Luminescent solute

Fig. 1.4 Absorption spectrum of a spent luminescence solution of firefly luciferin containing luciferase-oxyluciferin after dialysis in 0.1 M potassium phosphate, pH 7.8. Replotted from the data of Gates and DeLuca, 1975, with permission from Elsevier.

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... 

Recipe is from Schneppenheim et al. (1991). Premixed luminol substrate mix (Mast lmmunosystems, Amersham, Du Pont NEN Renaissance, or Kirkegaard Perry Lumi-GLO) may also be used. For selection of appropriate luminescent solutions, and for definition of abbreviations, see Table B3.4.1. 

Luminescence decay curves are also often used to verify that samples do not contain impurities. The absence of impurities can be established if the luminescence decay curve is exponential and if the spectrum does not change with time after pulsed excitation. However, in some cases, the luminescence decay curve can be nonexponential even if all of the luminescing solutes are chemically identical. This occurs for molecules with luminescence lifetimes that depend upon the local environment. In an amorphous matrix, there is a variation in solute luminescence lifetimes. Therefore, the luminescence decay curve can be used as a measure of the interaction of the solute with the solvent and as a probe of the micro-environment. Nag-Chaudhuri and Augenstein (10) used this technique in their studies of the phosphorescence of amino acids and proteins, and we have used it to study the effects of polymer matrices on the phosphorescence of aromatic hydrocarbons (ll). 

Colorless, non-luminescent solutions of [AuI C(NHMe)2 2](PF6)-0.5(acetone) become intensely luminescent when they are frozen in a liquid N2 bath [48]. Strikingly, the colors of the emission vary in different solvents and appear only after the solvent has frozen. The frozen acetonitrile solution produces a green-yellow luminescence, with dimethyl sulfoxide and pyridine the emission is different shades of blue, with acetone it is orange, but with dimethyl-formamide no luminescence is observed. The process is entirely reversible ... 

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. 

Sample solution is diluted 40 times by 600 mmol/L sodium hydroxide, the diluted sample solution 50 /iL is poured into the plate. After addition of a luminescence solution (40 mmol/L potassium hexacyanoferrate (II ) and 0.1 mmol/L potassium hexacyanoferrate (III) containing 5 /nmol/L luminol solution), luminescence intensity was measured. Calibration curve was obtained by measurement of the chemiluminescence intensity using GPL as a standard, and the relative amount of the glycated protein in hair was computed. 

Chemiluminescence intensities were obtained as follows xanthine oxidase (0.37 units/mL, 40 pL) was added to the mixture consisting of 20 mM Mops/0.2 M KCl (pH 7.2, 0.5 mL), 0.3 mM hypoxanthine (0.5 mL), and 25 mM probe in water at 25 °C, then the reaction mixture was placed in an Aloka Luminescence Reader BLR-301 and chemiluminescent intensity time curves were obtained at 25 °C. Immediately after xanthine oxidase was added, the chemiluminescence with maximum intensity was observed. The intensity of background chemiluminescence was measured before the addition of xanthine oxidase. Chemiluminescence spectra were obtained as follows the luminescence solution was placed in a JASCO FP-750DS spectrofluorometer and spectra were obtained without light-irradiation. 

Cu (CH3CN)4]C104 to the acetonitrile solution, the Cu ion is complexed by the tetrathia subunit, but the fluorescence spectrum is not altered at all. This indicates that the CT excited state does not involve the tetra-thia ring, which is now engaged in the coordination of the Cu centre. Moreover, on addition of NOBF4, oxidation to the [Cu kthiacyAn)] form occurs and fluorescence is almost completely quenched. On the other hand, on dissolution in acetonitrile of the isolated blue solid complex [Cu thiacyAn)]( 104)2/ a non-luminescent solution is obtained. 

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