RESUMO
Serial X-ray crystallography allows macromolecular structure determination at both X-ray free electron lasers (XFELs) and, more recently, synchrotron sources. The time resolution for serial synchrotron crystallography experiments has been limited to millisecond timescales with monochromatic beams. The polychromatic, "pink", beam provides a more than two orders of magnitude increased photon flux and hence allows accessing much shorter timescales in diffraction experiments at synchrotron sources. Here we report the structure determination of two different protein samples by merging pink-beam diffraction patterns from many crystals, each collected with a single 100 ps X-ray pulse exposure per crystal using a setup optimized for very low scattering background. In contrast to experiments with monochromatic radiation, data from only 50 crystals were required to obtain complete datasets. The high quality of the diffraction data highlights the potential of this method for studying irreversible reactions at sub-microsecond timescales using high-brightness X-ray facilities.
Assuntos
Cristalografia por Raios X/métodos , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/estatística & dados numéricos , Bases de Dados de Compostos Químicos/estatística & dados numéricos , Endopeptidase K/química , Desenho de Equipamento , Modelos Moleculares , Ficocianina/química , Conformação Proteica , Eletricidade Estática , Síncrotrons , Difração de Raios XRESUMO
X-ray beam stability is crucial for acquiring high-quality data at synchrotron beamline facilities. When the X-ray beam and defining apertures are of similar dimensions, small misalignments driven by position instabilities give rise to large intensity fluctuations. This problem is solved using extremum seeking feedback control (ESFC) for in situ vertical beam position stabilization. In this setup, the intensity spatial gradient required for ESFC is determined by phase comparison of intensity oscillations downstream from the sample with pre-existing vertical beam oscillations. This approach compensates for vertical position drift from all sources with position recovery times <6â s and intensity stability through a 5â µm aperture measured at 1.5% FWHM over a period of 8â hours.
Assuntos
Síncrotrons , Raios XRESUMO
The wild boar is an important source of trichinellosis for people in European countries as a large number of hunted animals escape veterinary control. In November 2012, uncooked sausages made with meat from wild boar were consumed by 38 persons in a village of the Lucca province (Tuscany region, Italy). Of them, 34 were serologically positive, 32 developed clinical signs and symptoms of trichinellosis, and two were asymptomatic. Trichinella britovi larvae were detected in vacuum-packed sausages made with the same batch of sausages consumed raw which had been prepared with meat from wild boar hunted in the Lucca province. As no case of trichinellosis had been reported in this region during the last 20 years, the regional public health authority considered the risk for this zoonosis to be negligible and put in place a surveillance programme on Trichinella spp. in indicator animals (mainly foxes and including wild boar for private consumption), by testing only a percentage of heads. The experience from this outbreak shows that the definition of a region with a negligible risk for Trichinella infection is not applicable to wild boar and stresses the need to test all Trichinella-susceptible wild animals intended for human consumption and to implement risk communication to consumers and hunters.
Assuntos
Surtos de Doenças , Doenças Transmitidas por Alimentos/microbiologia , Carne/microbiologia , Sus scrofa/microbiologia , Doenças dos Suínos/transmissão , Triquinelose/transmissão , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Feminino , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/epidemiologia , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Suínos , Doenças dos Suínos/epidemiologia , Trichinella/isolamento & purificação , Triquinelose/epidemiologia , Adulto JovemRESUMO
Sixteen specimens of female crucian carp, Carassius carassius (L.), during the breeding season, were investigated for post-mortem and full diagnostic examination during a mortality outbreak in a tributary stream of the Arno River in Tuscany in 2011. Necropsy highlighted the presence of a swollen anus and widespread haemorrhages in the body, fins, gills and eyes. Haemorrhages in internal organs and spleen granulomas were also observed. Bacteria isolated from the brain, kidney and spleen of affected fish were identified as A. sobria. Microscopic lesions observed in gills were characterized by necrosis of the secondary lamellae, congestion and multifocal lamellar fusion. The kidney showed necrosis, oedema, fibrin exudation and areas of haemorrhages, while in the spleen the main lesions were by multifocal necrosis of the lymphoid tissue. In the gills, transmission electron microscopy revealed herpesvirus-like particles, subsequently identified as Cyprinid herpesvirus-2 (CyHV-2) with a nested PCR protocol. Although it was not possible to attribute a pathogenic role to CyHV-2 in this mortality event, the identification of this herpesvirus in crucian carp increases the concern about its potential role in this species.
