Resonance Raman microspectroscopic characterization of eosinophil peroxidase in human eosinophilic granulocytes.

A resonance Raman microspectroscopic study is presented of eosinophil peroxidase (EPO) in human eosinophilic granulocytes. Experiments were carried out at the single cell level with laser excitation in Soret-, Qv-, and charge transfer absorption bands of the active site heme of the enzyme. The Raman signal obtained from the cells was almost exclusively due to EPO. Methods were developed to determine depolarization ratios and excitation profiles of Raman bands of EPO in situ. A number of Raman band assignments based on earlier experiments with isolated EPO have been revised. The results show that in agreement with literature on isolated eosinophil peroxidase, the prosthetic group of the enzyme in the (unactivated) cells is a high spin, 6-coordinated, ferric protoporphyrin IX. The core size of the heme is about 2.04 A. The proximal and distal axial ligands are most likely a histidine with the strong imidazolate character typical for peroxidases, and a weakly bound water molecule, respectively. The data furthermore indicate that the central iron is displaced from the plane of the heme ring. The unusual low wavenumber Raman spectrum of EPO, strongly resembling that of lactoperoxidase, intestinal peroxidase and myeloperoxidase, suggests that these mammalian peroxidases are closely related, and characterized by, as yet unspecified, interactions between the peripheral substituents and the protein, different from those found in other protoheme proteins.     

Distinct heme-substrate interactions of lactoperoxidase probed by resonance Raman spectroscopy: difference between animal and plant peroxidases

Resonance Raman scattering from cow milk lactoperoxidase (LPO) and its complexes with various electron donors and inhibitors was investigated. The Raman spectrum of LPO is strikingly close to that of hog intestinal peroxidase but distinctly dissimilar to that of horseradish peroxidase (HRP). The v10 frequency suggested the six-coordinate high-spin structure of heme for native LPO in contrast with the five-coordinate high-spin structure for HRP. For the v10 band, benzohydroxamic acid caused a frequency shift with HRP but not with LPO. Guaiacol, o-toluidine, and histidine brought about a frequency shift of the v4 mode for LPO but not for HRP. The frequency shift was restored upon removal of the substrate or inhibitor by dialysis. The down shift of the v4 frequency is considered to represent an appreciable donation of electrons from the substrate or inhibitor to the porphyrin LUMO and thus their direct interaction with the heme group. From the relative intensity of the shifted and unshifted v4 lines, the dissociation constant was determined to be Kd = 52 mM for guaiacol and Kd = 87 mM for histidine at pH 7.4. The binding of histidine was relatively retarded in the presence of sulfate anion (Kd = 150 mM for 0.53 M sulfate present), and imidazole alone yielded no frequency shift, indicating the binding of the carboxyl group of histidine to the protein cationic site on one hand and a weak charge-transfer interaction between the imidazole group and the heme group on the other.     

Interrogation of heme pocket environment of mammalian peroxidases with diatomic ligands

Recent studies demonstrate that myeloperoxidase (MPO), eosinophil peroxidase (EPO), and lactoperoxidase (LPO), homologous members of the mammalian peroxidase superfamily, can all serve as catalysts for generating nitric oxide- (nitrogen monoxide, NO) derived oxidants. These enzymes contain heme prosthetic groups that are ligated through a histidine nitrogen and use H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions. Here we show that heme reduction of these peroxidases results in distinct electronic and/or conformational changes in their heme pockets using a combination of rapid kinetics measurements, optical absorbance, and diatomic ligand binding studies. Addition of reducing agent to each peroxidase at ground state [Fe(III) state] causes immediate buildup of the corresponding Fe(II) complexes. Spectral changes indicate that two LPO-Fe(II) species are present in solution at equilibrium. Analyses of stopped-flow traces collected when EPO, MPO, or LPO solutions rapidly mixed with NO were accurately fit by single-exponential functions. Plots of the apparent rate constants as a function of NO concentration for all Fe(III) and Fe(II) forms were linear with positive intercepts, consistent with NO binding to each form in a simple reversible one-step mechanism. Fe(II) forms of MPO and LPO, but not EPO, displayed significantly lower affinity toward NO compared to Fe(III) forms, suggesting that heme reduction causes a dramatic change in the heme pocket electronic environment that alters the affinity and/or accessibility of heme iron toward NO. Optical absorbance spectra indicate that CO binds to the Fe(II) forms of both LPO and EPO, but not with MPO, and generates their respective low-spin six-coordinate complexes. Kinetic analyses indicate that the binding of CO to EPO is monophasic while CO binding to LPO is biphasic. Collectively, these results illustrate for the first time functional differences in the heme pocket environments of Fe(II) forms of EPO, LPO, and MPO toward binding of diatomic ligands. Our results suggest that, upon reduction, the heme pocket of MPO collapses, LPO adopts two spectroscopically and kinetically distinguishable forms (one partially open and the other relatively closed), and EPO remains open.     

