[Fluorescence quenching assay of ultratrace horseradish peroxidase using rhodamine dye].

In acetate buffer solution, the reaction of H2O2 with KI was catalyzed by horseradish peroxidase (HRP) to form I3-. The I3- combined respectively with rhodamine S(RhS), rhodamine 6G(Rh6G), rhodamine B(RhB) and butyl-rhodamine B(b-RhB) to form RhS-I3, Rh6G-I3, RhB-I3 and b-RhB-I3 association particles, resulting in the fluorescence quenching at 580, 580, 554 and 554 nm, respectively. The effect of pH value, rhodamine dye concentration, KI concentration, H2O2 concentration, reaction temperature and time on the fluorescence quenching intensity (deltaF) of the four catalytic systems was considered respectively. For the RhS, Rh6G, RhB and b-RhB catalytic systems, pH 4.6-3.2 x 10(-5) mol x L(-1) RhS-4 x 10(-3) mol x L(-1) KI-1.30 x 10(-5) mol x L(-1) H2O2-25 degrees C-20 min, 4.8-2.4 x 10(-5) mol x L(-1) Rh6G-4 x 10(-3) mol x L(-1) KI-2.59 x 10(-5) mol x L(-1) H2O2-25 degrees C-20 min, 4.6-1.6 x 10(-5) mol x L(-1) RhB-4 x 10(-3) mol x L(-1) KI-2.16 x 10(-5) mol x L(-1) H2O2-25 degrees C-20 min, and 4.6-1.6 x 10(-5) mol x L(-1) b-RhB-4 X 10(-3) mol x L(-1) KI-3.02 x 10(-5) mol x L(-1) H2O2-25 degrees C-20 min were chosen for use respectively. Under the optimal conditions, the HRP linear range was 8-6 400 pg x mL(-1) for the RhS catalytic system, 40-4 000 pg x mL(-1) for the Rh6G catalytic system, 32-3 200 pg x mL(-1) for the RhB catalytic system and 40-6 400 pg x mL(-1) for the b-RhB catalytic system, with a detection limit of 3.2, 3.0, 2.4 and 3.7 pg x mL(-1) HRP, respectively. The regress equation of the four catalytic systems was deltaF = 0.061 1c+39.6, deltaF = 0.047 2c+50.4, deltaF = 0.138 6c+34.2 and deltaF = 0.026 25c+36.72, with a correlation coefficient of 0.997 9, 0.999 0, 0.997 3 and 0.996 9, respectively. The RhS catalytic system was most sensitive, and was chosen for the determination of HRP. The influence of foreign substance on the RhS assay of 3.5 ng x mL(-1) HRP was examined, with a relative error of +/- 10%. A 3000-times L-glutamic acid, L-lysine, Ca2+, Mg2+, Cu2+, Fe3+, Zn2+ and vitamin B6, 1000-times HAS etc did not interfere with the assay. This showed that the assay has good selectivity. The RhS fluorescence quenching assay was applied to the determination of HRP in the solution of hepatitis B surface antibody labeling HRP, with satisfactory results.

[Resonance scattering spectral assay of papain enzymatic activity with sodium dodecyl benzene sulfonate].

In pH 6.5 phosphate buffer solutions, dodecyl benzene sulfonate (SDBS) was combined with casein to form association particles, which exhibited five Rayleigh scattering peaks at 470, 360, 400, 420 and 520 nm, respectively. Under suitable conditions, papain has catalytic effect on the hydrolysis of casein, and SDBS can stop the catalytic reaction and be combined with the excess casein to form association particles. The scattering peak at 470 nm decreased with the activity of papain. The delta I470 value was linear with the papain activity in the range of 0.048-4.8 USP x mL(-1). Its regress equation is delta I(SDBS) = 1.972c + 2.31, with a related coefficient of 0.9999 and detection limit of 0.020 USP x mL(-1). This new assay has been applied to the assay of the papain activity in food additive with satisfactory results.

Resonance scattering spectra of micrococcus lysodeikticus and its application to assay of lysozyme activity.

BACKGROUND: Several methods, including turbidimetric and colorimetric methods, have been reported for the detection of lysozyme activity. However, there is no report about the resonance scattering spectral (RSS) assay, which is based on the catalytic effect of lysozyme on the hydrolysis of micrococcus lysodeikticus (ML) and its resonance scattering effect. RESULTS: ML has 5 resonance scattering peaks at 360 400, 420, 470, and 520 nm with the strongest one at 470 nm. The concentration of ML in the range of 2.0x10(6)-9.3x10(8) cells/ml is proportional to the RS intensity at 470 nm (I(470 nm)). A new catalytic RSS method has been proposed for 0.24-40.0 U/ml (or 0.012-2.0 mug/ml) lysozyme activity, with a detection limit (3sigma) of 0.014 U/ml (or 0.0007 microg/ml). Saliva samples were assayed by this method, and it is in agreement with the results of turbidimetric method. The slope, intercept and the correlation coefficient of the regression analysis of the 2 assays were 0.9665, -87.50, and 0.9973, respectively. CONCLUSION: The assay has high sensitivity and simplicity.