Open access peer-reviewed chapter

Essence of Reducing Equivalent Transfer Powering Neutrophil Oxidative Microbicidal Action and Chemiluminescence

Written By

Robert C. Allen

Reviewed: September 17th, 2018 Published: November 5th, 2018

DOI: 10.5772/intechopen.81543

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Neutrophil leukocytes provide first-line phagocytic defense against infection. Phagocyte locomotion to the site of infection, identification, and phagocytosis of the infecting microbe results in metabolically driven O2-dependent combustive microbicidal action. NADPH oxidase activity controls this respiratory burst metabolism. Its flavoenzyme character allows semiquinone-mediated crossover from two reducing equivalents (2RE) to 1RE transfer, as is necessary for univalent reduction of O2 to the acid hydroperoxyl radical (HO2) and its conjugate base, superoxide anion (O2−). RE transfer dynamics is considered from the perspectives of quantum and particle physics, as well as frontier orbital interactions. Direct disproportionation of HO2-O2− yields electronically excited singlet molecular oxygen (1O2*) and hydrogen peroxide (H2O2). Myeloperoxidase catalyzes H2O2-dependent 2RE oxidation of chloride (Cl−) to hypochlorite (OCl−). Direct nonenzymatic reaction of OCl− with an additional H2O2 yields Cl−, H2O, and 1O2*. Thus, for two 2RE metabolized through NADPH oxidase, a total of three 1O2* are possible. H2O2, OCl−, and 1O2* generated are all singlet multiplicity reactants and can participate in spin-allowed combustive oxygenations yielding light emission, that is, luminescence or chemiluminescence. The sensitivity of luminescence for measuring neutrophil redox activities is increased several orders of magnitude by introducing chemiluminigenic probes. Probes can be selected to differentiate oxidase from haloperoxidase activities.


  • neutrophil
  • respiratory burst
  • reducing equivalent
  • combustion
  • frontier orbital
  • spin quantum number
  • NADPH oxidase
  • myeloperoxidase
  • Wigner spin conservation
  • Hund’s maximum multiplicity rule
  • boson
  • fermion

1. Introduction

There is a complicated hypothesis, which usually entails an element of mystery and several unnecessary assumptions. This is opposed by a more simple explanation, which contains no unnecessary assumptions. The complicated one is always the popular one at first, but the simpler one, as a rule, eventually is found to be correct. This process frequently requires 1020 years. The reason for this long time lag was explained by Max Planck. He remarked that scientists never change their minds, but eventually they die.

John H. Northrop, 1961 [1].

Appreciating why combustion is not spontaneous, how electrons are transferred biologically, and the unusual nature of oxygen reactivity were difficult for me as a student. So, in addition to biochemical studies, my mentor Richard Steele suggested I study the writings of Herzberg and others. Although challenging, such exposure shook open the door to other perspectives. Fundamental quantum and particle physics considerations were entertained with regard to oxygen and biologic electron transfer. My epiphany was in recognizing that the polymorphonuclear neutrophil, a leukocyte familiar to me from clinical laboratory experience, might realize the electronegative potential of oxygen for combustive microbicidal action by changing the spin multiplicity of oxygen. The following, taken from a symposium abstract presented in 1972, succinctly describes that position [2]. “Recently, a chemiluminescence (CL) has been observed when human polymorphonuclear leukocytes (PMN) phagocytize bacteria or particulate matter. The CL response correlates well with the stimulation of the hexose monophosphate shunt, which results in the generation of NADPH. The PMN possesses both CN-insensitive NADH and NADPH oxidases. Flavoproteins oxidases of this type are capable of univalent reduction of O2. The reduced oxygen ( · O2, · O2H) may then disproportionate yielding HOOH and singlet molecular oxygen 1O2. The PMN also possesses a CN-sensitive peroxidase, myeloperoxidase, which has microbicidal activity in the presence of HOOH and halide. In this reaction, the HOOH is reduced to OH with the oxidation of the halide to a reactive halogonium species. In cases where the halogonium formed is Cl+ or Br+, there is potential for further reaction with HOOH resulting in the generation of a haloperoxy anion. This unstable species can disintegrate to yield the original halide and 1O2. 1O2 has been demonstrated to be a potent microbicidal agent. Therefore, the biochemical generation of 1O2 by the PMN might be closely associated with microbicidal activity. The CL response may be the result of the relaxation of excited carbonyl groups generated via 1O2-mediated oxidations.”

Neutrophil leukocytes and monocytes play an essential role in innate phagocytic defense against infection. Immune surveillance mechanisms detect the presence of potentially pathologic microbes and generate the chemical signals that mobilize circulating neutrophils and prime the expression of receptors necessary for neutrophil navigation and phagocytosis. Contact of a primed neutrophil with activated endothelium is followed by neutrophil diapedesis into the tissue interstitial space, and locomotion to the site of infection guided by concentration gradients of complement anaphylatoxin, microbial products, cytokines, and lipid factors. Once an immunologically primed neutrophil contacts an opsonin-labeled pathogen, phagocytosis occurs. Phagocytosis is associated with a constellation of metabolic changes classically referred to as the “respiratory burst” [3]. This presentation focuses on the neutrophil redox mechanisms necessary for microbicidal action, especially the roles of NADPH oxidase and myeloperoxidase (MPO) in lethal microbicidal oxygenations. The Merriam-Webster dictionary defines combustion as a chemical reaction that occurs when oxygen combines with other substances to produce heat and usually light. By changing the spin multiplicity of oxygen from triplet to doublet, and then to singlet, neutrophils remove the spin barrier to direct oxygenation, enabling direct oxygen combustive microbicidal action with associated light emission, that is, chemiluminescence or luminescence [4].


2. Respiratory burst

The neutrophil “respiratory burst” describes the large increases in glucose consumption via the hexose monophosphate shunt (aka, pentose pathway) [5, 6], and in nonmitochondrial molecular oxygen (O2) consumption [7] associated with phagocytosis, and required for microbicidal action. Appreciating the underlying necessity for such metabolic changes provides perception into oxygen chemistry and biochemistry, radical reactivity and combustion in general. The character of electron transfer mediated by the dehydrogenases of the hexose monophosphate (HMP) shunt is common to cytoplasmic redox reactions. Such oxidation-reduction transfers typically involve movement of two reducing equivalents (2RE), that is, 2 electrons (e) and 2 protons (H+), from an organic substrate catalyzed by a dehydrogenase. In turn, the dehydrogenase mobilizes the 2RE by transfer to nicotinamide adenine dinucleotide (phosphate) NAD(P)+ generating its reduced form NAD(P)H. The cofactors NADPH and NADH serve as the cytoplasmic redox carriers for 2RE transfers between dehydrogenases and oxidases, and are common to various pathways of cytoplasmic metabolism. Consumption of 2RE carried by NADPH returns it to NADP+. Availability of NADP+ is rate limiting for HMP shunt dehydrogenase activity. Dehydrogenation is a type of oxidation that does not require or directly involve O2. Glucose-6-phosphate (G-6-P) dehydrogenase, the initiator enzyme of the HMP shunt removes a total of 2RE and transfers the 2RE to NADP+ producing NADPH. The point for emphasis is that 2RE are transferred, not one. Such 2RE transfer, sometimes referred to as hydride ion (H) transfer, is the rule for cytoplasmic redox reactions [8].

Respiratory burst metabolism results from the activation of NADPH oxidase. Like many oxidases, NADPH oxidase is a flavoenzyme. Flavoenzymes are mechanistically unique in that 2RE reduction, by cofactors such as NAD(P)H, is followed by a series of 1RE oxidations. In its 1RE form, the riboflavin prosthetic group of flavin adenine dinucleotide (FAD) is in the semiquinone state [9, 10]. This semiquinone capability, usually in combination with a cytochrome component, allows the oxidase to transition from 2RE transfer to 1RE transfers. As such, flavoenzymes are the junction enzymes where 2RE transfer proceeds as 1RE cytochrome transfers, for example, the mitochondrial electron transport system or the microsomal cytochrome-P450 mixed-function oxidase system [10, 11]. Flavoprotein oxidases are also capable of catalyzing the 1RE reduction of O2 [12, 13]. As such, phagocytosis-associated activation of NADPH oxidase opens the possibility for univalent, that is, 1RE, reduction of O2.