A theoretical three-dimensional model for lactoperoxidase and eosinophil peroxidase,built on the scaffold of the myeloperoxidase X-ray structure

 Lactoperoxidase (LPO), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) belong to the class of haloperoxidases, a group of mammalian enzymes able to catalyze the peroxidative oxidation of halides and pseudohalides, such as thiocyanate. They all play a key role in the development of antibacterial activity. The homology in their functional role is emphasized by the striking similarity of their primary structures. A theoretical model for the three-dimensional structure of LPO and EPO has been developed on the basis of the X-ray structure of MPO, a high degree of similarity having been found in their sequences. Evidence supporting the hypothesis of an ester linkage between heme and apoprotein in LPO and EPO, originally proposed by Hultquist and Morrison is discussed. Received: 2 May 1996 / Accepted: 25 July 1996     

Activation of lactoperoxidase by heme‐linked protonation and heme‐independent iodide binding

Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red‐shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe‐His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme‐linked protonation site. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 113–120, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Resonance Raman spectroscopy study of change of iron spin state in horseradish peroxidase C induced by removal of calcium

Resonance Raman spectroscopy is used to probe the effect of calcium depletion on the heme group of horseradish peroxidase C at pH 8. Polarized Raman spectra are recorded with an argon ion laser at eight different wavelengths to provide a sound database for a reliable spectral decomposition. Upon calcium depletion, the spectrum is indicative of a predominantly pentacoordinated high spin state of the heme iron coexisting with small fractions of hexacoordinated high and low spin states. The dominant quantum mixed spin state of native ferric horseradish peroxidase, which is characteristic for class III peroxidases, is not detectable in the spectrum of the enzyme with partial distal Ca(2+) depletion. The quenching of the quantum mixed spin state and the predominance of the pentacoordinated high spin state are likely to arise from distortions induced by distal calcium depletion, which translates into a weaker Fe-N(epsilon)(His) bond and a more tilted imidazole. A correlation is proposed between the lower enzyme activity and the elimination of the pentacoordinated quantum mixed state upon Ca(2+) depletion.     

Thiocyanate modulates the catalytic activity of mammalian peroxidases

We investigated the potential role of the co-substrate, thiocyanate (SCN-), in modulating the catalytic activity of myeloperoxidase (MPO) and other members of the mammalian peroxidase superfamily (lactoperoxidase (LPO) and eosinophil peroxidase (EPO)). Pre-incubation of SCN- with MPO generates a more complex biological setting, because SCN- serves as either a substrate or inhibitor, causing diverse impacts on the MPO heme iron microenvironment. Consistent with this hypothesis, the relationship between the association rate constant of nitric oxide binding to MPO-Fe(III) as a function of SCN- concentration is bell-shaped, with a trough comparable with normal SCN- plasma levels. Rapid kinetic measurements indicate that MPO, EPO, and LPO Compound I formation occur at rates slower than complex decay, and its formation serves to simultaneously catalyze SCN- via 1e- and 2e- oxidation pathways. For the three enzymes, Compound II formation is a fundamental feature of catalysis and allows the enzymes to operate at a fraction of their possible maximum activities. MPO and EPO Compound II is relatively stable and decays gradually within minutes to ground state upon H2O2 exhaustion. In contrast, LPO Compound II is unstable and decays within seconds to ground state, suggesting that SCN- may serve as a substrate for Compound II. Compound II formation can be partially or completely prevented by increasing SCN- concentration, depending on the experimental conditions. Collectively, these results illustrate for the first time the potential mechanistic differences of these three enzymes. A modified kinetic model, which incorporates our current findings with the mammalian peroxidases classic cycle, is presented.     

