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

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 O 2 -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 O 2 to the acid hydroperoxyl radical (HO 2 ) and its conjugate base, superoxide anion (O 2 − ). RE transfer dynamics is considered from the perspectives of quantum and particle physics, as well as frontier orbital interactions. Direct disproportionation of HO 2 -O 2 − yields electronically excited singlet molecular oxygen ( 1 O 2 *) and hydrogen peroxide (H 2 O 2 ). Myeloperoxidase catalyzes H 2 O 2 -dependent 2RE oxidation of chloride (Cl − ) to hypochlorite (OCl − ). Direct nonenzymatic reaction of OCl − with an additional H 2 O 2 yields Cl − , H 2 O, and 1 O 2 *. Thus, for two 2RE metabolized through NADPH oxidase, a total of three 1 O 2 * are possible. H 2 O 2 , OCl − , and 1 O 2 * 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.


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 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 O 2 . The reduced oxygen ( · O 2 − , · O 2 H) may then disproportionate yielding HOOH and singlet molecular oxygen 1 O 2 . 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 1 O 2 . 1 O 2 has been demonstrated to be a potent microbicidal agent. Therefore, the biochemical generation of 1 O 2 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 1 O 2 -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].

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, 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, m l , s, and m s [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 m s , and only two values are allowed. When m s = ½, the e − is described as spin up (↑); when m s = −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 m s = ½ (↑), the other orbital e − must have an m s = −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.

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 ( 2 OH) can extract 1RE from the HOMO of a singlet multiplicity nonradical substrate ( 1 substrate) yielding singlet multiplicity 1 H 2 O, but in the process the HOMO of the substrate is converted to a SOMO, that is, the substrate becomes a doublet multiplicity radical ( 2 substrate). 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].
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 3 O 2 . The high electronegativity of 3 O 2 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  the bi-fermionic, bi-radical nature of 3 O 2 that restricts its reactive potential. As per Table 1, the reaction of 3 O 2 with a bosonic 1 substrate 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 3 O 2 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, 3 O 2 can participate in and be a necessary reactant in radical propagation reactions.

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 (gp91 phox ) subunit associated with a smaller protein (p22 phox ). The C-terminal portion of gp91 phox 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 p67 phox 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 1 NADPH and facilitating 1RE reduction of 3 As illustrated in Figure 2, the product of 1RE reduction of 3 O 2 is the acid hydroperoxyl radical ( 2 HO 2 ) with an acid dissociation constant pK a of 4.8 [30]. For comparison, the pK a of 1 H 2 O 2 is 11.7. As the pH of the phagolysosomal space approaches the pK a , the ratio of 2 HO 2 to its conjugate base, the superoxide anion ( 2 O 2 − ) approaches unity, and acid disproportionation, that is, reaction of 2 HO 2 with 2 O 2 − , approaches maximum reaction rate. At unity, anionic repulsion is no longer a problem. The rate constant for the reaction is 4.5 × 10 5 M −1 s −1 at pH 7.0 and reaches a maximum of 8.5 × 10 7 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 1 H 2 O 2 and 1 O 2 *. As per Table 1, doublet-doublet annihilation yields single products [15,32]. The reaction is sufficiently exergonic to yield 1 O 2 * with an energy of 22.5 kcal/mol (94.1 kJ/mol) above ground state 3 O 2 .

Figure 2.
Schema illustrating the central role of membrane-associated NADPH oxidase in respiratory burst metabolism. In the activated state, the Michaelis constant (K M ) of the oxidase for NADPH is decreased. NADP + availability controls the activities of glucose-6-PO 4 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  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 3 O 2 . Transfer of an orbital electron couple is nonradical, bosonic, and singlet multiplicity. In an atmosphere that is 20.9% 3 O 2 , 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 3 O 2 , 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].

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].

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 pHdependent 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].
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 ( For the reaction, A red + B ox ↔ B red + D ox , the half reaction equations become: ΔE₀ = (RT / nF) ln K eq (8) K eq is the equilibrium constant. The change in potential (ΔE) can be expressed in terms of Gibbs free energy (ΔG).
The schema of Figure 3  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., Eh 2 o 2 − 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 − .
The plots of Figure 4 illustrate that increasing acidity increases the exergonicity of MPO-catalyzed 1 H 2 O 2 -dependent oxidation of halides. This is especially import for MPO-catalyzed oxidation of chloride. Conversely, increasing alkalinity increases the exergonicity of the nonenzymatic 1 OCl − -1 H 2 O 2 reaction yielding 1 O 2 * as depicted in    Since the reactants involved are all singlet multiplicity, the products of reaction, that is, 1 H 2 O, 1 Cl − , and 1 O 2 *, are all singlet multiplicity. This provides a spin symmetry explanation as to why pouring bleach ( 1 OCl − ) into 1 H 2 O 2 causes rapid reactive release of 1 O 2 * gas and a red chemiluminescence [23]. Caution, rapid release of gas is potentially explosive. When the concentration of 1 O 2 * is sufficiently high, 1 O 2 *-1 O 2 * collision with simultaneous relaxation yields red chemiluminescence. The relaxation of one 1 O 2 * emits a 1270 nm photon; simultaneous relaxation of two 1 O 2 * emits a 635 nm photon. As such, this red emission is second order with respect to 1 O 2 *, that is, dhν 635nm /dt = k[ 1 O 2 *] 2 , and is relatively short-lived.   The double dehydrogenation of 1 glucose-6-PO 4 produces 1 ribulose-5-PO 4 plus 1 CO 2 plus two 2RE, that is, two bosonic electron couples carried as 2NADPH. As illustrated in Figure 2, NADPH oxidase reduces four 3 O 2 in four one 1RE reduction steps, ultimately yielding two 1 O 2 * and two 1 H 2 O 2 . As illustrated in Figure 3, MPO uses one 1 H 2 O 2 for oxidation of Cl − to OCL − , and this OCL − reacts with the other 1 H 2 O 2 to generate an additional 1 O 2 *. Thus, two NADPH have the potential to drive the generation of three 1 O 2 *. Steinbeck et al. have reported experiments using glass beads coated with 9,10-diphenylanthracene, a 1 O 2 *-specific trap, for measurements of neutrophil 1 O 2 * production [43]. Neutrophils were allowed to phagocytose the beads for an hour. The endoperoxide trapped indicated that at least 11.3 ± 4.9 nmol 1 O 2 */1.25 × 10 6 neutrophils were produced. When the neutrophils were chemically activated with phorbol-12-myristate-13-acetate (PMA), at least 14.1 ± 4.1 nmol 1 O 2 */1.25 × 10 6 neutrophils were produced. Based on their trapping results, 1 O 2 * production accounted for at least 19 ± 5% of the total oxygen consumed. Although the quantities of 1 O 2 * measured using this difficult trapping approach are lower than expected; this study provides direct empirical evidence of significant neutrophil 1 O 2 * production.
Quantifying cellular production of 1 O 2 * by measuring the 1270 nm near-infrared photon emitted on 1 O 2 * relaxation to 3 O 2 is also problematic. Although highly specific for 1 O 2 *, this infrared proton emission approach is highly insensitive in biological system measurements. The fact that a 1270 nm photon is measured is proof that 1 O 2 * did not participate in chemical reaction. Considering the variety of reactive substrates available in biological milieux, electrophilic reaction is favored over relaxation.

Myeloperoxidase-binding specificity focuses combustive activity
1 O 2 * 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 1 O 2 * restricts its reactive possibilities [44]. In biological milieux, 1 O 2 * 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 1 O 2 *, 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 1 H 2 O 2 as a metabolic product. The green hemolysis associated with colonies of viridans streptococci on blood agar plates results from the production of 1 H 2 O 2 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 1 H 2 O 2 -producing Strep. viridans. Thus, LAB-produced 1 H 2 O 2 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 1 H 2 O 2 -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.