The molecular oxygen we breathe has unique physical-chemical characteristics. In its ground, that is, lowest energy state, oxygen is a diradical, paramagnetic molecule with triplet spin multiplicity [3O2; the preceding superscripted (3) indicates multiplicity]. These spin characteristics guarantee a tendency for 3O2 to participate in 1RE reduction yielding the doublet multiplicity hydroperoxyl radical (2HO2) and its conjugate base, the superoxide anion radical (2O2) [2, 4, 14, 15]. Such reduction does not produce radical character; it decreases such character.

2.1. Bosonic character of coupled fermionic electron transfer

Movement of 2RE is the transfer of an electron couple, that is, an orbital pair of electrons. Such 2RE transfers are the rule in cytoplasmic redox reactions. Considered from the perspective of particle physics, movement of a single electron (1RE) is quite different from paired electron (2RE) movement. Transfer of 1RE is a fermionic transfer. An electron is a fermion, and fermions have wave functions that are antisymmetric to exchange of particles; that is, Ψ (a, b) = −Ψ (b, a). Fermions anti-commute; that is, a × b ≠ b × a. Rotating a fermion through 360°, Ψ — 360° → −Ψ, changes the phase, but does not return the fermion to its original state. An additional 360° rotation, −Ψ —360° → Ψ, is required to return the antisymmetric particle to its original state [16]. Fermions obey Fermi-Dirac statistics.

A fermionic electron is defined by its five quantum numbers: n, l, ml, s, and ms [17]. The spin number, s, describes the intrinsic angular momentum of the electron independent of orbital motion, and has a value of ½ħ (abbreviated to ½). This quality has no analogy in classical physics. The total spin angular momentum, S, of an atom or molecule is expressed by the equation S = √[s(s + 1)]ħ. s gives rise to the quantum number ms, and only two values are allowed. When ms = ½, the e is described as spin up (↑); when ms = −½, the e is described as spin down (↓). The Pauli exclusion principle states that the total wave function for a system must be antisymmetric to the exchange of any pair of electrons. Differently stated, no two electrons of a given atom or molecule can have identical quantum numbers, and for two electrons to occupy an orbital, each electron must have opposite spins, that is, one orbital e must have an ms = ½ (↑), the other orbital e must have an ms = −½ (↓). Consequently, the total spin quantum number, S, for a filled orbital electron-couple is ½ + −½ = 0 (↑↓).

Bosons obey Bose-Einstein statistics, and have wave functions that are symmetric to exchange of a pair of particles; that is, Ψ (a, b) = Ψ (b, a). They obey ordinary commutation, that is, a × b = b × a. Rotating a boson through 360°, Ψ — 360°  Ψ, returns it to its original state. Bosons, for example, photons are symmetric particles with integral spin. Likewise, a spin-balanced composite of fermionic particles, for example, an alpha particle with an S of 0, is bosonic. With regard to biochemical redox reactions, the coupling of antisymmetric fermions, for example, the coupled electrons of an orbital pair, result in a S = 0 state with bosonic symmetry. The transfer of 2RE describes the movement of a coupled electron pair with an S = 0 and is in essence a bosonic transfer.

2.2. Bosonic versus fermionic frontier orbital interactions

Chemistry is about the frontier orbital interactions of atoms and molecules [18]. The focus of frontier orbital theory is on the initial orbital conditions of the reactants and on reactive transition to product(s) with emphasis on the highest occupied atomic or molecular orbital (HO(A)MO) and the lowest unoccupied atomic or molecular (LU(A)MO) orbital. The frontier orbital of a radical reactant is neither empty nor completely filled, and as such, is described as a singly occupied atomic or molecular orbital (SOAO or SOMO). Atomic and molecular orbitals, including frontier orbitals, can have bosonic or fermionic character [19, 20]. A HO(A)MO has an S = 0. Such an atom or molecule has singlet spin multiplicity with nonradical, diamagnetic character. A radical SO(A)MO has an S = ½ or −½, and has doublet spin multiplicity with radical, paramagnetic character.

The bosonic character of the HOMO of a nonradical reactant differs fundamentally from the fermionic character of the SOMO of a radical reactant. The fermionic nature of a SOMA limits overlap possibilities with bosonic HOMO. If such reaction occurs, the fermionic character must be preserved in the product. The electronegative Fenton radical (2OH) can extract 1RE from the HOMO of a singlet multiplicity nonradical substrate (1substrate) yielding singlet multiplicity 1H2O, but in the process the HOMO of the substrate is converted to a SOMO, that is, the substrate becomes a doublet multiplicity radical (2substrate). The symmetry of the reactants is preserved in the products. If a fermionic (doublet)-bosonic (singlet) reaction occurs, symmetry will be retained in the bosonic (singlet)-fermionic (doublet) products. Consistent with the Wigner-Witmer rules described in Table 1, spin symmetry is conserved [19, 20, 21, 22].

Reactants Products
Singlet + Singlet
bosonic + bosonic
Singlet + Doublet
bosonic + fermionic
Singlet + Triplet
bosonic + bi-fermionic
Doublet + Doublet
fermionic + fermionic
Doublet + Triplet
fermionic + bi-fermionic
Triplet + Triplet
bi-fermionic + bi-fermionic

Table 1.

Spin conservation rules.

Spin multiplicity states with regard to the bosonic-fermionic character of reactants and products.

The fermionic character of two radical reactants is eliminated in reactive bonding yielding a bosonic product. As described in Table 1, fermionic radical-radical, SOMO-SOMO reaction yields bosonic nonradical product. Simply stated, radicals tend to react with radicals, and such doublet-doublet annihilations yield nonradical, that is, bosonic, product. Such reaction is responsible for terminating radical chain propagation reactions.

Molecular oxygen in its ground state has unique triplet spin multiplicity [23]. Its two degenerate, that is, equal energy, frontier orbitals are each populated by a single electron. These two SOMO electrons obey Hund’s maximum multiplicity rule, that is, the electron in each degenerate SOMO will have the same spin [24]. As illustrated in Figure 1, the S value for molecular oxygen is ½ + ½ or −½ + −½, and thus, the multiplicity is triplet, that is, 2|1 or −1| + 1 = 3. This bi-radical, bi-fermionic character is responsible for the paramagnetic character of 3O2. The high electronegativity of 3O2 predicts potential for highly exergonic reactions with nonradical, singlet multiplicity organic molecules, but thermodynamic potential does not guarantee reactivity, and combustion is not spontaneous. Taking a different perspective, it is the bi-fermionic, bi-radical nature of 3O2 that restricts its reactive potential. As per Table 1, the reaction of 3O2 with a bosonic 1substrate molecules is spin symmetry restricted, and could only result in the improbable generation of a bi-fermionic, triplet multiplicity product(s). However, the reaction of bi-fermionic 3O2 with a fermionic (doublet multiplicity) radical can proceed via SOMO-SOMO overlap. As per Table 1, such a doublet-triplet reaction will generate a fermionic (doublet multiplicity) radical product. Thus, 3O2 can participate in and be a necessary reactant in radical propagation reactions.

Figure 1.

Triplet and electronically excited singlet molecular oxygen with emphasis on the π* (pi antibonding) frontier orbitals. The two π* are degenerate (same energy level). Hund’s maximum multiplicity rule predicts lowest energy is achieved when each SOMO electron has the same spin, that is, the triplet state (3O2). The electronic energy of 1O2* is 22.5 kcal/mol (94.2 kJ/mol) above 3O2.


3. NADPH oxidase

NADPH oxidase controls “respiratory burst” metabolism, microbicidal action, and chemiluminescence [15, 25]. The oxidase (Nox2) is a complex flavoenzyme, and a member of the Nox family of enzymes involved in various biochemical activities [26, 27, 28, 29]. More specifically, NADPH oxidase is a flavocytochrome enzyme composed of a large membrane-bound glycoprotein (gp91phox) subunit associated with a smaller protein (p22phox). The C-terminal portion of gp91phox subunit contains the NADPH and flavin adenine dinucleotide (FAD) binding sites and an N-terminal portion that binds two heme groups. The activation of the oxidase is complex and involves other components. Association with the p67phox component is essential for full activity. The present treatment will focus on the central role of the semiquinone state of the riboflavin component of FAD and heme involvement in splitting the 2RE from 1NADPH and facilitating 1RE reduction of 3O2.

As illustrated in Figure 2, the product of 1RE reduction of 3O2 is the acid hydroperoxyl radical (2HO2) with an acid dissociation constant pKa of 4.8 [30]. For comparison, the pKa of 1H2O2 is 11.7. As the pH of the phagolysosomal space approaches the pKa, the ratio of 2HO2 to its conjugate base, the superoxide anion (2O2) approaches unity, and acid disproportionation, that is, reaction of 2HO2 with 2O2, approaches maximum reaction rate. At unity, anionic repulsion is no longer a problem. The rate constant for the reaction is 4.5 × 105 M−1 s−1 at pH 7.0 and reaches a maximum of 8.5 × 107 M−1 s−1 at pH 4.8 [30, 31]. From the frontier orbital perspective, this is a SOMO-SOMO reaction that yields the nonradical (singlet multiplicity) products 1H2O2 and 1O2*. As per Table 1, doublet-doublet annihilation yields single products [15, 32]. The reaction is sufficiently exergonic to yield 1O2* with an energy of 22.5 kcal/mol (94.1 kJ/mol) above ground state 3O2.

Figure 2.

Schema illustrating the central role of membrane-associated NADPH oxidase in respiratory burst metabolism. In the activated state, the Michaelis constant (KM) of the oxidase for NADPH is decreased. NADP+ availability controls the activities of glucose-6-PO4 dehydrogenase and 6-phosphogluconate dehydrogenase of the HMP shunt. Each pass of the cycle generates two NADPH, that is, two 2RE. In the schema, the spin multiplicities of each molecule are indicated by the superscripted number preceding the molecular description, that is, 1, 2, and 3 for singlet, doublet, and triplet multiplicity, respectively.

In Figure 2, note that all reactions in the cytoplasmic milieu are singlet multiplicity nonradical reactions and that radical production is confined to the phagolysosomal milieu. The 2RE nature of cytoplasmic redox transfer provides a bosonic barrier to reaction with bi-fermionic 3O2. Transfer of an orbital electron couple is nonradical, bosonic, and singlet multiplicity. In an atmosphere that is 20.9% 3O2, the presence of a doublet multiplicity molecule is an opportunity for SOMO-SOMO overlap. The 2RE transfer from the HOMO of a reductant to the LUMO of an oxidant maintains the bosonic S = 0 condition.

The S = 0 condition is described by Dirac’s statement that “If a state has zero total angular momentum, the dynamical system is equally likely to have any orientation, and hence spherical symmetry occurs” [33]. In addition to providing protection from the reactive consequences of fermionic 1RE transfer in an atmosphere high in 3O2, 2RE transfer of a bosonic orbital electron couple may have additional advantage. Heisenberg’s uncertainty principle states that the uncertainty of momentum (Δp) multiplied by the uncertainty of position (Δx) is always equal to or greater than ½ħ, that is, ΔpΔx ≥ ½ħ [17]. With regard to 2RE transfer, the bosonic orbital electron couple has S = 0. Consequently, the positional uncertainty of the electron-couple must be proportionally large. The S = 0 nature of HOMO-LUMO redox transfer involving a 2RE orbital electron-couple opens the possibility that such transfer is facilitated by quantum tunneling. The nature of such transfer would be analogous to the emission of a bosonic S = 0 alpha particle from an atomic nucleus in alpha radiation decay [19, 20].


4. Myeloperoxidase

Myeloperoxidase (MPO) is a unique green cationic homo-dimeric glycosylated heme-a protein that is highly expressed in neutrophil leukocytes, making up about 5% of its dry mass [34, 35]. It is also synthesized to a lesser degree in monocytes and serves as a cellular marker for both neutrophils and monocytes. MPO synthesis occurs only during the promyelocyte phase of neutrophil development [36]. During the promyelocyte phase, MPO and other cationic lysosomal proteins are synthesized and stored in the azurophilic (aka primary) granules. Each mitotic division during the following myelocyte phase of development dilutes the azurophilic granule content per neutrophil by a half. Under normal conditions of hematopoietic production, these myelocytic phase mitoses are the rule, but under condition of neutrophil inflammatory consumption or G-CSF-stimulated marrow production, the promyelocyte pool is expanded, and there are fewer mitoses in the myelocyte phase of development. Neutrophils released into the circulation following a few days of myelopoietic stimulation show the effect of decreased myelocyte mitoses. These neutrophils are significantly increased in size due to greater azurophilic granule retention, and the MPO activity per neutrophil is severalfold higher than normal [37].

4.1. Electrochemistry of halide oxidation-reduction

MPO, like eosinophil peroxidase, lactoperoxidase and thyroperoxidase, is a haloperoxidase (XPO). However, MPO is unique in its ability to catalyze the pH-dependent oxidation of chloride [38, 39, 40]. Based on the Allen scale, fluorine (F) is the most electronegative element with a value of 4.19, followed by oxygen with a value of 3.61, then chlorine with a value of 2.87, bromine with a value of 2.69, and iodine with a value of 2.36 [41].

With regard to chloride oxidation, the Nernstian electrochemical possibilities and limitations are as follows [11, 42].

E = E RT / nF ln reduced / oxidized E1

where E is observed potential (in volts), E₀ is the standard potential (in volts), R is the gas constant, T is the absolute temperature, F is a faraday (23 kcal/absolute volt equivalent), and n is the number of electrons/gram equivalent transferred.

Also, appreciate that hydrogen ion concentration, [H+], has an effect on redox chemistry.

E = RT / F ln H + / P H2 1 / 2 E2

PH2 is the partial pressure of H2 gas

E = 2 . 3RT / F log H + = 0.06 log H + = 0.06 pH E3

For the reaction, Ared + Box ↔ Bred + Dox, the half reaction equations become:

E = E A RT / nF ln A red / A ox E4
E = E B RT / nF ln B red / B ox E5
E B E A = RT / nF ln B red / B ox ln A red / A ox E6
ΔE = RT / nF ln A ox B red / ln A red B ox E7
ΔE = RT / nF ln K eq E8

Keq is the equilibrium constant. The change in potential (ΔE) can be expressed in terms of Gibbs free energy (ΔG).

Δ G 0 = RT ln K eq E9
ΔG 0 = nF ΔE E10

The schema of Figure 3 depicts the MPO-catalyzed H2O2 oxidation of Cl to HOCl. Chloride serves as the reductant and undergoes a 2RE oxidization yielding a chloronium intermediate (Cl+) that reacts with 1H2O to generate hypochlorous acid with a pKa of 7.5.

1 Cl 1 Cl + + 2RE E11
1 Cl + + 1 H 2 O 1 HOCl + H + E12

Figure 3.

Schema depicting myeloperoxidase-catalyzed H2O2-dependent oxidation of chloride to hypochlorite, and its reaction with a second H2O2 to generate 1O2*. The spin multiplicity of each molecule is indicated by the superscripted number preceding the molecular description.

Note that 1H2O2 is the oxidant for the MPO-catalyzed reaction undergoing 2RE reduction yielding two waters. One 1H2O is consumed in the reaction described by Eq. (12).

