Analysis of kinetics of dihydroethidium fluorescence with superoxide using xanthine oxidase and hypoxanthine assay

Abstract

Superoxide (O(2) (-)) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O(2) (-) is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O(2) (-) generation and a O(2) (-) sensitive fluorescent dye dihydroethidium (DHE) for O(2) (-) measurement. To quantify O(2) (-) and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE-O(2) (-) interaction to understand the effect of O(2) (-) self-dismutation and to quantify DHE-O(2) (-) reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O(2) (-) interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169 ± 0.059 × 10(3) M(-1) s(-1) for DHE-O(2) (-) reaction that is ~100× slower than the reported value of 2.6 ± 0.6 × 10(5) M(-1) s(-1). In addition, the O(2) (-) self-dismutation has significant effect on DHE-O(2) (-) interaction. A slower reaction rate of DHE with O(2) (-) is more reasonable for O(2) (-) measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O(2) (-). Results suggest that an accurate measurement of O(2) (-) production rate may be difficult due to competitive interference for many factors; however O(2) (-) concentration may be quantified.

Figure 1. Measured DHE and EOH fluorescence as a function of DHE concentration

Figure 1A and B shows the DHE and EOH fluorescence with respect to time for DHE concentration of 2, 5, 10, 20, and 50 M with XOD concentration of 1.5 mU/ml and HX concentration of 0.25 mM for 120 min.

Figure 2. Measured DHE and EOH fluorescence as a function of XOD concentration

Figure 2A and B shows the DHE and EOH fluorescence for XOD concentration of 1.0, 1.5, 2.5 and 5 mU/ml for HX (=0.25 mM) and DHE (=5 mM) for 120 min.

Figure 3. Measured DHE and EOH fluorescence as a function of HX concentration

Figure 3A and B shows the DHE and EOH fluorescence for HX concentration of 0.0625, 0.125 and 0.25 mM with XOD (=1.5 mU/ml) and DHE (=5 M) for 120 min.

Figure 4. O₂⁻ generation rate by HX-XOD system determined by measuring ferrocytochrome c at 550 nm (EC=21 mM⁻¹cm⁻¹)

Figure 4A shows the absorbance of ferrocytochrome c for XOD concentration of 1.0, 1.5, 2.5 and 5 mU/ml for HX (=0.25 mM). Figure 4B shows the absorbance of ferrocytochrome c for HX concentration of 0.0625, 0.125 and 0.25 mM with XOD (=1.5 mU/ml).

Figure 5. Standard curve of DHE concentration vs. DHE fluorescence

Figure 5 shows DHE relative fluorescence unit (RFU) vs. DHE concentration for standard curve.

Figure 6. Measured and Predicted DHE concentration profiles using kinetic analysis

Figure 6A-C shows the kinetic model results (solid lines) for experimental data (discrete symbols) of varying DHE concentration, XOD concentration and HX concentrations, respectively.

Figure 7. Predicted O₂⁻, EOH, and DHE concentration profiles using kinetic analysis

Figure 7 shows the predicted concentration profiles from the kinetic model for DHE and O₂⁻ reaction for 120 min. DHE and EOH concentration are shown on left axis. O₂⁻ concentration is shown on right axis. Figure A–D shows DHE and O₂⁻ reaction under O₂⁻ formation of 8 μM/min and DHE of 50μM, and Figure E–F shows DHE and O₂⁻ reaction under O₂⁻ formation of 1.465μM/min and DHE of 5μM. Figure A and B shows the profile for without and with O₂⁻ self-dismutation, respectively for 2.6 × 10⁵ M⁻¹s⁻¹ reaction rate constant of DHE and O₂⁻ reaction. Figure C and E shows the profile for without O₂⁻ self-dismutation for 2.17 × 10³ M⁻¹s⁻¹ reaction rate constant of DHE and O₂⁻ reaction. Figure D and F shows the profile for with O₂⁻ self-dismutation for 2.17 × 10³ M⁻¹s⁻¹ reaction rate constant of DHE and O₂⁻ reaction