Citation: Allen, R.C.
Haloperoxidase-Catalyzed Luminol
Luminescence. Antioxidants 2022, 11,
518. https://doi.org/10.3390/
antiox11030518
Academic Editor: Ernst Malle
Received: 27 January 2022
Accepted: 4 March 2022
Published: 8 March 2022
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antioxidants
Article
Haloperoxidase-Catalyzed Luminol Luminescence
Robert C. Allen
Department of Pathology, Creighton University, Omaha, NE 68178, USA; robertallen@creighton.edu;
Tel.: +1-402-350-3193
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 (H
2
O
2
), but myeloperoxidase (MPO) and eosinophil
peroxidase (EPO) are second order for H
2
O
2
. 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 H
2
O
2
is
consistent with the non-enzymatic reaction of hypohalite with a second H
2
O
2
to produce singlet
molecular oxygen (
1
O
2
*) for luminol dioxygenation. Alternatively, luminol dehydrogenation by
hypohalite followed by reaction with H
2
O
2
yields dioxygenation consistent with the same reaction
order. Haloperoxidase action, Cl
-
, and Br
-
are specifically quantifiable as luminol luminescence in
an acidic milieu.
Keywords: haloperoxidase; myeloperoxidase; eosinophil peroxidase; horseradish peroxidase; halide
oxidation; singlet molecular oxygen; luminol luminescence; chemiluminescence; reaction order;
kinetic analysis
1. Introduction
Myeloperoxidase (MPO) enzymatic action produces light emission or chemilumi-
nescence. Such luminescence is native, i.e., no chemiluminigenic substrate is needed,
requires H
2
O
2
, halide, and acidic pH [1,2], and correlates with the requirements for MPO
microbe killing described by Klebanoff [3]. Both luminescence and microbicidal action are
hydrogen peroxide (H
2
O
2
), chloride (Cl
-
), and acid-dependent. Light emission implies
haloperoxidase-catalyzed combustive oxygenation. MPO catalyzes H
2
O
2
oxidization of
halide to hypohalite, e.g., Cl
-
oxidation to hypochlorite (OCl
-
). The non-enzymatic reac-
tion of hypohalite with a second H
2
O
2
produces electronically excited singlet molecular
oxygen (
1
O
2
*) [4–6]. Both H
2
O
2
and OCl
-
are singlet multiplicity reactants necessitating
a single multiplicity product [7,8]. The relaxation of
1
O
2
* to its triplet ground state (
3
O
2
)
requires intersystem crossing, and as such,
1
O
2
* has about a microsecond lifetime [9,10].
This lifetime is sufficient for reactivity, but such reaction is restricted to within a radius of
about 0.3 microns (micrometer) of its nascence.
Spin conservation and frontier orbital considerations restrict the direct reaction of
ground-state triplet multiplicity oxygen (
3
O
2
) with singlet multiplicity biomolecules. Thus,
combustion is not spontaneous, and the large exergonicity that would result from such
action is unrealized. Spin and frontier orbital considerations do not limit
1
O
2
* reaction with
biomolecules. The electrophilic reactivity of
1
O
2
* drives oxygenation of organic molecules,
and a fraction of the reaction products will have electronically excited singlet multiplicity
carbonyl functions that relax by emitting photons in the visible spectrum.
Antioxidants 2022, 11, 518. https://doi.org/10.3390/antiox11030518 https://www.mdpi.com/journal/antioxidants