Fl3 11 spectrofluorometer

Fluorescence emission spectra of two solutions were generated with excitation wavelength of 350 nm. The first solution contained 5 µM ANS in buffer (10 mM Tris-HCl, pH 8, and 200 mM NaCl). The second solution was similar and contained protein at a final concentration of 5 µM. Samples were equilibrated at room temperature. The fluorescence emission spectra were recorded (Horiba Jobin Yvon FL3-11 Spectrofluorometer). The cuvette length was 1 cm. Measurements were conducted at 4, 25, 37, and 50°C. Baseline corrections were made with buffer lacking protein and ANS.

The aryl hydrocarbon hydroxylase (AHH) activity was measured by the fluorometric method described for human cultured lymphocytes by Gurtoo et al. (1975) (link) with modifications made in Atlas et al. (1976) (link). Briefly, the incubation mixture consisted of 50 mM Tris–HCl, pH 8.5, 0.36 mM NADPH, 0.42 mM NADH (4.2 mM), 3 mM MgCl₂, 0.7 mg/ml BSA, 0.2 M sucrose, and DDW, and 10–15 μl lymphocytes (∼2.5 mg/ml, ∼5 × 104 cells/ml) in a total volume of 0.25 ml. The reaction was initiated by adding 2 mM benzo(a)pyrene followed by incubation at 37°C in a shaking incubator for 1 h. The reaction was stopped by the addition of 0.75 mL cold acetone:hexane (1:3) mixture. Aliquots of 0.25 ml were taken from the organic upper phase [containing the Hydroxy-Benzo-Pyrene (OH-BP) reaction product], added to 0.75 ml 1 M NaOH. The lower fraction containing water soluble OH-BP was transferred in a new Eppendorf tube. Fluorescence emission was measured at 596 nm under excitation at 515 nm using a Horiba Jobin Yvon FL3-11 Spectrofluorometer and supplied software. The AHH activity was expressed in picomol OH-BP/min/mg protein.