1 H 2 O 2 + 2RE 2 1 H 2 O E13

The reactants and products of this MPO-catalyzed redox reaction are exclusively singlet multiplicity, that is, nonradical [2, 15].

As depicted in Figure 4, increasing acidity, that is, lowering pH, increases the ΔE (i.e., Eh2o2 − Ex) and the Gibbs free energy for all halides. The exergonicity of MPO-catalyzed 2RE dehydrogenation of Cl increases with increasing acidity. The required potentials for the various halides are consistent with their electronegativities. Dehydrogenation of Cl is more difficult than Br, but dehydrogenation of I is relatively easy. Whereas MPO is capable of dehydrogenating Cl, Br, and I, eosinophil peroxidase (EPO), lactoperoxidase, and thyroperoxidase are only capable of dehydrogenating Br and I.

Figure 4.

Graph A plots changes in potential (ΔE) and graph B plots change in Gibbs free energy against pH for various halides. From bottom to top, the plotted lines represent chloride (lowest), bromide (middle) and iodide (highest).

The plots of Figure 4 illustrate that increasing acidity increases the exergonicity of MPO-catalyzed 1H2O2-dependent oxidation of halides. This is especially import for MPO-catalyzed oxidation of chloride. Conversely, increasing alkalinity increases the exergonicity of the nonenzymatic 1OCl-1H2O2 reaction yielding 1O2* as depicted in Figure 5. The combined 1H2O2-driven haloperoxidase plus 1H2O2-driven 1OCl generation of 1O2* can be considered as a net disproportionation reaction, as depicted in Figure 6. 1H2O2 is the reactant common to both MPO-catalyzed reaction of Figure 4 and the chemical reaction of Figure 5. The Gibbs free energies shown in Figure 6 have been adjusted to reflect the energy conserved in electronically excited 1O2*. The overall net free energy is independent of the halide employed and independent of pH.

Figure 5.

Graph A plots changes in potential (ΔE) and graph B plots change in Gibbs free energy with respect to pH for various halides for the reaction of 1H2O2 with 1OCl. In graph B the Gibbs free energies are adjusted for the 22.5 kcal mol−1 retained as the electronic energy of 1O2*, that is, the difference separating 3O2 from 1O3*.

Figure 6.

Plot of free energy against pH for the net 1H2O2 disproportionation reaction as described in Figure 2. The free energy results are expressed with (ΔG = −27.8 kcal mol−1) and without (ΔG = −50.3 kcal mol−1) adjustment for the energy electronically conserved in oxygen excitation (ΔG = −22.5 kcal mol−1).

Since the reactants involved are all singlet multiplicity, the products of reaction, that is, 1H2O, 1Cl, and 1O2*, are all singlet multiplicity. This provides a spin symmetry explanation as to why pouring bleach (1OCl) into 1H2O2 causes rapid reactive release of 1O2* gas and a red chemiluminescence [23]. Caution, rapid release of gas is potentially explosive. When the concentration of 1O2* is sufficiently high, 1O2*-1O2* collision with simultaneous relaxation yields red chemiluminescence. The relaxation of one 1O2* emits a 1270 nm photon; simultaneous relaxation of two 1O2* emits a 635 nm photon. As such, this red emission is second order with respect to 1O2*, that is, d635nm/dt = k[1O2*]2, and is relatively short-lived.

The double dehydrogenation of 1glucose-6-PO4 produces 1ribulose-5-PO4 plus 1CO2 plus two 2RE, that is, two bosonic electron couples carried as 2NADPH. As illustrated in Figure 2, NADPH oxidase reduces four 3O2 in four one 1RE reduction steps, ultimately yielding two 1O2* and two 1H2O2. As illustrated in Figure 3, MPO uses one 1H2O2 for oxidation of Cl to OCL, and this OCL reacts with the other 1H2O2 to generate an additional 1O2*. Thus, two NADPH have the potential to drive the generation of three 1O2*. Steinbeck et al. have reported experiments using glass beads coated with 9,10-diphenylanthracene, a 1O2*-specific trap, for measurements of neutrophil 1O2* production [43]. Neutrophils were allowed to phagocytose the beads for an hour. The endoperoxide trapped indicated that at least 11.3 ± 4.9 nmol 1O2*/1.25 × 106 neutrophils were produced. When the neutrophils were chemically activated with phorbol-12-myristate-13-acetate (PMA), at least 14.1 ± 4.1 nmol 1O2*/1.25 × 106 neutrophils were produced. Based on their trapping results, 1O2* production accounted for at least 19 ± 5% of the total oxygen consumed. Although the quantities of 1O2* measured using this difficult trapping approach are lower than expected; this study provides direct empirical evidence of significant neutrophil 1O2* production.

Quantifying cellular production of 1O2* by measuring the 1270 nm near-infrared photon emitted on 1O2* relaxation to 3O2 is also problematic. Although highly specific for 1O2*, this infrared proton emission approach is highly insensitive in biological system measurements. The fact that a 1270 nm photon is measured is proof that 1O2* did not participate in chemical reaction. Considering the variety of reactive substrates available in biological milieux, electrophilic reaction is favored over relaxation.

4.2. Myeloperoxidase-binding specificity focuses combustive activity

1O2* is a potent electrophilic reactant with a high probability for participation in spin-allowed reaction with electron-dense biological substrates. The lifetime of metastable electronically excited 1O2* restricts its reactive possibilities [44]. In biological milieux, 1O2* has a reactive lifetime of about 4–6 microseconds [45, 46]. This lifetime restricts reactivity to within a radius of about 0.2–0.3 μm (microns) from its point of generation. In the case of MPO generation of 1O2*, these temporal and spatial restrictions can be advantageous.

MPO selectively binds to all gram-negative bacteria and most gram-positive bacteria tested, but MPO binding is weak for gram-positive lactic acid bacteria (LAB) [44, 47]. LAB are common members of the normal flora of the mouth, vagina, and colon, and include streptococci, lactobacilli, and bifidobacteria. These LAB cannot synthesize cytochromes and produce lactic acid as a metabolic end product. They are typically microaerophilic, and often produce 1H2O2 as a metabolic product. The green hemolysis associated with colonies of viridans streptococci on blood agar plates results from the production of 1H2O2 by the streptococci. When a pathogen, such as Staphylococcus aureus or Escherichia coli, is contacted with a nonpathogen LAB, such as Streptococcus viridans, the pathogen overwhelmingly inhibits the LAB, but when a small quantity of MPO is added to a mixture, the pathogen is inhibited allowing LAB dominance. This phenomenon repeats even when erythrocytes are added to the mix at a ratio of 10 erythrocytes per bacteria. MPO selectively binds to the S. aureus and E. coli with essentially no binding to 1H2O2-producing Strep. viridans. Thus, LAB-produced 1H2O2 drives MPO microbicidal action that is restricted to the surface of the MPO-bound pathogen. MPO combustive microbicidal action is focused on the pathogen with minimum damage to the 1H2O2-producing LAB, and without hemolytic damage to the added erythrocytes, that is, no bystander injury.

Specificity of MPO binding results in specificity of microbicidal action. Binding specificity allows synergistic MPO-LAB interaction and suppression of pathogens. It also suggests a role for MPO in the selection and maintenance of LAB in the normal flora [48]. Healthy human adults release about a hundred billion MPO-rich neutrophils into the circulating blood each day. The circulating lifetime of the neutrophil is reportedly less than a day. The neutrophils then leave the blood and enter a tissue and body cavity phase lasting a few days [36]. Migration of MPO-rich neutrophils into the mouth and vagina is well-known [49, 50]. When quantified, the neutrophil count of the mouth is proportional to the blood neutrophil count. These spaces typically provide an acidic milieu. Neutrophil disintegration with MPO release may provide LAB with a selective advantage in such body spaces.


