DNP fluorescence

The evidence accumulated in the literature suggests that the structure of surfactant adsorbed layers is, in some respects, analogous to that of surfactant micelles. Fluorescence probing techniques - e.g., pyrene and dinaphtylpropane (DNP) fluorescence probes are used to investigate the structure of adsorbed layer of a surfactant - give information on the polarity of the microenvironment in the adsorbed... 

Fig. 52 DNP fluorescence spectra in (A) SDS micellar solution and (B) SDS-alumina slurry [SDS]/[DNP] refers to the micellized or adsorbed SDS to added DNP...

Pyrene and dinaphthylpropane (DNP) fluorescence and nitroxide ESR probes have been successfully used to investigate the structure of the adsorbed layer of sodium dodecyl sulfate at the alumina-water interface [11,12], The fluorescence fine structure of pyrene yielded information on the polarity of the microenvironment in the adsorbed layer. Intramolecular excimer formation of DNP was used to measure the microfiuidity of this environment. The results indicate the presence of highly organized surfactant aggregates at the solid-liquid interface, formed by the association of hydrocarbon chains. 

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. 

The use of dansyl derivatives is not recommended for routine analysis of free amino acids but is very suitable in the identification of an unknown amino acid that has been selectively extracted from the original sample and is present in small quantities. Both kinds of derivative can be easily separated by chromatography or electrophesis and no locating reagent is required for either because the DNP derivatives are themselves yellow in colour and the dansyl derivatives are fluorescent. 

Tatsu et a/.(106) reported a novel immunosensor using immobilized liposomes doped with carboxyfluorescein and dinitrophenyl (DNP) hapten on the tip of an optical fiber. On complement-mediated immunolysis by anti-DNP-antibody, the fluorescent signal of the liberated carboxyfluorescein was measured. 

Moreover, uncoupHng experiments using Escherichia coli cells were described. An addition of glucose to stationary E. coli cells leads to an increase of fluorescence intensity in the region of NAD(P)H, because it is formed in the glycolysis. This increase was stopped abruptly by addition of 2,4 dinitrophenol (DNP), which effects a decrease in the fluorescence signal according to the theory of uncoupled oxidative phosphorylation. The dynamics of this process. 

The reaction of 2,4-dinitrofluorobenzene (DNFB) (Sanger s reagent [10]) with amino acids is another useful technique which is often employed for the analysis of N-terminal amino acids by TLC and column chromatography after derivatization. The reaction involved in product formation is shown in Fig.4.6. The separated derivatives are determined by measuring the quenching of fluorescence on TLC plates or by UV analysis after column chromatography. The generalized absorption curves of dinitrophenyl (DNP)-amino acids in acidic and alkaline solutions are shown in Fig. 4.7. 

FIGURE 6.4. (a) An epifluorescence image of a 4 x 4 membrane array, (b) A quantitative plot of fluorescence intensity vs. cholesterol content at each DNP ligand concentration. 

FIGURE 10.9 (a) Bulk-phase epifluorescence image of Alexa 594 dye-labeled anti-DNP inside 12 bilayer-coated microchannels. Starting from the left-hand side, the antibody concentrations in these channels are 13.2, 8.80, 5.87, 3.91, 2.61, 1.74, 1.16, 0.77, 0.52, 0.34, 0.23, and 0.15 mM. A line scan of fluorescence intensity across the 12 microchannels is plotted in (b) [1029]. Reprinted with permission from the American Chemical Society. 

Methods that utilized derivatives (DNP and 7-hydroxyquinoline) combined with colorimetric or fluorimetric detection were not specific for acrolein and consistently did not correlate with those obtained from bioassays. Certain direct methods of detection (nuclear magnetic resonance (NMR), fluorescence, and differential pulse polarography) gave the best correlation to the bioassay results (see discussion of analysis of environmental samples in Section 6.2). 

The compound l-fluoro-2,4-dinitrobenzene (FDNB) reacts with free amino, imidazole, and phenolic groups at neutral to alkaline pH to yield the corresponding, colored dinitrophenyl (DNP) compounds. Thus, FDNB will react with the free, unprotonated a-amino groups on amino acids, as well as with the side chains of lysine, histidine, and tyrosine (Fig. 6-1). Dansyl chloride is another compound that is known to react with the unprotonated, N-terminal amino groups of peptides. De-rivatization of peptides with this compound yields fluorescent products that provide a very sensitive method of detection of the amino acid derivatives (Fig. 6-2). 

Fig. 10.6. Sensitivity of Rcelp to chloromethyl ketones and peptidyl (acyloxy)methyl ketones. Reporters based orr Ras (A) arrd a-factor (B) arrd were used to measure the impact of TPCK, TLCK, Phe-Lys AOMK (FKBK), and Phe-Ala AOMK (FABK) on the activity of yeast Rcelp as measured through fluorescence output (A) or a biological readout assay (B) DMSO is the solvent control. A schematic for each assay is shown on the left of the panel, and a graphical representation of data collected with the assay is shown on the right. Numbers on top of each bar in the graph reflect the percent activity observed relative to the DMSO-treated control. ABZ is aminobenzoic acid DNP is dinitrophenol. Data is reproduced in part with permission from Ref. [74].

MCA-[K(DNP)35]-BigET(18-34) Quenched Fluorescent substrates MCA-DIIW VNTPEHVVPYGLGK(DNP)-NH2 Low... 

The DNP-amino acids, after separation into individual spots on the chromatographic plate, can be eluted from the scraped off area by adding 4 ml of water to the material in a small tube. The tube is heated at 50° in a water bath for 15 minutes and centrifuged to clear the solution. The color is read against known standards at 360 nm. Direct estimation of DNP-, PTH-, and DANS-amino acids separated on the thin-layer plate can be performed by fluorescence and fluorescence quenching techniques (P8). It is also possible to convert unmodified amino acids, separated on a silica gel G chromatographic plate, into DNP-amino acids by in situ conversion as was described in Section 4.7.18. The DNP-derivatives can then be developed in the second dimension and the spots analyzed quantitatively. 

Information on microviscosity is obtained by studying the excimer forming capabilities of suitable fluorescent probes. The excimer, which is a complex of a ground state and excited state monomer, has a characteristic emission frequency. The intramolecular excimer formation for example, of 1,3-dinaphthyl propane (DNP), is a sensitive function of the microviscosity of its neighborhood. This property, expressed as the ratio of the excimer and monomer yield (/e//m) for DNP, has been determined for dodecyl sulfonate solutions and its adsorbed layer for the various regions of the adsorption isotherm (Fig. 4.18) (Somasundaran et al., 1986). Comparing the ratios thus obtained to the /e//m values of DNP in mixtures of ethanol and glycerol of known viscosities, a microviscosity value of 90 to 120 cPs is obtained for the adsorbed layer in contrast to a value of 8 cPs for micelles. The constancy of microviscosity as reported by DNP is indicative of the existence of a condensed surfactant assembly (solloids) that holds the probe. 

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