Assuntos
Carpas/microbiologia , Carpas/virologia , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas/veterinária , Infecções por Herpesviridae/veterinária , Aeromonas/fisiologia , Animais , Linhagem Celular , Feminino , Doenças dos Peixes/microbiologia , Doenças dos Peixes/patologia , Doenças dos Peixes/virologia , Brânquias/patologia , Infecções por Bactérias Gram-Negativas/complicações , Infecções por Bactérias Gram-Negativas/patologia , Herpesviridae/isolamento & purificação , Herpesviridae/ultraestrutura , Infecções por Herpesviridae/complicações , Infecções por Herpesviridae/patologia , Itália , Rim/patologia , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Baço/patologiaRESUMO
Proteins are dynamic molecules whose function in virtually all biological processes requires conformational motion. Direct experimental probes of protein structure in solution are needed to characterize these motions. Anomalous scattering from proteins in solution has the potential to act as a precise molecular ruler to determine the positions of specific chemical groups or atoms within proteins under conditions in which structural changes can take place free from the constraints of crystal contacts. In solution, anomalous diffraction has two components: a set of cross-terms that depend on the relative location of the anomalous centers and the rest of the protein, and a set of pure anomalous terms that depend on the distances between the anomalous centers. The cross-terms are demonstrated here to be observable and to provide direct information about the distance between the anomalous center and the center of mass of the protein. The second set of terms appears immeasurably small in the context of current experimental capabilities. Here, we outline the theory underlying anomalous scattering from proteins in solution, predict the anomalous differences expected on the basis of atomic coordinate sets, and demonstrate the measurement of anomalous differences at the iron edge for solutions of myoglobin and hemoglobin.
Assuntos
Hemoglobina A/química , Mioglobina/química , Difração de Raios X/métodos , Animais , Humanos , SoluçõesRESUMO
Specific ligation states of hemoglobin are, when crystallized, capable of taking on multiple quaternary structures. The relationship between these structures, captured in crystal lattices, and hemoglobin structure in solution remains uncertain. Wide-angle X-ray solution scattering (WAXS) is a sensitive probe of protein structure in solution that can distinguish among similar structures and has the potential to contribute to these issues. We used WAXS to assess the relationships among the structures of human and bovine hemoglobins in different liganded forms in solution. WAXS data readily distinguished among the various forms of hemoglobins. WAXS patterns confirm some of the relationships among hemoglobin structures that have been defined through crystallography and NMR and extend others. For instance, methemoglobin A in solution is, as expected, nearly indistinguishable from HbCO A. Interestingly, for bovine hemoglobin, the differences between deoxy-Hb, methemoglobin and HbCO are smaller than the corresponding differences in human hemoglobin. WAXS data were also used to assess the spatial extent of structural fluctuations of various hemoglobins in solution. Dynamics has been implicated in allosteric control of hemoglobin, and increased dynamics has been associated with lowered oxygen affinity. Consistent with that notion, WAXS patterns indicate that deoxy-Hb A exhibits substantially larger structural fluctuations than HbCO A. Comparisons between the observed WAXS patterns and those predicted on the basis of atomic coordinate sets suggest that the structures of Hb in different liganded forms exhibit clear differences from known crystal structures.
Assuntos
Carboxihemoglobina/química , Metemoglobina/química , Espalhamento a Baixo Ângulo , Regulação Alostérica , Animais , Bovinos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Soluções , Difração de Raios XRESUMO
AIMS: Multicentre evaluation of biochemical and molecular methods for the identification of Vibrio parahaemolyticus. METHODS AND RESULTS: For the biochemical identification methods, API 20E and API 20NE and Alsina's scheme were evaluated in intra- and interlaboratory tests in order to determine the accuracy and concordance of each method. Both in intra- and interlaboratory tests, the Alsina's scheme showed the highest sensitivity (86% of correct identifications in the interlaboratory test). False-positive results were obtained by all methods (specificity was 95% for API 20E, 73% for API 20NE and 84% for Alsina's scheme) and concordance varied from 65% of API 20NE to 84% of API 20E. For the molecular identifications, polymerase chain reaction (PCR) for the detection of toxR gene, tl gene and pR72H fragment were tested on 30 strains by two laboratories. The PCR for toxR showed the highest inclusivity (96%), exclusivity (100%) and concordance (97%). CONCLUSIONS: Among the biochemical identification methods tested, the Alsina's scheme gave more reliable results; however, in order to avoid false-positive results, all the biochemical identifications should be confirmed by means of molecular methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Availability of an efficient identification method of Vibrio parahaemolyticus to use in official control of fisheries products.
Assuntos
Vibrio parahaemolyticus/isolamento & purificação , Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Reações Falso-Positivas , Genes Bacterianos/genética , Reação em Cadeia da Polimerase/métodos , Fatores de Transcrição/genética , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismoRESUMO
A chemical genetics approach to functional analysis of gene products utilizes high-throughput target-based screens of compound libraries to identify ligands that modulate the activity of proteins of interest. Candidates are further screened using functional assays designed specifically for the protein--and function--of interest, suffering from the need to customize the assay to each protein. An alternative strategy is to utilize a probe to detect the structural changes that usually accompany binding of a functional ligand. Wide-angle X-ray scattering from proteins provides a means to identify a broad range of ligand-induced changes in secondary, tertiary, and quaternary structure. The speed and accuracy of data acquisition, combined with the label-free targets and binding conditions achievable, indicate that WAXS is well suited as a moderate-throughput assay in the detection and analysis of protein-ligand interactions.
Assuntos
Conformação Proteica , