Luminescent solute concentration

A sample of aequorin (purity > 80%) is first luminesced by adding a sufficient amount of Ca2+. To the spent luminescence solution, ammonium sulfate is dissolved to a concentration of 1M, and then the solution is added onto a column of Butyl Sepharose 4. The apoaequorin adsorbed on the column is eluted stepwise with buffer solutions containing decreasing concentrations of (NH4)2S04 starting from 1M. Apoaequorin is eluted at a (NH4)2SC>4 concentration lower than 0.1 M. The apoaequorin eluted is regenerated with coelenter-azine in the presence of 5 mM EDTA and 2 mM 2-mercaptoethanol... 

It has been observed in many cases that the luminescence quantum yield in solution decreases as the solute concentration increases, and this applies also to the quantum yields of photochemical reactions. There must therefore be some process which can be written as... 

As the concentration of a potentially luminescent solute is increased, the frequency of encounters between solute molecules is increased. This often results in the formation of solute complexes at the expense of monomeric solute molecules. Obviously, such interactions will affect the fluorescence expected from a given solution based strictly on the formal concentration of monomers and can seriously affect the results of a fluorimetric analysis. 

The discussion of luminescence has, up to the present, been based on the properties of dilute solutions in which the analyte molecules were presumed not to interact with one another. It has already been established that at high absorbance at the wavelength of excitation, deviations from linearity of the fluorescence in-tensity-versus-concentration relationship may occur because of the exponential variation of luminescence intensity with concentration. However, over a wide range of solute concentrations, solute-solute interactions may also account for loss of luminescence intensity with increasing solute concentration. 

Free Bp is fluorescent in solution although its quantum yield is low. However, cocrystal 1 does not fluoresce or phosphoresce because the two phenyls in Bp are not co-planar. Compared to 1, cocrystals 2-6 emit strong phosphorescence, and their phosphorescence excitation and emission characteristics are listed in Table 5. On one hand, the introduction of haloperfluorobenzenes into the crystal dilutes the concentration of phosphors (Nap, Phe, and Pyr), thus preventing luminescence from concentration- or aggregation-quenching. On the other hand, the strong spin-orbital coupling of heavy atom iodine/bromine makes the forbidden Si-Ti and Ti-Sq transitions possible. 

The aqueous micellai solutions of some surfactants exhibit the cloud point, or turbidity, phenomenon when the solution is heated or cooled above or below a certain temperature. Then the phase sepai ation into two isotropic liquid phases occurs a concentrated phase containing most of the surfactant and an aqueous phase containing a surfactant concentration close to the critical micellar concentration. The anionic surfactant solutions show this phenomenon in acid media without any temperature modifications. The aim of the present work is to explore the analytical possibilities of acid-induced cloud point extraction in the extraction and preconcentration of polycyclic ai omatic hydrocai bons (PAHs) from water solutions. The combination of extraction, preconcentration and luminescence detection of PAHs in one step under their trace determination in objects mentioned allows to exclude the use of lai ge volumes of expensive, high-purity and toxic organic solvents and replace the known time and solvent consuming procedures by more simple and convenient methods. 

Fig. 4.1.3 Absorption spectra of aequorin (A), spent solution of aequorin after Ca2+-triggered luminescence (B), and the chromophore of aequorin (C). Fluorescence emission spectrum of the spent solution of aequorin after Ca2+-triggered bioluminescence, excited at 340 nm (D). Luminescence spectrum of aequorin triggered with Ca2+ (E). Curve C is a differential spectrum between aequorin and the protein residue (Shimomura et al., 1974b) protein concentration 0.5 mg/ml for A and B, 1.0 mg/ml for C. From Shimomura and Johnson, 1976.

Fig. 4.1.5 The time course of aequorin luminescence measured with various concentrations of Ca2+. Calcium acetate solution (5 ml) was added to 10 pi of aequorin solution to give the final Ca2+ concentrations of 10 2 M (A), 10-4 M (B), 10-5 M (C), 10 6 M (D), and 10 7 M (E) at 25°C. The dashed line (F) represents the light emitted following the addition of deionized distilled water that had been redistilled in quartz. The concentration of EDTA derived from the aequorin sample was 10 7 M (final cone.). From Shimomura et al., 1963b, with permission from John Wiley Sons Ltd.

Fig. 4.1.7 Relationship between the concentration of Ca2+ and the initial maximum intensity of luminescence when 2.5 ml of 2mM sodium acetate (ultrapure grade) containing the indicated amount of calcium acetate was added to 5pJ of aequorin stock solution, at 25°C. The aequorin stock solution contained 0.7mg of aequorin in 1 ml of 2 mM sodium acetate containing 10-5 M EDTA. When no Ca2+ was added the maximum intensity was 1.1 x 109 quanta/s. From Shimomura and Johnson, 1976.