5. Microbicidal combustion and chemiluminescence

Reactions of 1O2* with singlet multiplicity substrates (1Sub) are spin-allowed and highly exergonic. The exergonicities of most biochemical reactions are sufficient for rotational and vibrational excitation, but not electronic excitation. Dioxygenation reactions are sufficiently exergonic for electronic excitation. Oxygenations producing singlet multiplicity endoperoxide and dioxetane intermediates are excellent candidates for luminescence [51]. The disintegrations of such intermediates generate nπ* electronically excited products, that is, an electron from the nonbonding (n) orbital of oxygen populates the pi antibonding (π*) orbital of the carbonyl. Singlet multiplicity nπ* excited molecules have short lifetimes. Electronic transition from the π* of the carbonyl to the n of oxygen with photon emission is spin-allowed.

In addition to direct reaction of 1O2* with 1Sub, other singlet multiplicity reactants such as 1OCl can react with 1Sub to yield chloramine products (1Sub-Cl) or dehydrogenated products (1Sub-2RE). Such products can in turn react with 1H2O2 yielding endoperoxide or dioxetane intermediates with subsequent disintegration to nπ*-excited carbonyl products relaxing by photon emission [52, 53]. The fundamental principle is that all reactants and products are singlet multiplicity nonradicals.

Dioxygenations yielding intermediate endoperoxide and dioxetanes disintegrate yielding an nπ* electronically excited carbonyl. Figure 7 illustrates the energy and orbital differences that characterize the carbonyl states. Physical generation of a nπ* electronically excited carbonyl occurs when a fluorescent compound in its ground state absorbs a photon of appropriate energy. Because the ground state of the carbonyl is singlet, an electronically excited singlet multiplicity carbonyl undergoes rapid spin-allowed relaxation to ground state with a lifetime of less than 10−8 second [51]. Fluorescence describes photon-generated excitation followed by photon emission. Chemiluminescence or luminescence describes chemically generated electronic excitation followed by photon emission.

Figure 7.

Orbital diagram plot depicting the nπ* electronically excited singlet state and the singlet ground state of a carbonyl. The gray dashed brackets indicate the carbonyl with the participating carbon and oxygen atoms shown on to the left and right, respectively. In the carbonyl diagram on the left, the nπ* notation indicates that an electron of the nonbonding (n) orbital of the carbonyl oxygen atom has been excited to the pi antibonding (π*) orbital of the carbonyl. Although excited, the electrons remained paired and the excited state is singlet multiplicity. Electron relaxation from π*-to-n yields photon emission.

The metabolic changes of the respiratory burst describe the movement of RE required to change the spin multiplicity of 3O2 from triplet to doublet (2HO2), and ultimately to singlet, that is, 1H2O2 and 1O2*. MPO catalyzes the 2RE oxidation of 1Cl to 1HOCl followed by chemical reaction with a second 1H2O2 to generate 1O2*. Changing the bi-fermionic 3O2 to bosonic 1O2*eliminates the spin barrier to direct dioxygenation of bosonic singlet multiplicity biological molecules. If intermediate endoperoxides and dioxetanes are generated, their disintegration yields electronically excited nπ* carbonyl functions that relax by photon emission. By changing the spin multiplicity of oxygen, neutrophil leukocytes realize its electronegative potential for combustive microbicidal action. Such combustion generates electronically excited products emitting light in the visible range of the spectrum.


6. Chemiluminigenic probes

The native chemiluminescence of neutrophils is proportional to respiratory burst activity [4, 54]. Since the luminescence resulting from microbicidal combustion is proportional to dioxygenations, especially those yielding endoperoxide and dioxetane intermediates, it follows that native neutrophil luminescence is influenced by the molecular composition of the microbe combusted. Native luminescence from phagocytosing neutrophils can be detected using less than a million neutrophils. For perspective, a milliliter of normal human blood contains about 4 million neutrophils. The native luminescence product of neutrophil combustive action is of low intensity. However, electronic excitation and the resultant luminescence is unambiguous evidence of neutrophil combustive dioxygenation action. Native luminescence has been usefully applied to measurement of neutrophil metabolic defects, e.g., chronic granulomatous disease [54, 55], and neutrophil responsiveness to humoral immune factors, such as complement and immunoglobulins [56].

Inclusion of high quantum yield chemiluminigenic substrates as probes (CLP) of neutrophil dioxygenation activities greatly increases the sensitivity and, to some degree, the specificity for detecting such activities [52, 57, 58]. With regard to increasing sensitivity, a CLP must be susceptible to neutrophil dioxygenation activities. This is achieved when endoperoxide or dioxetane intermediate are produced. The breakdown of such intermediates yields electronically excited nπ* carbonyl functions that relax by light emission. Use of a CLP typically increases the sensitivity for detecting dioxygenation activity by several orders of magnitude. Selecting a CLP with reactive specificity also provides information with regard to the nature of neutrophil activity measured.

6.1. Probing reductive oxygenation activity with lucigenin

Phagocytic or chemical activation of neutrophil respiratory burst metabolism can be tested using the dye nitro-blue tetrazolium (NBT) [59]. The NBT reaction measures neutrophil reduction activity, not neutrophil oxidation activity. A positive NBT result requires neutrophil respiratory burst activity resulting in reduction of the tetrazolium ring of the dye to a dark blue water-insoluble formazan precipitate. NBT is a large complex nitrogen heterocyclic compound with abundant resonance and electron delocalization possibilities. That NBT reduction might be linked to neutrophil univalent reduction of molecular oxygen was considered, and we observed that adding a small grain of potassium superoxide (KO2) to a solution of NBT resulted in immediate reduction of the dye to a dark blue formazan precipitate [15]. Normal neutrophils reduce NBT upon activation of NADPH oxidase. The neutrophils of chronic granulomatous disease patients have defective NADPH oxidase, and as such, are incapable of NBT reduction [60].

Lucigenin (aka, bis-N-methylacridinium nitrate, or dimethyl biacridinium nitrate (1DBA+2)) is a heterocyclic organic compound known to generate chemiluminescence as a product of base-catalyzed peroxidation [61]. If sufficiently alkaline, singlet multiplicity 1lucigenin reacts with the conjugate base of peroxide (1HO2) producing a dioxetane (1lucigenin-dioxetane) intermediate that disintegrates to a nπ*-excited carbonyl function that relaxes to ground state by π*-to-n transition with photon emission. The pKa of 1H2O2 is 11.7. As previously considered, 1H2O2 is the sum product of two RE reductions of 3O2. Consequently, lucigenin chemiluminescence is the product of reductive dioxygenation. Both lucigenin and peroxide are singlet multiplicity reactants. Spin restriction is not a problem. Alkalinity favors the formation of 1HO2 and dioxygenation yielding a dioxetane.

Lucigenin is a heterocyclic compound with resonance and electron delocalization possibilities, and can undergo one RE reduction yielding a doublet multiplicity product (2lucigenin+RE+). Such reduction may involve 2O2 or some other 1RE reductant. The product radical, 2lucigenin+RE+, can now react with 2O2 by SOMO-SOMO overlap, that is, a doublet-doublet annihilation, producing a singlet multiplicity product, the 1lucigenin-dioxetane intermediate. As depicted in Figure 8, the disintegration of this unstable dioxetane yields chemiluminescence [52, 58, 62, 63].

Figure 8.

Oxygenating reactions yielding lucigenin chemiluminescence. Spin multiplicity is shown by the superscript value that precedes the reactant, and 1RE indicates one reducing equivalent.

Reduction of lucigenin by 2RE, that is, by a bosonic orbital electron couple, maintains singlet multiplicity. Such a reduced 1lucigenin+2RE can react with 1O2*, but not 3O2, to produce chemiluminescence [64]. As shown in Figure 8, the state of lucigenin reduction determines the deoxygenating agent required. All reactions shown satisfy the spin conservation rules.

