Growth of Plasmodium falciparum in response to a rotating magnetic field.

BACKGROUND: Plasmodium falciparum is the deadliest strain of malaria and the mortality rate is increasing because of pathogen drug resistance. Increasing knowledge of the parasite life cycle and mechanism of infection may provide new models for improved treatment paradigms. This study sought to investigate the paramagnetic nature of the parasite's haemozoin to inhibit parasite viability. RESULTS: Paramagnetic haemozoin crystals, a byproduct of the parasite's haemoglobin digestion, interact with a rotating magnetic field, which prevents their complete formation, causing the accumulation of free haem, which is lethal to the parasites. Plasmodium falciparum cultures of different stages of intraerythrocytic growth (rings, trophozoites, and schizonts) were exposed to a magnetic field of 0.46 T at frequencies of 0 Hz (static), 1, 5, and 10 Hz for 48 h. The numbers of parasites were counted over the course of one intraerythrocytic life cycle via flow cytometry. At 10 Hz the schizont life stage was most affected by the rotating magnetic fields (p = 0.0075) as compared to a static magnetic field of the same strength. Parasite growth in the presence of a static magnetic field appears to aid parasite growth. CONCLUSIONS: Sequestration of the toxic haem resulting from haemoglobin digestion is key for the parasites' survival and the focus of almost all existing anti-malarial drugs. Understanding how the parasites create the haemozoin molecule and the disruption of its creation aids in the development of drugs to combat this disease.

Role of Ionic Strength and pH in Modulating Thermodynamic Profiles Associated with CO Escape from Rice Nonsymbiotic Hemoglobin 1.

Type 1 nonsymbiotic hemoglobins are found in a wide variety of land plants and exhibit very high affinities for exogenous gaseous ligands. These proteins are presumed to have a role in protecting plant cells from oxidative stress under etiolated/hypoxic conditions through NO dioxygenase activity. In this study we have employed photoacoustic calorimetry, time-resolved absorption spectroscopy, and classical molecular dynamics simulations in order to elucidate thermodynamics, kinetics, and ligand migration pathways upon CO photodissociation from WT and a H73L mutant of type 1 nonsymbiotic hemoglobin from Oryza sativa (rice). We observe a temperature dependence of the resolved thermodynamic parameters for CO photodissociation from CO-rHb1 which we attribute to temperature dependent formation of a network of electrostatic interactions in the vicinity of the heme propionate groups. We also observe slower ligand escape from the protein matrix under mildly acidic conditions in both the WT and H73L mutant (τ = 134 ± 19 and 90 ± 15 ns). Visualization of transient hydrophobic channels within our classical molecular dynamics trajectories allows us to attribute this phenomenon to a change in the ligand migration pathway which occurs upon protonation of the distal His73, His117, and His152. Protonation of these residues may be relevant to the functioning of the protein in vivo given that etiolation/hypoxia can cause a decrease in intracellular pH in plant cells.

A Study of the Dynamics of the Heme Pocket and C-helix in CooA upon CO Dissociation Using Time-Resolved Visible and UV Resonance Raman Spectroscopy.

CooA is a CO-sensing transcriptional activator from the photosynthetic bacterium Rhodospirillum rubrum that binds CO at the heme iron. The heme iron in ferrous CooA has two axial ligands: His77 and Pro2. CO displaces Pro2 and induces a conformational change in CooA. The dissociation of CO and/or ligation of the Pro2 residue are believed to trigger structural changes in the protein. Visible time-resolved resonance Raman spectra obtained in this study indicated that the ν(Fe-His) mode, arising from the proximal His77-iron stretch, does not shift until 50 µs after the photodissociation of CO. Ligation of the Pro2 residue to the heme iron was observed around 50 µs after the photodissociation of CO, suggesting that the ν(Fe-His) band exhibits no shift until the ligation of Pro2. UV resonance Raman spectra suggested structural changes in the vicinity of Trp110 in the C-helix upon CO binding, but no or very small spectral changes in the time-resolved UV resonance Raman spectra were observed from 100 ns to 100 µs after the photodissociation of CO. These results strongly suggest that the conformational change of CooA is induced by the ligation of Pro2 to the heme iron.

