Cary 100 bio

The APX assay was performed according to a method described by Mishra et al. [36 (link)], with modifications. The reaction mixture (1 mL) consisted of 500 μL 50 mM phosphate buffer (pH 7.0), 200 μL 0.1 mM H₂O₂, 150 μL 0.5 mM sodium ascorbate, 50 μL 0.1 mM EDTA, and 100 μL enzyme. A decrease in absorbance as a result of ascorbate oxidation was measured at 290 nm (Cary 100 Bio, Varian, Australia) for 5 min at 20 °C against a blank in which the enzyme was replaced with phosphate buffer. An extinction coefficient of 2.8 mM⁻¹cm⁻¹ was used to calculate enzyme activity.
A modified method described by Zieslin and Ben-Zaken [37 ] was used for the determination of GPX. The reaction mixture contained 50 μL 0.2 M H₂O₂, 100 μL 50 mM guaiacol, 340 μL distilled H₂O, 500 μL 80 mM phosphate buffer (pH 5.5), and 10 enzymes. An increase in absorbance as a result of tetraguaiacol formation was measured at 470 nm (Cary 100 Bio, Varian, Australia) for 3 min at 30 °C. The blank contained all reagents except for the enzyme, which was replaced with phosphate buffer. An extinction coefficient of 26.6 mM⁻¹cm⁻¹ was used to calculate enzyme activity.
Protein concentration determination was done according to Bradford [38 (link)] using γ-globulin as a standard in order to calculate specific enzyme activities.

To determine melting temperatures, T_m, UV absorption was recorded at or 295 nm as a function of temperature using a Varian UV–visible spectrophotometer (Cary 100 Bio). Fluorescence measurements of 2-aminopurine (2AP) (ex 310 nm, em 370 nm) were performed using a Varian spectrophotometer (Cary Eclipse). In a typical experiment, oligonucleotide samples were mixed and diluted into the desired buffers in optical cuvettes. The solutions were incubated at 95 °C for a few minutes in the cell holder prior to ramping to the desired starting temperature. The melting experiments were performed at a heating rate of 1 °C/min.

A Cary 100 Bio UV–visible spectrophotometer was used to record the absorbance spectra, at 25°C, pH 7.0. Experiments were performed in a 1-ml phosphate buffer solution (200 mM, pH 7.4), and then supplemented with fixed amount of (CN)₂-Cbi (10 µM) followed by increasing concentrations of HOCl. Reaction completion was assured after 2 hours of incubation, and methionine (5-fold of the final HOCl concentration) was added to eliminate excess HOCl, and absorbance changes were recorded from 300 to 700 nm.