The radical product of 1RE reduction of lucigenin, 2lucigenin+RE+, can react with the radical product of NADPH oxidase, 2O2, resulting in intermediate dioxetane formation with breakdown to a nπ* electronically excited carbonyl with relaxation by light emission, and as such, lucigenin can be applied as a chemiluminigenic probe for measurement of NADPH oxidase activity [52, 58, 63]. MPO haloperoxidase activity does not yield lucigenin-luminescence.

Chicken blood phagocytes, that is, heterophil leukocytes, have oxidase activity, but are deficient in haloperoxidase. Chemical or phagocytic stimulation of these heterophil leukocytes results in lucigenin-dependent luminescence responses comparable to those observed from human neutrophils under similar test conditions and using similar stimuli [58, 65]. However, the luminol-dependent luminescence responses of MPO-deficient chicken heterophils are a hundredfold lower than those observed from MPO-rich human neutrophils. In addition, azide (N3), a known inhibitor of MPO, inhibits the luminol-dependent luminescence responses of MPO-rich human neutrophil. Azide shows no inhibitory action against the luminol or the lucigenin luminescence responses of MPO-deficient chicken heterophils [66]. These chicken heterophil results plus the previously described macrophage results [57] experimentally support the position that luminol provides a very sensitive measure of MPO activity. However, the weaker luminol-luminescence measured is evidence for haloperoxidase-independent oxidase activity.

6.2. Probing oxygenation activities with cyclic hydrazides

Luminol chemiluminescence is a well-established phenomenon, but the mechanisms responsible for luminol-luminescence are diverse [67]. Luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) is a nonradical, cyclic hydrazide [68]. Luminol dioxygenation is thought to involve an intermediate endoperoxide with disintegration yielding the nπ* electronically excited aminophthalate that relaxes by photon emission. Albrecht first described the blood-catalyzed luminol-luminescence [69]. Like lucigenin, alkalinity and 1H2O2 are required, but luminol-luminescence has an additional requirement for a catalyst, for example, blood or peroxidase. To appreciate how these CLS differ, compare, and contrast the net reactions responsible for luminol-luminescence and lucigenin-luminescence. Luminol-luminescence is a dioxygenation:

1 luminol + 3 O 2 X 1 aminophthalate + 1 N 2 + Photon E14

The reaction of 1luminol with 3O2 (Eq. (14)) is not spin allowed, but reaction with 1O2* (Eq. (15)) is spin allowed producing nπ* electronically excited 1aminophthalate* plus 1N2, and ultimately, ground state 1aminophthalate plus a photon.

1 luminol + 1 O 2 1 aminophthalate + 1 N 2 + Photon E15

Lucigenin-luminescence is a reductive dioxygenation.

1 lucigenin + 1 H 2 O 2 2 1 N methylacrodone + Photon E16

As per Eq. (16), lucigenin-luminescence requires the spin-allowed reactive addition of molecular oxygen plus 2RE, that is, 1H2O2. The product of this reductive dioxygenation is a dioxetane intermediate that breaks down to one ground state 1N-methylacridone and one nπ*electronically excited 1N-methylacridone*. Relaxation of the 1N-methylacridone* yields a photon.

Luminol dioxygenation is not reductive. The net dioxygenation incorporates molecular oxygen to produce an endoperoxide intermediate with the breakdown release of 1N2 and formation of a nπ*electronically excited aminophthalate. As indicated by Eq. (14), 1luminol does not react with ground state oxygen. Spin conservation and frontier orbital overlap problems restrict such direct reaction. As illustrated in Figure 1, the frontier orbitals of 3O2 are its two degenerates π* SOMOs. Hund’s maximum multiplicity rule is satisfied when the electrons of each SOMO have the same spin. Each of the two π* orbitals of 3O2 have fermionic character that restricts overlap with the bosonic frontier orbitals of luminol. By contrast, the frontier π* orbitals of 1O2* are bosonic and include one LUMO π* orbital and one HOMO π* orbital. Overlap of the LUMO of 1O2* with the HOMO of 1luminol satisfies the symmetry requirements for reaction.

There are three mechanistic possibilities for 1luminol reactions yielding luminescence. The fermionic (doublet multiplicity/radical) pathway requires two steps as illustrated by Eqs. (17) and (18).

1 luminol + 1 H 2 O 2 peroxidase 2 luminol 1RE + 1 H 2 O E17

The radical 2luminol-1RE can participate in SOMO-SOMO reaction with superoxide (2O2) yielding singlet multiplicity electronically excited aminophthalate (1aminophthalate*) that relaxes with photon emission.

2 luminol 1RE + 2 O 2 1 aminophthalate + 1 N 2 + Photon E18

The bosonic (singlet multiplicity/nonradical) pathway can occur by a single reaction as illustrated by Eq. (19),

1 luminol + 1 O 2 1 aminophthalate + 1 N 2 + Photon E19

The bosonic (singlet multiplicity/nonradical) pathway can also occur by a two-step reaction as illustrated by Eqs. (20) and (21).

1 luminol + 1 OCl 1 luminol 2RE + 1 Cl E20
1 luminol 2RE + 1 H 2 O 2 1 aminophthalate + 1 N 2 + Photon E21

Although luminol is versatile with regard to reactive mechanism, dioxygenation is ultimately required for chemiluminescence. In an alkaline milieu, classical peroxidase or hemoglobin can catalyze 1H2O2-dependent luminol-luminescence. The peroxidase-catalyzed mechanism of luminol-luminescence described by Dure and Cormier illustrates the kinetics of the fermionic pathway [70]. For such reaction, a classical peroxidase is first oxidized by 1H2O2, that is, 2RE are transferred to 1H2O2 producing two 1H2O as described in Eq. (22).

peroxidase + 1 H 2 O 2 Cpx 1 2RE + 2 1 H 2 O E22

This 2RE oxidized peroxidase, referred to as complex 1 (Cpx 1), can now readily oxidize 1luminol by removing 1RE producing 2luminol-1RE, as per Eq. (23).

1 luminol + Cpx 1 2RE Cpx 2 RE + 2 luminol 1RE E23

The reaction of complex 2 (Cpx 2-RE) with another 1luminol is slow and rate limiting with regard to luminescence, but this reaction is necessary for regeneration of the starting peroxidase, as per Eq. (24).

1 luminol + Cpx 2 RE peroxidase + 2 luminol 1RE E24

Disproportionation of the two radical 2luminol-1RE can proceed as a spin allowed SOMO-SOMO reaction, that is, a doublet-doublet annihilation, yielding the nonradical 1luminol (starting reactant) and nonradical 2RE-oxidized luminol (1luminol-2RE).

2 2 luminol 1RE 1 luminol + 1 luminol 2RE E25

As per Eq. (21), the spin-allowed reaction of 1luminol-2RE with 1H2O2 yields electronically excited aminophthalate (1aminophthalate*) that relaxes by photon emission.

Metalloenzymes and cytochromes are suited to 1RE transfers and under proper reaction conditions can catalyze the 1RE oxidation of a 1substrate producing 2substrate-1RE. The 1H2O2-dependent oxidation of peroxidase to Cpx 1-2RE allows it to catalyze the initial fermionic 1RE oxidation of luminol in an alkaline milieu. Hemoglobin has peroxidase activity under alkaline conditions, thus explaining the sensitivity of luminol-luminescence for detecting the presence of blood erythrocytes by alkaline peroxide methods. Luminol-luminescence by the classical plant peroxidase-catalyzed reactions of Eqs. (22)(25) is sensitive to pH, decreasing with increasing acidity. Acidification of the reaction milieu to a pH of about 5 ± 1 effectively eliminates classical peroxidase-catalyzed luminol luminescence. This is quantitatively demonstrated in the Michaelis-Menten enzyme kinetic analyses of luminol-luminescence for myeloperoxidase and horse radish peroxidase presented in Table 2 [71].

Substrate [S], variable (conc. range) pH Substrates, constant Michaelis-Menten kinetics
H2O2, mM Cl, mEq/L Br, mEq/L Luminol, μM M-M equation Km ± SE Vmax ± SE
Haloperoxidase: Myeloperoxidase H2O2 (0.01–1.4 mM) 5.0 variable 90 0 77 v = Vmax[S]2/(Km + [S])2 2.82 ± 0.05 3900 ± 1
H2O2 (0.01–1.4 mM) 5.0 variable 0 4.5 77 v = Vmax[S]2/(Km + [S])2 0.58 ± 0.03 2932 ± 2
Cl (0.2–7.7 mEq/L) 5.0 2.27 variable 0 45 v = Vmax[S]/Km + [S] 7.60 ± 2.60 1105 ± 253
Br (14–882 μEq/L) 5.0 2.27 0 variable 45 v = Vmax[S]/Km + [S] 0.68 ± 0.05 2280 ± 96
Luminol (0.0018–15 μM) 4.9 2.27 90 0 variable v = Vmax[S]/Km + [S] 8.80 ± 0.77 1490 ± 70
Luminol (0.0018–0.47 μM) 7.0 2.27 90 0 variable v = Vmax[S]/Km + [S] 0.10 ± 0.02 3252 ± 219
Classical Peroxidase: Horse Radish Peroxidase H2O2 (0.01–1.4 mM) 5.0 variable 90 0 77 v = Vmax[S]/Km + [S] 31.02 ± 0.05 280 ± 55
H2O2 (0.01–1.4 mM) 5.0 variable 0 4.5 77 v = Vmax[S]/Km + [S] 17.49 ± 3.84 156 ± 34
Cl (0.2–900 mEq/L) 5.0 2.27 variable 0 45 v = Vmax[S]/Km + [S] 0.0 ± 0.7 0 ± 0
Br (14–882 μEq/L 5.0 2.27 0 variable 45 v = Vmax[S]/Km + [S] 6.23 ± 2.68 3 ± 0
Luminol (0.0147–30 μM) 4.9 2.27 0 0 variable v = Vmax[S]2/(Km + [S])2 0.0 ± 0.0 0 ± 0
Luminol (0.0018–7.5 μM) 7.0 2.27 0 0 variable v = Vmax[S]2/(Km + [S])2 0.90 ± 0.17 189 ± 2

Table 2.

Michaelis-Menten enzyme kinetic analyses of classical peroxidase (horse radish peroxidase) and haloperoxidase (myeloperoxidase) activities with regard to H2O2, halide (Cl- or Br), luminol, and pH.

Reaction milieu was 50 mM acetate buffer (pH 5.0, 4.9) or phosphate buffer (pH 7.0) in a 0.3 mL volume. The indicated conc. of Cl or Br was added in a 0.3 mL volume.

The enzymes, 78 pmol MPO and 10 pmol HRP as indicated, were added in a 0.1 mL volume. The final concentration was 78 nM for MPO and 10 nM for HRP.

The luminescence reaction was initiated by injecting the indicated concentration of H2O2 in a 0.3 mL volume. The final volume was 1.0 mL.

Chemiluminescence velocity (v) and Vmax are expressed as peak kilocounts of relative light units (RLU × 10–3) per sec measured during the initial 20 sec post H2O2 injection.

Alkaline pH favors the fermionic luminol-luminescence reactions catalyzed by plant peroxidase, hemoglobin, and heavy metals. The pKa of 1H2O2 is 11.75. The ferricyanide-catalyzed luminol luminescence reaction is most efficient in the pH range from 10.4 to 10.8 [72]. In Table 2, note that no significant luminescence is observed from HRP-catalyzed luminol reaction at pH 4.9. The maximum luminescence velocity (Vmax) values are low and standard errors (SE) are high. However, a relatively weak but significant luminescence is observed at pH 7.0, that is, Michaelis-Menten analysis of the HRP luminescence shows a low Vmax, but an acceptable SE.

Of special note, Michaelis-Menten kinetic analysis indicates that the HRP-catalyzed luminol-luminescence velocity is first order with respect to H2O2 concentration, but second order with respect to luminol concentration, that is, the luminescence velocity is directly proportional to the square of the luminol concentration. These results are consistent with those reported by Dure and Cormier [70], and with the fermionic radical reactive pathway described in Eqs. (22)(25) and Eq. (21).

Although luminol solubility becomes a problem at low pH, acidity favors the bosonic haloperoxidase luminol-luminescence catalyzed by MPO. Note that bosonic, haloperoxidase-catalyzed luminol luminescence is first order with respect to luminol, chloride, or bromide, but second order with respect to H2O2, that is, luminescence activity is proportional to the square of the H2O2 concentration.

The MPO-catalyzed luminol-luminescence kinetic finding is the opposite of those observed for HRP-catalyzed luminol-luminescence, and are consistent with the bosonic reactive pathway for luminol-luminescence via 1O2* reaction described in Eq. (19) or the sequential bosonic pathway described in Eqs. (20), (21). By either pathway, and consistent with the second order findings, two 1H2O2 are required for luminol dioxygenation.

Under alkaline conditions, luminol-luminescence provides high sensitivity for detection of classical peroxidase catalysts or1H2O2, but relatively low specificity. Under acid conditions, the luminol-luminescence provides a method for specific quantification of haloperoxidase-dependent dioxygenation activity. In Table 2, note that Cl or Br is required for MPO-catalyzed luminol-luminescence, that the requirement is first order with respect to halide, and that the Michaelis constant (KM) for the more electronegative Cl is expectedly greater that for Br. Haloperoxidase activity is exclusively bosonic. Reactants are all singlet multiplicity, involving HOMO-LUMO frontier orbital interaction.

Luminol was the first, and remains the most common, chemiluminigenic probe used for measurement of phagocyte oxygenation activities. Its original application was an attempt to amplify the relatively weak native luminescence signal from stimulated macrophages. Comparing the luminol-luminescence responses of neutrophils with those of macrophages illustrates that the MPO-rich neutrophils responses are several magnitudes greater than the luminol-luminescence responses from MPO-deficient macrophage [57].

Comparing MPO-rich human neutrophils with the MPO-deficient heterophile leukocytes of chickens further illustrates how chemiluminigenic probing can be used as a sensitive method for quantifying and differentiating the oxygenating activities of phagocytes [58, 65]. The luminol-dependent activities of MPO-positive human neutrophil leukocytes are a hundredfold higher than those of MPO-negative chicken heterophil leukocytes. Despite the diminution in luminol-luminescence, dioxygenation activity is still quantifiable from MPO-negative phagocytes. Such activity is not inhibited by the MPO inhibitor azide (N3) [66]. In the absence of haloperoxidase, luminol-luminescence most probably reflects the type of fermionic oxidase-dependent reactions described in reactions Eqs. (17)(18).


7. Circulating neutrophils reflect the state of inflammation

Under normal conditions, large numbers of neutrophils are produced by the hematopoietic marrow and released into the circulating blood each day, highlighting the importance of neutrophils for innate host protection against infection. To accomplish its microbicidal role, neutrophils undergo specific degranulation and mobilization of appropriate membrane receptors in response to a constellation of microbial peptides, complement activation products, cytokines, interleukins, and lipid activators. Such activities prepare neutrophils for phagocytosis, but do not directly trigger respiratory burst activity [73]. Priming actuates neutrophil locomotion and increases neutrophil recognition of and phagocytic response to opsonin-labeled microbes [56, 74, 75].

Activation of systemic inflammation in response to infection directly affects circulating blood neutrophils. The chemical signals of inflammation alter the state of neutrophil alert. As such, the state of neutrophil priming reflects the state of host immune activation [76]. Selective in vitro measurement of unprimed and maximally-primed circulating blood neutrophil activities by sensitive chemiluminigenic probing allows rapid multi-metric analysis using less than a half drop of anticoagulated whole blood. Analysis of such blood neutrophil luminescence metrics using classification statistical approaches, especially discriminant function analysis, allows assessment of the in vivo state of immune activation. The state of neutrophil priming gauges the state of host systemic inflammation [77, 78].