Microbicidal combustion and chemiluminescence
Reactions of 1 O 2 * with singlet multiplicity substrates ( 1 Sub) 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 1 O 2 * with 1 Sub, other singlet multiplicity reactants such as 1 OCl − can react with 1 Sub to yield chloramine products ( 1 Sub-Cl) or dehydrogenated products ( 1 Sub -2RE ). Such products can in turn react with 1 H 2 O 2 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.
The metabolic changes of the respiratory burst describe the movement of RE required to change the spin multiplicity of 3 O 2 from triplet to doublet ( 2 HO 2 ), and ultimately to singlet, that is, 1 H 2 O 2 and 1 O 2 *. MPO catalyzes the 2RE oxidation of 1 Cl − to 1 HOCl followed by chemical reaction with a second 1 H 2 O 2 to generate 1 O 2 *. Changing the bi-fermionic 3 O 2 to bosonic 1 O 2 *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.

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.

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 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 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 (KO 2 ) 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 ( 1 DBA +2 )) is a heterocyclic organic compound known to generate chemiluminescence as a product of base-catalyzed peroxidation [61]. If sufficiently alkaline, singlet multiplicity 1 lucigenin reacts with the conjugate base of peroxide ( 1 HO 2 − ) producing a dioxetane ( 1 lucigenin-dioxetane) intermediate that disintegrates to a nπ*-excited carbonyl function that relaxes to ground state by π*-to-n transition with photon emission. The pK a of 1 H 2 O 2 is 11.7. As previously considered, 1 H 2 O 2 is the sum product of two RE reductions of 3 O 2 . 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 1 HO 2 − 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 ( 2 lucigenin +RE + ). Such reduction may involve 2 O 2 − or some other 1RE reductant. The product radical, 2 lucigenin +RE + , can now react with 2 O 2 − by SOMO-SOMO overlap, that is, a doublet-doublet annihilation, producing a singlet multiplicity product, the 1 lucigenin-dioxetane intermediate. As depicted in Figure 8, the disintegration of this unstable dioxetane yields chemiluminescence [52,58,62,63].
Reduction of lucigenin by 2RE, that is, by a bosonic orbital electron couple, maintains singlet multiplicity. Such a reduced 1 lucigenin +2RE can react with 1 O 2 *, but not 3 O 2 , 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, 2 lucigenin +RE + , can react with the radical product of NADPH oxidase, 2 O 2 − , 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 (N 3 − ), a known inhibitor of MPO, inhibits the luminol-dependent luminescence responses of MPOrich 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.
Luminol dioxygenation is not reductive. The net dioxygenation incorporates molecular oxygen to produce an endoperoxide intermediate with the breakdown release of 1 N 2 and formation of a nπ*electronically excited aminophthalate. As indicated by Eq. (14), 1 luminol 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 3 O 2 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 3 O 2 have fermionic character that restricts overlap with the bosonic frontier orbitals of luminol. By contrast, the frontier π* orbitals of 1 O 2 * are bosonic and include one LUMO π* orbital and one HOMO π* orbital. Overlap of the LUMO of 1 O 2 * with the HOMO of 1 luminol satisfies the symmetry requirements for reaction.
There are three mechanistic possibilities for 1 luminol reactions yielding luminescence. The fermionic (doublet multiplicity/radical) pathway requires two steps as illustrated by Eqs. (17) and (18) The bosonic (singlet multiplicity/nonradical) pathway can also occur by a twostep reaction as illustrated by Eqs. (20) and (21) 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 1 H 2 O 2 -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 1 This 2RE oxidized peroxidase, referred to as complex 1 (Cpx 1), can now readily oxidize 1 luminol by removing 1RE producing 2 luminol -1RE , as per Eq. (23).
Metalloenzymes and cytochromes are suited to 1RE transfers and under proper reaction conditions can catalyze the 1RE oxidation of a 1 substrate producing 2 substrate -1RE . The 1 H 2 O 2 -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].
Alkaline pH favors the fermionic luminol-luminescence reactions catalyzed by plant peroxidase, hemoglobin, and heavy metals. The pKa of 1 H 2 O 2 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 (V max ) 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 V max , but an acceptable SE.
Of special note, Michaelis-Menten kinetic analysis indicates that the HRPcatalyzed luminol-luminescence velocity is first order with respect to H 2 O 2 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 H 2 O 2 , that is, luminescence activity is proportional to the square of the H 2 O 2 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 1