Laser-induced inactivation of Plasmodium falciparum.

BACKGROUND: Haemozoin crystals, produced by Plasmodium during its intra-erythrocytic asexual reproduction cycle, can generate UV light via the laser-induced, non-linear optical process of third harmonic generation (THG). In the current study the feasibility of using haemozoin, constitutively stored in the parasite's food vacuole, to kill the parasite by irradiation with a near IR laser was evaluated. METHODS: Cultured Plasmodium parasites at different stages of development were irradiated with a pulsed NIR laser and the viability of parasites at each stage was evaluated from their corresponding growth curves using the continuous culture method. Additional testing for germicidal effects of haemozoin and NIR laser was performed by adding synthetic haemozoin crystals to Escherichia coli in suspension. Cell suspensions were then irradiated with the laser and small aliquots taken and spread on agar plates containing selective agents to determine cell viability (CFU). RESULTS: Parasites in the late-trophozoites form as well as trophozoites in early-stage of DNA synthesis were found to be the most sensitive to the treatment with -4-log reduction in viability after six passes through the laser beam; followed by parasites in ring phase (-2-log reduction). A -1-log reduction in E. coli viability was obtained following a 60 min irradiation regimen of the bacteria in the presence of 1 µM synthetic haemozoin and a -2-log reduction in the presence of 10 µM haemozoin. Minimal (≤ 15%) cell kill was observed in the presence of 10 µM haemin. CONCLUSIONS: Laser-induced third-harmonic generation by haemozoin can be used to inactivate Plasmodium. This result may have clinical implications for treating severe malaria symptoms by irradiating the patient's blood through the skin or through dialysis tubing with a NIR laser.

Cryoradiolytic reduction of crystalline heme proteins: analysis by UV-Vis spectroscopy and X-ray crystallography.

The X-ray crystallographic analysis of redox-active systems may be complicated by photoreduction. Although radiolytic reduction by the probing X-ray beam may be exploited to generate otherwise short-lived reaction intermediates of metalloproteins, it is generally an undesired feature. Here, the X-ray-induced reduction of the three heme proteins myoglobin, cytochrome P450cam and chloroperoxidase has been followed by on-line UV-Vis absorption spectroscopy. All three systems showed a very rapid reduction of the heme iron. In chloroperoxidase the change of the ionization state from ferric to ferrous heme is associated with a movement of the heme-coordinating water molecule. The influence of the energy of the incident X-ray photons and of the presence of scavengers on the apparent reduction rate of ferric myoglobin crystals was analyzed.

Correlation of empirical magnetic susceptibility tensors and structure in low-spin haem proteins.

Experimental magnetic susceptibility tensors are reported for eight haems c with bis-His coordination. These data, obtained by fitting the dipolar shifts of backbone protons in the tetrahaem cytochromes c(3) from Desulfovibrio vulgaris and D. gigas, are analysed together with published values for other haem proteins. The x and y axes are found to rotate in the opposite sense to the axial ligands and are also counter-rotated with respect to the frontier molecular orbitals of the haem. The magnetic z-axis is close to the normal to the haem plane in each case. The magnitudes of the magnetic anisotropies are used to derive crystal field parameters and the rhombic splitting, V, is correlated with the dihedral angle between the axial ligands. Hence, it is apparent that the axial ligands are the dominant factor in determining the variation in magnetic properties between haems, and it is confirmed that "high g(max)" EPR signals are a reliable indicator of near-perpendicular ligands. These results are in full agreement with the analysis of non-Curie effects and electronic structure in the His-Met coordinated cytochromes c and C(551). Collectively, they show that the orientations of axial ligands to the haem may be estimated from single-crystal EPR data, from (13)C NMR shifts of the haem substituents, or from NMR dipolar shifts of the polypeptide.

N2 laser-induced oxidation of hemoproteins in red blood cell lysate.

Irradiation of red blood cell lysate with a N(2) laser (337 nm) was observed to induce oxidation of hemoproteins. This process showed a strong dependence on the concentration of red blood cell lysate and the dose of radiation. Studies of mechanisms and experiments with deoxygenated red blood cell lysate rule out involvement of any reactive oxygen species and suggest that the process is not a photodynamic reaction.