I gratefully acknowledge all who assisted in my education, especially my deceased mother Gladys L. Puig, my undergraduate professor Dr. Walter L. Scott Jr., my deceased mentor Dr. Richard H. Steele, and my friend and colleague Dr. Randolph M. Howes.


Conflict of interest

I am the inventor of pending and issued patents related to diagnostic applications of chemiluminescence for quantifying neutrophil function and for gauging systemic immune activation, and patents related to therapeutic applications of haloperoxidases.


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  45. 45. Skovsen E, Snyder JW, Lambert JD, Ogilby PR. Lifetime and diffusion of singlet oxygen in a cell. Journal of Physical Chemistry B. 2005;109(18):8570-8573
  46. 46. Redmond RW, Kochevar IE. Spatially resolved cellular responses to singlet oxygen. Photochemistry and Photobiology. 2006;82(5):1178-1186
  47. 47. Allen RC, Stephens JT Jr. Reduced-oxidized difference spectral analysis and chemiluminescence-based Scatchard analysis demonstrate selective binding of myeloperoxidase to microbes. Luminescence. 2011;26(3):208-213
  48. 48. Allen RC, Stephens JT Jr. Role of lactic acid bacteria-myeloperoxidase synergy in establishing and maintaining the normal flora in man. Food and Nutrition Sciences. 2013;4:67-72
  49. 49. Wright DG, Meierovics AI, Foxley JM. Assessing the delivery of neutrophils to tissues in neutropenia. Blood. 1986;67(4):1023-1030
  50. 50. Cauci S, Guaschino S, De Aloysio D, Driussi S, De Santo D, Penacchioni P, et al. Interrelationships of interleukin-8 with interleukin-1beta and neutrophils in vaginal fluid of healthy and bacterial vaginosis positive women. Molecular Human Reproduction. 2003;9(1):53-58
  51. 51. Turro NJ, Ramamurthy V, Scaiano JC. Principles of Molecular Photochemistry. Sausalito: University Science Books; 2009
  52. 52. Allen RC. Biochemiexcitation: Chemiluminescence and the study of biological oxygenation. In: Adam W, Cilento G, editors. Chemical and Biological Generation of Excited States. New York: Academic Press; 1982. pp. 310-344
  53. 53. Kearns DR, Khan AU. Sensitized photooxygenation reactions and the role of singlet oxygen. Photochemistry and Photobiology. 1969;10(3):193-210
  54. 54. Allen RC, Stjernholm RL, Reed MA, Harper TB, Gupta S, Steele RH, et al. Correlation of metabolic and chemiluminescent responses of granulocytes from three female siblings with chronic granulomatous disease. Journal of Infectious Diseases. 1977;136(4):510-518
  55. 55. Allen RC, Mills EL, McNitt TR, Quie PG. Role of myeloperoxidase and bacterial metabolism in chemiluminescence of granulocytes from patients with chronic granulomatous disease. Journal of Infectious Diseases. 1981;144(4):344-348
  56. 56. Allen RC. Evaluation of serum opsonic capacity by quantitating the initial chemiluminescent response from phagocytizing polymorphonuclear leukocytes. Infection and Immunity. 1977;15(3):828-833
  57. 57. Allen RC, Loose LD. Phagocytic activation of a luminol-dependent chemiluminescence in rabbit alveolar and peritoneal macrophages. Biochemical and Biophysical Research Communications. 1976;69(1):245-252
  58. 58. Allen RC. Phagocytic leukocyte oxygenation activities and chemiluminescence: A kinetic approach to analysis. Methods in Enzymology. 1986;133:449-493
  59. 59. Park BH, Fikrig SM, Smithwick EM. Infection and Bitroblue-tetrazolium reduction by neutrophils: A diagnostic aid. The Lancet. 1968;292(7567):532-534
  60. 60. Ochs HD, Igo RP. The NBT slide test: A simple screening method for detecting chronic granulomatous disease and female carriers. The Journal of Pediatrics. 1973;83(1):77-82
  61. 61. Gleu K, Petsch W. Die Chemiluminescenz der dimethyl-diacridyliumsalze. Angewandte Chemie. 1935;48(3):57-59
  62. 62. Totter JR. The quantum yield of the chemiluminescence of dimethylbiacridinium nitrate and the mechanism of its enzymatically induced chemiluminescence. Photochemistry and Photobiology. 1964;3:231-241
  63. 63. Allen RC. Lucigenin chemiluminescence: A new approach to the study of polymorphonuclear leukocyte redox activity. In: Bioluminescence and Chemiluminescence Basic Chemistry and Analytical Applications. New York: Academic Press; 1981. pp. 63-73
  64. 64. McCapra F, Hann RA. The chemiluminescent reaction of singlet oxygen with 10, 10′-dimethyl-9, 9′-biacridylidene. Journal of the Chemical Society D: Chemical Communications. 1969;9:442-443
  65. 65. Merrill GA, Bretthauer R, Wright-Hicks J, Allen RC. Oxygenation activities of chicken polymorphonuclear leukocytes investigated by selective chemiluminigenic probes. Laboratory Animal Science. 1996;46(5):530-538
  66. 66. Merrill GA, Bretthauer R, Wright-Hicks J, Allen RC. Effects of inhibitors on chicken polymorphonuclear leukocyte oxygenation activity measured by use of selective chemiluminigenic substrates. Comparative Medicine. 2001;51(1):16-21
  67. 67. Roswell DF, White EH. The chemiluminescence of luminol and related hydrazides. Methods in Enzymology. 1978;57:409-423
  68. 68. Lind J, Merenyi G, Eriksen TE. Chemiluminescence mechanism of cyclic hydrazides such as luminol in aqueous solutions. Journal of the American Chemical Society. 1983;105(26):7655-7661
  69. 69. Albrecht HO. Über die chemiluminescenz des aminophthalsäurehydrazids. Zeitschrift für Physikalische Chemie. 1928;136U:321-330
  70. 70. Dure LS, Cormier MJ. Studies on the bioluminescence of Balanoglossus biminiensis extracts. Journal of Biological Chemistry. 1964;239:2351-2359
  71. 71. Allen RC. Haloperoxidase Acid Optimum Chemiluminescence Assay System. USPO. Patent US005556758A1996. pp. 1-46
  72. 72. Seitz RW. Chemiluminescence detection of enzymically generated peroxide. Methods in Enzymology. 1978;57:445-462
  73. 73. Allen RC, Stevens DL. The circulating phagocyte reflects the in vivo state of immune defense. Current Opinion in Infectious Diseases. 1992;5(3):389-398
  74. 74. Madonna GS, Allen RC. Shigella sonnei phase I and phase II: Susceptibility to direct serum lysis and opsonic requirements necessary for stimulation of leukocyte redox metabolism and killing. Infection and Immunity. 1981;32(1):153-159
  75. 75. Allen RC, Lieberman MM. Kinetic analysis of microbe opsonification based on stimulated polymorphonuclear leukocyte oxygenation activity. Infection and Immunity. 1984;45(2):475-482
  76. 76. Stevens DL, Bryant AE, Huffman J, Thompson K, Allen RC. Analysis of circulating phagocyte activity measured by whole blood luminescence: Correlations with clinical status. Journal of Infectious Diseases. 1994;170(6):1463-1472
  77. 77. Taylor F, Haddad P, Kinasewitz G, Chang A, Peer G, Allen RC. Luminescence studies of the phagocyte response to endotoxin infusion into normal human subjects: Multiple discriminant analysis of luminescence response and correlation with phagocyte morphologic changes and release of elastase. Journal of Endotoxin Research. 2000;6(1):3-15
  78. 78. Allen RC, Dale DC, Taylor FB. Blood phagocyte luminescence: Gauging systemic immune activation. Methods in Enzymology. 2000;305:591-629

Written By

Robert C. Allen

Reviewed: September 17th, 2018 Published: November 5th, 2018