Proteins Quenching

The injector, columns and valves reside in a low temperature chamber to minimize the loss of deuterium by back exchange (Fig. 12.2). The quenched protein solution is pumped in series through a column containing an immobilized protease and a trap column to capture the peptide fragments. The gradient pump is activated following digestion and the peptides captured on the trap column are eluted and separated over an analytical reverse-phase HPLC column directly into the mass spectrometer. 

During the steady-state production of NADH from lactate and NAD+, the enzyme has quenched protein fluorescence, and enhanced NADH... 

Phase 1. An instantaneous (<1 msec) formation of 0.1-0.3 mole NADH per subunit. This instantaneously formed compound has absorbance at 340 nm, little NADH fluorescence, and quenched protein fluorescence. No proton is liberated. 

Welder F, McCorquodale EM, Colyer CL. Proteinase assay hy capiUary electrophoresis employing fluorescence-quenched protein-dye conjugates. Electrophoresis 2002 23 1585-90. 

A wide variety of molecules can act as quenchers (Chter 8), and they permit devdopment of sensors based on collisional quenching. Boizo[h]fluotoanthene was found to be highly sensitive to sulfur dioxide. Oxygen interfered with the measurements but was 26-fold less efficient as a quencher than SOz- Halogenated anesthetics ate known to quench protein fluraescence and can be detected by collisional quenching of anthracoie and petylene. Carbazole is quenched by a wide variety of chlorinated hydrocarbons. NO. which serves as a signal for blood vessel dilation, is also a collisional quencher. ... 

Adding TEMPO appears to effectively quench protein radieals and thus exert a proteetive aetion. New compounds that release NO under visible light irradiation (nitrosamine) are reported. Further reaetions for functional group introduction or elaboration are the hydrojgramination of algmes under visible light photocatalysis and the photocatalytic acylation of amines. ... 

Kolubayev T, Geacintov N E, Paillotin G and Breton J 1985 Domain sizes in chloroplasts and chlorophyll-protein complexes probed by fluorescence yield quenching induced by singlet-triplet exciton annihilation Biochimica Biophys. Acta 808 66-76... 

The temperature of a simulation depends on your objectives. You might use high temperatures to search for additional conformations of a molecule (see Quenched Dynamics on page 78). Room temperature simulations generally provide dynamic properties of molecules such as proteins, peptides, and small drug molecules. Low temperatures (<250 K) often promote a molecule to a lower energy conformation than you could obtain by geometry optimization alone. 

In turn, 1O2 is a very electrophilic excited state species of molecular oxygen that interacts efficiently with electron-rich molecules, such as aminoadd residues of proteins like histidine, metionine, tryptophan, tyrosine, etc., by both physical and chemical quenching processes, eqns. 9 and 10 (Davies, 2003 Bisby et al., 1999). 

In addition, Montenegro et al., (2007) determined that the photosensitized RF-mediated degradation of vitamins A, D3, and RF itself in skimmed milk was strongly reduced by the addition of small amounts of lycopene-gum arabic-sucrose microcapsules, prepared by spray-drying. Under these conditions, the bulk properties of the skimmed milk were unmodified. The main photoprotection mechanism of the milk vitamins was the efficient quenching of the 3Rf by the protein moiety of GA. Small contributions (<5%) to the total photoprotection percentage was due to both inner filter effect and 1O2 quenching by the microencapsulated lycopene. 

Fig. 9. The MoFe protein cycle of molybdenum nitrogenase. This cycle depicts a plausible sequence of events in the reduction of N2 to 2NH3 + H2. The scheme is based on well-characterized model chemistry (15, 105) and on the pre-steady-state kinetics of product formation by nitrogenase (102). The enzymic process has not been chsiracter-ized beyond M5 because the chemicals used to quench the reactions hydrolyze metal nitrides. As in Fig. 8, M represents an aji half of the MoFe protein. Subscripts 0-7 indicate the number of electrons trsmsferred to M from the Fe protein via the cycle of Fig. 8.

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-... 

Similarly, the rate of inhibition of phosphoenzyme formation by diethylpyrocarbonate (DEPC) was much slower than the loss of ATPase activity [368], Even when the reaction approached completion with more than 90% inhibition of ATP hydrolysis, about 70% of the Ca -ATPase could still be phosphorylated by ATP (2.3nmoles of E P/mg protein). The remaining 30% of E P formation and the corresponding ATPase activity was not reactivated by hydroxylamine treatment, suggesting some side reaction with other amino acids, presumably lysine. When the reaction of the DEPC-modified ATPase with P-ATP was quenched by histidine buffer (pH 7.8) the P-phosphoenzyme was found to be exceptionally stable under the same conditions where the phosphoenzyme formed by the native ATPase underwent rapid hydrolysis [368]. The nearly normal phosphorylation of the DEPC-trea-ted enzyme by P-ATP implies that the ATP binding site is not affected by the modification, and the inhibition of ATPase activity is due to inhibition of the hydrolysis of the phosphoenzyme intermediate [368]. This is in contrast to an earlier report by Tenu et al. [367], that attributed the inhibition of ATPase activity by... 

Additional evidence for conformational changes in the transporter has come from measurement of the intrinsic fluorescence of the protein tryptophan residues, of which there are six, in the presence of substrates and inhibitors of transport. The fluorescence emission spectrum of the transporter has a maximum at about 336 nm, indicating the presence of tryptophan residues in both non-polar environments (which would emit maximally at about 330 nm) and in polar environments (which would emit at 340-350 nm) [154], The extent of quenching by the hydrophilic quencher KI indicates that more than 75% of the fluorescence is not available for quenching, and so probably stems from tryptophan residues buried within the hydrophobic interior of the protein or lipid bilayer [155]. Fluorescence is quenched... 

Mooradian (1993) has studied the antioxidant properties of 14 steroids in a non-membranous system in which the fluorescence of the protein phycoerythrin was measured in the presence of a lipid peroxyl radical generator (ABAP). Oxidation of the protein produces a fluorescent species. Quenching of fluorescence by a test compound indicates antioxidant activity. Oestrone, testosterone, progesterone, androstenedione, dehydroepian-drosterone, cortisol, tetrahydrocortisone, deoxycorti-... 

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. 

Fig. 13 DNA-protein CT reactions. The DNA-bound protein, methyltransferase Hhal (mutant Q237W), flips a base out of the DNA double helix and inserts a trytophan side chain leaving the /r-stack largely unperturbed. This intercalated trytophan moiety transfers an electron to [Ru(bpy )(dppz)(phen)]3+, generated by flash quench, over 50 A away. Adapted from [164]... 

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