The photodynamic and non-photodynamic actions of porphyrins.

Porphyrias are a family of inherited diseases, each associated with a partial defect in one of the enzymes of the heme biosynthetic pathway. In six of the eight porphyrias described, the main clinical manifestation is skin photosensitivity brought about by the action of light on porphyrins, which are deposited in the upper epidermal layer of the skin. Porphyrins absorb light energy intensively in the UV region, and to a lesser extent in the long visible bands, resulting in transitions to excited electronic states. The excited porphyrin may react directly with biological structures (type I reactions) or with molecular oxygen, generating excited singlet oxygen (type II reactions). Besides this well-known photodynamic action of porphyrins, a novel light-independent effect of porphyrins has been described. Irradiation of enzymes in the presence of porphyrins mainly induces type I reactions, although type II reactions could also occur, further increasing the direct non-photodynamic effect of porphyrins on proteins and macro-molecules. Conformational changes of protein structure are induced by porphyrins in the dark or under UV light, resulting in reduced enzyme activity and increased proteolytic susceptibility. The effect of porphyrins depends not only on their physico-chemical properties but also on the specific site on the protein on which they act. Porphyrin action alters the functionality of the enzymes of the heme biosynthetic pathway exacerbating the metabolic deficiencies in porphyrias. Light energy absorption by porphyrins results in the generation of oxygen reactive species, overcoming the protective cellular mechanisms and leading to molecular, cell and tissue damage, thus amplifying the porphyric picture.

The photodynamic and non-photodynamic actions of porphyrins

Porphyrias are a family of inherited diseases, each associated with a partial defect in one of the enzymes of the heme biosynthetic pathway. In six of the eight porphyrias described, the main clinical manifestation is skin photosensitivity brought about by the action of light on porphyrins, which are deposited in the upper epidermal layer of the skin. Porphyrins absorb light energy intensively in the UV region, and to a lesser extent in the long visible bands, resulting in transitions to excited electronic states. The excited porphyrin may react directly with biological structures (type I reactions) or with molecular oxygen, generating excited singlet oxygen (type II reactions). Besides this well-known photodynamic action of porphyrins, a novel light-independent effect of porphyrins has been described. Irradiation of enzymes in the presence of porphyrins mainly induces type I reactions, although type II reactions could also occur, further increasing the direct non-photodynamic effect of porphyrins on proteins and macromolecules. Conformational changes of protein structure are induced by porphyrins in the dark or under UV light, resulting in reduced enzyme activity and increased proteolytic susceptibility. The effect of porphyrins depends not only on their physico-chemical properties but also on the specific site on the protein on which they act. Porphyrin action alters the functionality of the enzymes of the heme biosynthetic pathway exacerbating the metabolic deficiencies in porphyrias. Light energy absorption by porphyrins results in the generation of oxygen reactive species, overcoming the protective cellular mechanisms and leading to molecular, cell and tissue damage, thus amplifying the porphyric picture

Photoacoustic calorimetry of proteins.

We have described two examples of time-resolved photoacoustic calorimetry for the study of heme protein transient intermediates. Before photoacoustic calorimetry, determining thermodynamic information on short-lived intermediates was difficult. Along with being sensitive to enthalpic and volume changes, photoacoustic calorimetry can detect conformational changes in a time-resolved manner. In complex protein systems, the interpretation of the structural origins of a conformational change is sometimes difficult. Site-directed mutagenesis has been used successfully to identify the residues that play important roles in the ligand binding to both Mb and cytochrome P450cam. In both systems the hydration state of salt bridges gave rise to volume changes that were identified through mutagenesis of the residues involved. With its increasing popularity and the power of site-directed mutagenesis, time-resolved photoacoustic calorimetry is fast becoming a technique to probe conformational dynamics in proteins.

Mechanistic studies on uroporphyrin I-induced photoinactivation of some heme-enzymes.

