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. 2022 Mar 8;11(3):518.
doi: 10.3390/antiox11030518.

Haloperoxidase-Catalyzed Luminol Luminescence

Affiliations

Haloperoxidase-Catalyzed Luminol Luminescence

Robert C Allen. Antioxidants (Basel). .

Abstract

Common peroxidase action and haloperoxidase action are quantifiable as light emission from dioxygenation of luminol (5-amino-2,3-dihydrophthalazine-1,4-dione). The velocity of enzyme action is dependent on the concentration of reactants. Thus, the reaction order of each participant reactant in luminol luminescence was determined. Horseradish peroxidase (HRP)-catalyzed luminol luminescence is first order for hydrogen peroxide (H2O2), but myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are second order for H2O2. For MPO, reaction is first order for chloride (Cl-) or bromide (Br-). For EPO, reaction is first order for Br-. HRP action has no halide requirement. For MPO and EPO, reaction is first order for luminol, but for HRP, reaction is greater than first order for luminol. Haloperoxidase-catalyzed luminol luminescence requires acidity, but HRP action requires alkalinity. Unlike the radical mechanism of common peroxidase, haloperoxidases (XPO) catalyze non-radical oxidation of halide to hypohalite. That reaction is second order for H2O2 is consistent with the non-enzymatic reaction of hypohalite with a second H2O2 to produce singlet molecular oxygen (1O2*) for luminol dioxygenation. Alternatively, luminol dehydrogenation by hypohalite followed by reaction with H2O2 yields dioxygenation consistent with the same reaction order. Haloperoxidase action, Cl-, and Br- are specifically quantifiable as luminol luminescence in an acidic milieu.

Keywords: chemiluminescence; eosinophil peroxidase; halide oxidation; haloperoxidase; horseradish peroxidase; kinetic analysis; luminol luminescence; myeloperoxidase; reaction order; singlet molecular oxygen.

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Conflict of interest statement

Exoxemis, Inc produces MPO and EPO and owns intellectual property (IP) regarding the diagnostic and therapeutic applications of haloperoxidases. I am the inventor of IP described in patent US005556758A that was filed in 1994 and issued in 1996. I continue to consult for Exoxemis, Inc but I do not own this IP.

Figures

Figure 1
Figure 1
Plot of luminescence velocity against H2O2 concentration using 78 nM MPO with 100 mEq/L Cl and 51 µM luminol in 50 mM acetate buffer at pH 5.0. Reaction was initiated by contact mixing of all components. The final volume was 0.9 mL.
Figure 2
Figure 2
Luminescence velocity plotted against H2O2 under conditions as described in Figure 1. (A) depicts the plot of velocity against H2O2 to a concentration of 6.3 mM. (B) shows the standard double reciprocal (Lineweaver–Burk) plot of 1/velocity (1/(kcts/s)) against 1/[H2O2] (1/mM). (C) shows that linearity is achieved when the reciprocal of velocity (1/(kcts/s)) is plotted against the 1/[H2O2]2 (1/(mM)2). (D) shows that linearity is also achieved when the reciprocal of the square root of the velocity (1/√velocity) (1/√(kcts/s)) is plotted against 1/[H2O2] (1/mM).
Figure 3
Figure 3
Plots of MPO-catalyzed luminol luminescence against halide concentration expressed in milliequivalents per liter (mEq/L). (A) shows the hyperbolic relationship of velocity to increasing [Cl] (mEq/L). (B) presents the double reciprocal plot of 1/velocity (1/(kcts/s)) against 1/[Cl] (1/(mEq/L)) showing proper linearity. (C) depicts the hyperbolic response to increasing [Br] (mEq/L). (D) shows the double reciprocal plot of 1/velocity (1/(kcts/s)) against 1/[Br] (1/(mEq/L)) showing suitable linearity. Cl and Br concentrations were varied with a constant concentration of 78 nM MPO, 51 µM luminol, and 6.3 mM H2O2 in 50 mM acetate buffer pH 5. Reaction was initiated by mixing of reactants. The final volume was 1.0 mL.
Figure 4
Figure 4
Plots of EPO catalyzed luminol luminescence against halide concentration. (A) shows very low velocity to all [Cl] (mEq/L) tested. (B) shows the double reciprocal plot of 1/velocity (1/(kcts/s)) against 1/[Cl] (1/(mEq/L)) with a coefficient of determination (R2) of 0.19. (C) depicts the hyperbolic response to increasing [Br] (mEq/L). (D) shows the double reciprocal plot of 1/velocity (1/(kcts/s)) against 1/[Br] (1/(mEq/L)) showing proper linearity. Cl and Br concentrations were varied using a constant concentration of 39 nM EPO, 51 µM luminol, and 6.3 mM H2O2 in 50 mM acetate buffer pH 5. Reaction was initiated by mixing of reactants. The final volume was 1.0 mL. Background luminescence in the absence of enzyme is about 1.5 kcts/s.
Figure 5
Figure 5
Plots of luminescence velocity against luminol concentration for MPO, EPO, and HRP. The top graphics present the results using 100 nM MPO with 100 mEq/L Cl (A), the middle graphics present the results using 100 nM EPO with 10 mEq/L Br (C), and the bottom graphics present the results using 100 nM HRP without halide (E). The plots to the right show double reciprocal plots of 1/velocity (1/(kcts/s)) against 1/[luminol] (1/(µM)) for MPO (B) and EPO (D). For HRP, the double reciprocal plots show 1/√velocity (1/√(kcts/s)) against 1/[luminol] (1/(µM)) (F). MPO and EPO activities were measured in 50 mM acetate buffer at pH 5. HRP activity was measured in 50 mM phosphate buffer at pH 7. Reaction was initiated by mixing reactants with 2.5 mM H2O2. The final volume was 1.0 mL.
Figure 6
Figure 6
Luminol luminescence velocity plotted against MPO, EPO, and HRP concentration under acid and alkaline conditions. Varying concentrations of MPO (A,B), EPO, (C,D) and HRP (E,F) activities were measured in 50 mM acetate buffer at pH 5.0 (A,C,E) and in 50 mM phosphate buffer at pH 8.2 (B,D,F). MPO, EPO, and HRP were measured with 100 mEq/L Cl and with 2.5 mEq/L Br. The luminol concentration was 50 µM. Reaction was initiated by mixing reactants with 2.5 mM H2O2. The final volume was 1.0 mL. Velocity was measured as kilocounts collected during the initial 10 s post mixing and expressed as kcts/10 s.

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