Fig. 4.1.8 Influence of various calcium chelators on the relationship between Ca2 " concentration and the luminescence intensity of aequorin, at 23-25°C (panel A) in low-ionic strength buffers (I < 0.005) and (panel B) with 150 mM KC1 added. Buffer solutions (3 ml) of various Ca2+ concentrations, pH 7.05, made with or without a calcium buffer was added to 2 pi of 10 pM aequorin solution containing 10 pM EDTA. The calcium buffer was composed of the free form of a chelator (1 or 2mM) and various concentrations of the Ca2+-chelator (1 1) complex to set the Ca2+ concentrations (the concentration of free chelator was constant at all Ca2+ concentrations). The curves shown are obtained with 1 mM MOPS (A), 1 mM gly-cylglycine ( + ), 1 mM citrate (o), 1 mM EDTA plus 2mM MOPS ( ), 1 mM EGTA plus 2 mM MOPS ( ), 2 mM NTA plus 2 mM MOPS (V), and 2 mM ADA plus 2 mM MOPS (A). In the chelator-free buffers, MOPS and glycylglycine, Ca2+ concentrations were set by the concentration of calcium acetate. Reproduced with permission, from Shimomura and Shimomura, 1984. the Biochemical Society.

Fig. 5.8 Influence of pH, temperature, NaCl concentration, and the concentration of coelenterazine on the light intensity of luminescence reaction catalyzed by the luciferases of Heterocarpus sibogae, Heterocarpus ensifer, Oplophorus gracilirostris, and Ptilosarcus gruneyi. Buffer solutions used 20 mM MOPS, pH 7.0, for Ptilosarcus luciferase and 20 mM Tris-HCl, pH 8.5, for all other luciferases, all with 0.2 M NaCl, 0.05% BSA, and 0.3 p,M coelenterazine, at 23°C, with appropriate modifications in each panel. Various pH values are set by acetate, MES, HEPES, TAPS, CHES, and CAPS buffers.

Fig. 6.2.4 Change in the absorption spectrum of pholasin (14.5 p,M) caused by the luminescence reaction catalyzed by Pholas luciferase (1.1 p.M). The curve shown is the differential spectrum between a cell containing the mixture of pholasin and Pholas luciferase (0.9 ml in the sample light path) and two cells containing separate solutions of pholasin and the luciferase at the same concentrations (in the reference light path), all in 0.1 M Tris-HCl buffer, pH 8.5, containing 0.5 M NaCl. Four additions of ascorbate (3 iM) were made to the sample mixture to accelerate the reaction. The spectrum was recorded after 120 min with a correction for the base line. From Henry and Monny, 1977, with permission from the American Chemical Society.

Luciferase-catalyzed luminescence of luciferin. Odontosyllis luciferin emits light in the presence of Mg2+, molecular oxygen and luciferase. The relationship between the luminescence intensity and the pH of the medium shows a broad optimum (Fig. 7.2.8). The luminescence reaction requires a divalent alkaline earth ion, of which Mg2+ is most effective (optimum concentration 30 mM). Monovalent cations such as Na+, K+, and NH have little effect, and many heavy metal ions, such as Hg2+, Cu2+, Co2+ and Zn2+, are generally inhibitory. The activity of crude preparations of luciferase progressively decreases by repeated dialysis and also by concentrating the solutions under reduced pressure. However, the decreased luciferase activity can be completely restored to the original activity by the addition of 1 mM HCN (added as KCN). The relationship between the concentration of HCN and the luciferase activity is shown in Fig. 7.2.9. Low concentrations of h and K3Fe(CN)6 also enhance luminescence, but their effects are only transient. 

Fig. 7.2.9 Influence of cyanide and iodine on the Odontosyllis luciferin-luciferase luminescence reaction. Luciferin solution (0.1 ml) was first mixed with a HCN solution (0.1ml), and then the mixture was injected into 8 ml of 20 mM magnesium acetate containing luciferase. The concentrations of HCN shown in the figure are the final concentrations. In the control experiment, HCN was omitted. In the experiment labeled added at 0.5 min, 0.1 ml of HCN solution was injected to the control mixture 0.5 min after the start of the luminescence reaction to give a final concentration of 0.1 mM HCN. Arrows indicate the injection of a solution of I2-3KI to the control mixture to give a final concentration of 0.1 mM I2. From Shimomura et al., 1963d, with permission from John Wiley Sons Ltd.

Fig. 7.3.3 Relationship between the concentration of H2O2 and the peak intensity of luminescence, when 0.2 ml of a H2O2 solution was injected into a mixture of 0.575 ml of 0.1 M potassium phosphate (pH 7.5), 0.025 ml of a solution of Diplocardia luciferin, and 0.2 ml of luciferase solution (0.12 mg). 1 LU = 109 quanta/s. From Bellisario et al., 1972, with permission from the American Chemical Society.

Fig. 10.2.2 Influence of the concentrations of ATP, Mg2-1- and Ca2+ on the maximum luminescence intensity of the photoprotein of the millipede Luminodestnus. The luminescence reaction was started by mixing a solution of the photoprotein (A280 0.3, 10 pi) with 2 ml of 10mM Tris-HCl buffer, pH 8.3, containing either 1 mM MgCb plus various concentrations of ATP or 0.05 mM ATP plus various concentrations Mg2+ or Ca2+. From Shimomura, 1981, with permission from the Federation of the European Biochemical Societies.

These values equal 2.0, 1.05, and 0.5, respectively, for PP, DPAcN, and PPA. It is possible that the contribution of excited states caused by n - it transitions accounts basically for a bathochromic luminescence of some PCSs and for a shift of the maxima in the luminescence spectra of polymers of this kind when proceeding from the solution to the solid phase. PCS solutions reveal concentration-quenching accom-... 

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