Aerobic and anaerobic studies have demonstrated that uroporphyrin I-induced inactivation of delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase was dependent on oxygen and mediated by reactive oxygen species. The mechanism of photoinactivation of those heme-enzymes from human erythrocytes by uroporphyrin I by u.v. light was investigated. Enzymes of the heme pathway were preincubated in the presence of specific scavengers for several reactive oxygen species and then exposed to uroporphyrin I and u.v. light. Upon exposure of the enzymes to the porphyrin under u.v. light, and in an aerobic atmosphere, the percentage of enzyme activities with respect to the corresponding controls were 50.2 +/- 5.1 (SD, n = 6), 25.3 +/- 3.0 (SD, n = 6), 25.9 +/- 2.8 (SD, n = 6) and 49.7 +/- 7.5 (SD, n = 8) for delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase, respectively. The presence of sodium azide, histidine or superoxide dismutase did not protect the enzymes against the effects of uroporphyrin I. However, both cysteine and potassium ferrycyanide prevented the enzyme photoinactivation induced by uroporphyrin I. In the presence of either catalase or GSH, the enzyme photoinactivation was lower. Ethanol, glucose and dimethylsulfoxide had no effect on enzyme activity, while ion chelators had variable effects. This study shows that the type II mechanism is not the predominant reaction mediating the uroporphyrin I effect and enzyme photoinactivation would involve an electron transfer. Hydrogen peroxide and hydroxyl radicals could possibly mediate the uroporphyrin I-induced enzyme photoinactivation.

Protein dynamics. Comparative investigation on heme-proteins with different physiological roles.

We report the low temperature carbon monoxide recombination kinetics after photolysis and the temperature dependence of the visible absorption spectra of the isolated alpha SH-CO and beta SH-CO subunits from human hemoglobin A in ethylene glycol/water and in glycerol/water mixtures. Kinetic measurements on sperm whale (Physeter catodon) myoglobin and previously published optical spectroscopy data on the latter protein and on human hemoglobin A, in both solvents, (Cordone, L., A. Cupane, M. Leone, E. Vitrano, and D. Bulone. 1988. J. Mol. Biol. 199:312-218) are taken as reference. Low temperature flash photolysis data are analyzed within the multiple substates model proposed by Frauenfelder and co-workers (Austin, R. H., K. W. Beeson, L. Eisenstein, H. Frauenfelder, and I. C. Gunsalus. 1975. Biochemistry. 14:5355-5373). Within this model a distribution of activation enthalpies for ligand binding accounts for the structural heterogeneity of the protein, while the preexponential factor, containing also the entropic contribution to the free energy of the process, is considered to be constant for all conformational substates. Optical spectra are deconvoluted in gaussian components and the temperature dependence of the moments of the resulting bands is analyzed, within the harmonic Frank-Condon approximation, to obtain information on the stereodynamic properties of the heme pocket. The kinetic and spectral parameters thus obtained are found to be protein dependent also with respect to their sensitivity to changes in the composition of the external medium. A close correlation between the kinetic and spectral features is observed for the proteins examined under all experimental conditions studied. The results reported are discussed in terms of differences in the heme pocket structure and in the conformational heterogeneity among the various proteins, as related to their different capability to accommodate constraints imposed by the external medium.

[The role of the complex formation between cytochrome c and NAD in the manifestation of the photoprotective effect of the dinucleotide in relation to hemeprotein]. / O roli kompleksoobrazovaniia mezhdu tsitokhromom c i NAD v proiavlenii fotozashchitnogo éffekta dinukleotida po otnosheniiu k gembelku.

UV irradiation of free ferricytochrome solutions, pH 8, induces photorecovery of protein molecules. Hemoproteide photorecovery does not occur after irradiation of the ferricytochrome c/NAD mixture, pH 6 and 8: dinucleotide exerts a photoprotective effect with respect to ferricytochrome. This NAD effect is not observed after exposure of the ferricytochrome c/NAD system, pH 4. With this pH value, each component of the above mixture is eluted from a gel chromatographic column by its peak, whereas with pH 6 and 8, NAD and ferricytochrome c leave the column as one fraction. This indicates that the photoprotective effect of the coenzyme manifests itself upon formation of complex with hemoprotein.

Photodynamic and non-photodynamic action of several porphyrins on the activity of some heme-enzymes.

