexes and (d) phenol red absorbence monitoring two phases of proton liberation. (For detail see text and Whitaker ef n/., 1974.)...

Figure 8.1 Simulation of protein fluorescence quenching (F) during NADH binding to lactate dehydrogenase. The curve for (1 — a) rqnesents the exponential time course of a pseudo first order reaction calculated from the fluorescence change as described in the text. The displaced trace is an exponential with the amplitude and time constant of the fluorescence change.

Contrary to the observations with TNP-ATP, however, Mg enhanced eosin fluorescence, whereas ions decreased the fluorescence enhancement of Mg and caused a fluorescence quench in the absence of added ions [99]. The fluorescence enhancement caused by Mg was explained by an increase in the number of eosin-binding sites. Fuller [98] on the other hand, has challenged this explanation and argued that only an increase in enhancement factor (i.e., movement of the fluorophore to a more hydrophobic region of the protein) can explain the Mg -induced fluorescence increase. 

Solubilization of an active H,K-ATPase is also a prerequisite for reconstitution of the enzyme into liposomes. With these H,K-ATPase proteoliposomes it is then possible to study the transport characteristics of pure H,K-ATPase, without the interference of residual protein contamination that is usually present in native vesicular H,K-ATPase preparations. Rabon et al. [118] first reported the reconstitution of choleate or n-octylglucoside solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. The enzyme was reconstituted asymmetrically into the proteoliposomes with 70% of the pump molecules having the cytoplasmic side extravesicular. In the presence of intravesicular K, the proteoliposomes exhibited an Mg-ATP-dependent H transport, as monitored by acridine orange fluorescence quenching. Moreover, as seen with native H,K-ATPase vesicles, reconstituted H,K-... 

Proteases are enzymes that break peptide bonds in proteins. As such they lend themselves to a variety of homogeneous assay techniques. Most employ labeling both ends of the substrate with a different tag, and looking for the appearance (disappearance) of the signal generated in the intact substrate (product). As an example, for a fluorescence quench assay, the N-terminal of a peptide is labeled with DNP and the C-terminal with MCA. As such, the peptide is fluorescently silent since the fluorescence from DNP is quenched by absorption by the MCA. Another very popular donor/acceptor pair is EDANS 5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and DABCYL 4-(4-dimethylaminophenylazo)benzoic acid) (a sulfonyl derivative (DABSYL) [27], Upon peptide cleavage, the two products diffuse, and due to a lack of proximity, the fluorescence increases. 

Callis PR, Petrenko A, Muino PL, Tusell JR (2007) Ab initio prediction of tryptophan fluorescence quenching by protein electric field enabled electron transfer. J Phys Chem B 111(35) 10335-10339... 

Callis PR, Liu T (2006) Short range photoinduced electron transfer in proteins QM-MM simulations of tryptophan and flavin fluorescence quenching in proteins. Chem Phys 326 (l) 230-239... 

Eftink, M.R. and Chiron, C.A. (1976) Exposure of tryptophanyl residues in proteins quantitative determination by fluorescence quenching studies. Biochemistry 15, 672-679. 

Fan C, Plaxco KW, Heeger AJ (2002) High-efficiency fluorescence quenching of conjugated polymers by proteins. J Am Chem Soc 124 5642-5643... 

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