Redox thermodynamics of lactoperoxidase and eosinophil peroxidase

Eosinophil peroxidase (EPO) and lactoperoxidase (LPO) are important constituents of the innate immune system of mammals. These heme enzymes belong to the peroxidase-cyclooxygenase superfamily and catalyze the oxidation of thiocyanate, bromide and nitrite to hypothiocyanate, hypobromous acid and nitrogen dioxide that are toxic for invading pathogens. In order to gain a better understanding of the observed differences in substrate specificity and oxidation capacity in relation to heme and protein structure, a comprehensive spectro-electrochemical investigation was performed. The reduction potential (E°′) of the Fe(III)/Fe(II) couple of EPO and LPO was determined to be −126 mV and −176 mV, respectively (25 °C, pH 7.0). Variable temperature experiments show that EPO and LPO feature different reduction thermodynamics. In particular, reduction of ferric EPO is enthalpically and entropically disfavored, whereas in LPO the entropic term, which selectively stabilizes the oxidized form, prevails on the enthalpic term that favors reduction of Fe(III). The data are discussed with respect to the architecture of the heme cavity and the substrate channel. Comparison with published data for myeloperoxidase demonstrates the effect of heme to protein linkages and heme distortion on the redox chemistry of mammalian peroxidases and in consequence on the enzymatic properties of these physiologically important oxidoreductases.     

Evidence for a hydroxide intermediate in cytochrome c oxidase

A transient intermediate of cytochrome c oxidase has been generated by exposing the enzyme to a laser beam in the presence of oxygen. This intermediate develops when the enzyme is simultaneously reduced photoreductively and oxidized chemically, thereby forcing it to turn over. Under these conditions a form of the enzyme is generated with a line at 477 cm-1 in the resonance Raman spectrum, which we attribute to an Fe-OH stretching mode based on oxygen and hydrogen isotopic substitution. This hydroxide intermediate relaxes back to the resting state of the enzyme upon removal from the laser beam. Hydroxide intermediates have been postulated many times in the past in proposed catalytic mechanisms. The data reported here supply the first evidence for the existence of such an intermediate and a method for stabilizing it.     

Resonance Raman investigation of an enzyme-inhibitor complex.

The resonance Raman spectrum has been recorded for two different binary complexes formed between 2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene (zincon) and liver alcohol dehydrogenase. The shifts in the zincon spectrum upon complexation with enzyme in one complex are similar to those in model compounds containing azo or formazyl linkages upon complexation of these with zinc. The results are interpreted in terms of complexation of zincon to a zinc atom at the enzyme active site. Since zincon is a coenzyme competitive inhibitor, it is probably bound at or near the coenzyme binding site; the results of this study, therefore, are useful in understanding the chemistry of zinc at the enzyme active site.     

Protein radical formation during lactoperoxidase-mediated oxidation of the suicide substrate glutathione: immunochemical detection of a lactoperoxidase radical-derived 5,5-dimethyl-1-pyrroline N-oxide nitrone adduct

A novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts has been developed. In this study, we employed this new approach, which combines the specificity of spin trapping and the sensitivity of antigen-antibody interactions, to investigate protein radical formation from lactoperoxidase (LPO). When LPO reacted with GSH in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct using enzyme-linked immunosorbent assay and Western blotting. The formation of this nitrone adduct depended on the concentrations of GSH, LPO, and DMPO as well as pH values, and GSH could not be replaced by H(2)O(2). The level of this nitrone adduct was decreased significantly by azide, catalase, ascorbate, iodide, thiocyanate, phenol, or nitrite. However, its formation was unaffected by chemical modification of free cysteine, tyrosine, and tryptophan residues on LPO. ESR spectra showed that a glutathiyl radical was formed from the LPO/GSH/DMPO system, but no protein radical adduct could be detected by ESR. Its formation was decreased by azide, catalase, ascorbate, iodide, or thiocyanate, whereas phenol or nitrite increased it. GSH caused marked changes in the spectrum of compound II of LPO, indicating that GSH binds to the heme of compound II, whereas phenol or nitrite prevented these changes and reduced compound II back to the native enzyme. GSH also dose-dependently inhibited the peroxidase activity of LPO as determined by measuring 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation. Taken together, these results demonstrate that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO.     

