Hydroxyl radical generation: the effect of bicarbonate, dioxygen and buffer concentration on pH-dependent chemiluminescence.

Enhanced chemiluminescence (CL) induced by HO* was detected using the primary enhancers luminol (Lum), isoluminol (ILum) and lucigenin (Luc), in the absence of HCO3-, at pH 9.5, 10.0 and 10.5 but not between pH 4.0 and 9.0. This was confirmed using nine different HO* generators. FeCl3/NTA/H2O2 was the only HO* generator that was able to generate singular HO* which was obtained entirely from the Fenton reaction. However, this was so only at pH 10.0 since at all other pHs multiple ROS were produced. This was confirmed by the chemical detection of the fluorescent hydroxylated product of terephthalic acid in the absence of O2. No HO* Lum-CL pH optima coincided with the O2*- mediated Lum-CL optima found at pH 8.0 and 9.0. Bicarbonate had an enhancing effect on Lum-CL which was 70-2700% at pH 10.0 for the different HO* generators. This was due to the conversion of the radical-electron from HO* to CO3*-, making CL detection more efficient since less HO* were lost initially before detection. Methyl-cypridine-luciferin analogue (MCLA) elicited CL in the pH range 4.0-10.0 with the same set of generators in the absence of HCO3-. The iron-containing generators had their different MCLA-CL optima at pH 4.5, 5.0 or 6.0, excluding those overlapping with the O2(*-)-mediated CL optima. The two copper-containing generators had optima at the same pHs, viz, 7.0 and 10.5. Again, FeCl3/NTA/H2O2 was the only HO* generator able to produce singular HO* by the Fenton reaction. However, whereas Lum-CL was able to detect singular HO* only at pH 10.0, MCLA-CL detected it at pH 5.0 and 5.5. Therefore, MCLA is the most suitable CL enhancer for physiological assessments since it is the most sensitive enhancer and has HO* CL optima nearer to physiological pH than the other probes. The HCO3- enhancement of MCLA-CL was even greater than that of Lum-CL, since increases of 114-fold and 37-fold, respectively, were obtained at these HO*-specific pH optima for FeCl3/NTA/H2O2. Therefore, bicarbonate concentration is as important a parameter as pH when the enhanced CL of a non-cellular system is determined. Hydrogen peroxide was not able to elicit CL directly but, due to trace metal contamination, it produced artifactual CL due to HO* formation. High H2O2 levels, which prevent spontaneous O2*- dismutation, helped to establish the overlapping pH optima of CL mediated by O2*- and HO* which were artifactually produced either by O2*- via H2O2 and trace metals or by perferryl intermediates, respectively. Due to spontaneous dismutation to H2O2, only 22% of the O2*- produced by HX/XO could be detected by enhanced CL.