The action of porphyrins, uroporphyrin I and III (URO I and URO III), pentacarboxylic porphyrin I (PENTA I), coproporphyrin I and III (COPRO I and COPRO III), protoporphyrin IX (PROTO IX) and mesoporphyrin (MESO), on the activity of human erythrocytes delta-aminolevulinic acid dehydratase, porphobilinogenase, deaminase and uroporphyrinogen decarboxylase in the dark and under UV light was investigated. Both photoinactivation and light-independent inactivation was found in all four enzymes using URO I as sensitizer. URO III had a similar action as URO I on porphobilinogenase and deaminase and PROTO IX exerted equal effect as URO I on delta-aminolevulinic acid dehydratase and uroporphyrinogen decarboxylase. Photodynamic efficiency of the porphyrins was dependent on their molecular structure. Selective photodecomposition of enzymes by URO I, greater specificity of tumor uptake by URO I and enhanced porphyrin synthesis by tumors from delta-aminolevulic acid, with predominant formation of URO I, underline the possibility of using URO I in detection of malignant cells and photodynamic therapy.

[Dissipative functions of processes of electromagnetic radiation interaction with biological objects]. / Dissipativnye funktsii protsessov vzaimodeistviia élektromagnitnogo izlucheniia s biologichekimi ob''ektami.

Entropy generation rate inside the biological systems due to internal irreversible processes of the interaction with electromagnetic radiation is calculated for the processes of chemical free-energy increase. The irreversibility of several processes (photosynthesis in plants, eyesight of man, bioresonance effects of high frequencies of microwaves) is estimated under concrete experimental conditions. It is shown that the irreversible of five biological processes differs very much (by 10(8) times).

Green hemoprotein of erythrocytes: methemoglobin superoxide transferase.

Influences of base (pH 10), heat (50 degrees C), microwave radiation (2450 MHz, 103 +/- 4 W/kg), and hydrogen peroxide (5.6 mM) generated by glucose oxidase on oxidation of human oxyhemoglobin to methemoglobin were examined. Conversion of oxyhemoglobin to methemoglobin was followed by the difference in absorbancy of 540 or 542 nm and 576 nm wavelength light versus time. Fresh basic hemolysates auto-oxidized on heating with a zero order rate constant, implying that hemoglobin or another protein saturated with oxyhemoglobin catalyzed the oxidation. Simultaneous microwave irradiation inhibited thermally induced auto-oxidation on the average by 28.6%. However, there was great variability among samples and a decrease in auto-oxidation with aging of individual samples. The auto-oxidation rate was independent of initial oxyhemoglobin concentration. Oxidation of partially purified oxyhemoglobin by hydrogen peroxide was not influenced by microwave irradiation. Adding green hemoprotein isolated from human erythrocytes to the oxyhemoglobin/glucose oxidase reaction mixture yielded absorption spectra (500-600 nm) that were a combination of oxyhemoglobin, deoxyhemoglobin, and methemoglobin spectra. Green hemoprotein was labile in hemolysates but stable in a partially purified ferric form. These results imply that thermally unstable reduced green hemoprotein can reverse oxidation of oxyhemoglobin by hydrogen peroxide and could mediate the thermally induced and microwave inhibited auto-oxidation of oxyhemoglobin.

Interaction of radiation-generated radicals with myoglobin in aqueous solution. III. Effects of oxygen and catalase on the product distribution in solutions of ferrimyoglobin containing N2O.

The gamma-radiolysis of aqueous solutions of ferrimyoglobin in the presence of N2O at pH 7.3 has been examined as a function of added catalase and oxygen. Changes in the nature of the heme group have been monitored by visible absorption spectrophotometry and analysed quantitatively by a multiple wavelength method based on Beer's Law. Simple chemical analyses have been used to confirm qualitative identification of the product derivatives. As observed previously, the ferriheme is reduced by indirect globin-mediated action initiated by OH/H. The yield of reduced product decreases as [O2] increases. Conversion to ferrimyoglobin through the participation of H2O2 derived from irradiated water and from protein-mediated processes in oxygenated solution, is eliminated by the presence of catalase. Formation of a hemichrome form of ferrimyoglobin is apparent at higher doses in the presence of O2. These results demonstrate that oxygen plays an important role in controlling the nature and extent of redox that manifests ultimately on the heme group of ferrimyoglobin as a result of the initial interaction of OH/H.