Age and genotype dependent erythropoietin protection in COVID-19

Erythropoietin (EPO) is the main mediator of erythropoiesis and an important tissue protective hormone that appears to mediate an ancestral neuroprotective innate immune response mechanism at an early age. When the young brain is threatened-prematurity, neonatal hyperbilirubinemia, malaria- EPO is hyper-secreted disproportionately to any concurrent anemic stimuli. Under eons of severe malarial selection pressure, neuroprotective EPO augmenting genetic determinants such as the various hemoglobinopathies, and the angiotensin converting enzyme (ACE) I/D polymorphism, have been positively selected. When malarial and other cerebral threats abate and the young child survives to adulthood, EPO subsides. Sustained high ACE and angiotensin II (Ang II) levels through the ACE D allele in adulthood may then become detrimental as witnessed by epidemiological studies. The ubiquitous renin angiotensin system (RAS) influences the α-klotho/fibroblast growth factor 23 (FGF23) circuitry, and both are interconnected with EPO. Here we propose that at a young age, EPO augmenting genetic determinants through ACE D allele elevated Ang II levels in some or HbE/beta thalassemia in others would increase EPO levels and shield against coronavirus disease 2019, akin to protection from malaria and dengue fever. Human evolution may use ACE2 as a “bait” for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to gain cellular entry in order to trigger an ACE/ACE2 imbalance and stimulate EPO hypersecretion using tissue RAS, uncoupled from hemoglobin levels. In subjects without EPO augmenting genetic determinants at any age, ACE2 binding and internalization upon SARS-CoV-2 entry would trigger an ACE/ACE2 imbalance, and Ang II oversecretion leading to protective EPO stimulation. In children, low nasal ACE2 Levels would beneficially augment this imbalance, especially for those without protective genetic determinants. On the other hand, in predisposed adults with the ACE D allele, ACE/ACE2 imbalance, may lead to uncontrolled RAS overactivity and an Ang II induced proinflammatory state and immune dysregulation, with interleukin 6 (IL-6), plasminogen activator inhibitor, and FGF23 elevations. IL-6 induced EPO suppression, aggravated through co-morbidities such as hypertension, diabetes, obesity, and RAS pharmacological interventions may potentially lead to acute respiratory distress syndrome, cytokine storm and/or autoimmunity. HbE/beta thalassemia carriers would enjoy protection at any age as their EPO stimulation is uncoupled from the RAS system. The timely use of rhEPO, EPO analogs, acetylsalicylic acid, bioactive lipids, or FGF23 antagonists in genetically predisposed individuals may counteract those detrimental effects.     

Purification and characterization of human salivary peroxidase

Human salivary peroxidase (SPO) has been purified to homogeneity by subjecting human parotid saliva to immunoaffinity, cation exchange, and affinity chromatography. These procedures resulted in a 992-fold purification of the enzyme. When purified SPO was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), three Coomassie stainable bands were apparent, all of which stained positive for enzyme activity. The apparent molecular weights of the three bands were 78,000, 80,000, and 280,000 as analyzed by SDS-PAGE. Reduction with 2-mercaptoethanol resulted in a decreased mobility of these bands, and enzyme activity could no longer be detected on the gels. The SPO preparation had the characteristic peroxidase heme spectrum in the range 405-420 nm. The ratio between the absorbance of the Soret band (412 nm) and the absorbance at 280 nm was 0.81. The enzyme activity was inhibited by the classical peroxidase inhibitors cyanide and azide. Salivary peroxidase is similar to bovine lactoperoxidase (LPO) in amino acid composition, in ultraviolet and visible spectrum, in reaction with cyanide, in susceptibility to 2-mercaptoethanol inactivation, and in thermal stability. The two enzymes differ in carbohydrate composition and content. SPO contains 4.6% and LPO 7% total neutral sugars. The ratio of glucosamine to galactosamine is 2:1 in SPO and 3:1 in LPO. SPO contains mannose, fucose, and galactose in a molar ratio of 1.5:1.5:1.0, while the ratio was 14.9:0.5:1.0 in LPO. Glucose was present in both preparations in minor amounts. The concentration of azide required for 50% inhibition of enzyme activity was 20-fold greater for LPO than for SPO.(ABSTRACT TRUNCATED AT 250 WORDS)     

A vibrational structure of 7,8-dihydrobiopterin bound to dihydroneopterin aldolase

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7, 8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and glycolaldehyde. An inhibitor of the enzyme, 7,8-dihydrobiopterin, free in solution and bound in its complex with the enzyme has been studied by Raman difference spectroscopy. By using isotopically labeled 7,8-dihydrobiopterin and normal mode analyses based on ab initio quantum mechanic methods, we have positively identified some of the Raman bands in the enzyme-bound inhibitor, particularly the important N5=C6 stretch mode. The spectrum of the enzyme-bound inhibitor shows that the pK(a) of N5 is not significantly increased in the complex. This result suggests that N5 of 7,8-dihydroneopterin is not protonated before the bond cleavage of 7,8-dihydroneopterin during the DHNA-catalyzed reaction as has been suggested. Our results also show that the N5=C6 stretch mode of 7, 8-dihydrobiopterin shifts 19 cm(-)(1) upon binding to DHNA. Various possibilities on how the enzyme can bring about such large frequency change of the N5=C6 stretch mode are discussed.     

Binding of thiocyanate to lactoperoxidase: 1H and 15N nuclear magnetic resonance studies

The binding of thiocyanate to lactoperoxidase (LPO) has been investigated by 1H and 15N NMR spectroscopy. 1H NMR of LPO shows that the major broad heme methyl proton resonance at about 61 ppm is shifted upfield by addition of the thiocyanate, indicating binding of the thiocyanate to the enzyme. The pH dependence of line width of 15N resonance of SC15N- in the presence of the enzyme has revealed that the binding of the thiocyanate to the enzyme is facilitated by protonation of an ionizable group (with pKa of 6.4), which is presumably distal histidine. Dissociation constants (KD) of SC15N-/LPO, SC15N-/LPO/I-, and SC15N-/LPO/CN- equilibria have been determined by 15N T1 measurements and found to be 90 +/- 5, 173 +/- 20, and 83 +/- 6 mM, respectively. On the basis of these values of KD, it is suggested that the iodide ion inhibits the binding of the thiocyanate but cyanide ion does not. The thiocyanate is shown to bind at the same site of LPO as iodide does, but the binding is considerably weaker and is away from the ferric ion. The distance of 15N of the bound thiocyanate ion from the iron is determined to be 7.2 +/- 0.2 A from the 15N T1 measurements.     

Biochemical evidence for heme linkage through esters with Asp-93 and Glu-241 in human eosinophil peroxidase. The ester with Asp-93 is only partially formed in vivo.

The covalent heme attachment has been extensively studied by spectroscopic methods in myeloperoxidase and lactoperoxidase (LPO) but not in eosinophil peroxidase (EPO). We show that heme linkage to the heavy chain is invariably present, whereas heme linkage to the light chain of EPO is present in less than one-third of EPO molecules. Mass analysis of isolated heme bispeptides supports the hypothesis of a heme b linked through two esters to the polypeptide. Mass analysis of heme monopeptides reveals that >90% have a nonderivatized methyl group at the position of the light chain linkage. Apparently, an ester had not been formed during biosynthesis. The light chain linkage could be formed by incubation with hydrogen peroxide, in accordance with a recent hypothesis of autocatalytic heme attachment based on studies with LPO (DePillis, G. D., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano P. R. (1997) J. Biol. Chem. 272, 8857-8860). By sequence analysis of isolated heme peptides after aminolysis, we unambiguously identified the acidic residues, Asp-93 of the light chain and Glu-241 of the heavy chain, that form esters with the heme group. This is the first biochemical support for ester linkage to two specific residues in eosinophil peroxidase. From a parallel study with LPO, we show that Asp-125 and Glu-275 are engaged in ester linkage. The species with a nonderivatized methyl group was not found among LPO peptides.     

Denaturation and renaturation of myeloperoxidase. Consequences for the nature of the prosthetic group.

The effects of the chaotropic agent, guanidine HCl, on the chlorinating activity, optical absorption, EPR, and resonance Raman spectra of myeloperoxidase have been studied. In the presence of the agent the Soret optical absorption of the reduced enzyme (lambda max = 474 nm) is blue shifted to 448 nm, a position similar to heme alpha-containing enzymes. The chlorinating activity of the enzyme disappears, and EPR spectra show a loss of intensity of the rhombic high spin heme signals (gx = 6.9; gy = 5.4) and the appearance of a more axial high spin signal (gx = gy = 6.0). Surprisingly the effects of guanidine HCl are partly reversible. Upon decreasing the concentration of the chaotropic agents by dilution, both the chlorinating activity and the original optical spectrum of native reduced enzyme (lambda max = 474 nm) are partly restored. The resonance Raman spectra of denatured cyanomyeloperoxidase are less complicated than those of native myeloperoxidase, which have been interpreted previously to suggest an iron chlorin chromophore. The multiple lines in the oxidation state marker region are not seen in the spectra of the denatured species. The changes suggest that upon denaturation the macrocycle is converted into a more symmetric structure. Since the effects on the optical absorption spectrum are reversible we speculate that, in the native enzyme, an apparent porphyrin macrocycle undergoes a reversible interaction with amino acid residues in the protein which creates an asymmetry in the electronic distribution of the macrocycle. Comparison of the Raman spectra of denatured cyanomyeloperoxidase with those of analogous heme alpha model complexes suggests the presence of a formyl group in the denatured species; our data, however, demonstrate that the chromophore structure is not identical to heme alpha and may contain a different C beta substitution on the ring macrocycle.     

Possible involvement of delta-6-desaturase in control of melanoma growth by gamma-linolenic acid.

This study examined the effects of linoleic acid (LA) and gamma-linolenic acid (GLA) on BL6 melanoma growth in cell culture and of safflower oil (SFO) which contains LA and evening primrose oil (EPO) which contains GLA, on melanoma growth when grown in mice. The delta-6-desaturase activity of the melanoma cells in the two systems was also examined and an attempt made to relate the activity of the enzyme to the effects of GLA on cell and tumour growth. LA and GLA were found to be equipotent in inhibiting growth of the in vitro cultured BL6 cells which were found to contain an appreciable level of delta-6-desaturase activity. EPO was however found to be a more potent promoter of in vivo melanoma growth in mice than SFO. Melanomas grown in mice were found to lack delta-6-desaturase activity suggesting that the EPO diet, by providing GLA, was able to compensate for the loss of enzyme activity in the melanomas. The possibility that melanomas in mice have a requirement for GLA for growth while in in vitro cultured cells excess GLA inhibits the growth of the cells through an increase in lipid peroxidation is discussed.     

Lactoperoxidase: structural insights into the function,ligand binding and inhibition

Lactoperoxidase (LPO) is a member of a large group of mammalian heme peroxidases that include myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The LPO is found in exocrine secretions including milk. It is responsible for the inactivation of a wide range of micro-organisms and hence, is an important component of defense mechanism in the body. With the help of hydrogen peroxide, it catalyzes the oxidation of halides, pseudohalides and organic aromatic molecules. Historically, LPO was isolated in 1943, nearly seventy years ago but its three-dimensional crystal structure has been elucidated only recently. This review provides various details of this protein from its discovery to understanding its structure, function and applications. In order to highlight species dependent variations in the structure and function of LPO, a detailed comparison of sequence, structure and function of LPO from various species have been made. The structural basis of ligand binding and distinctions in the modes of binding of substrates and inhibitors have been analyzed extensively.     

A computer-controlled laser Raman spectrophotometer with interactive-graphics data analysis.

The computerization of a laser Raman spectrophotometer is described which permits automated operation of the instrument for signal averaging. The use of an interactive-graphics computer terminal for the rapid reduction of digitized data is discussed and illustrated by the acquisition and analysis of the Raman spectrum from the enzyme, protocatechuate 3,4-dioxygenase.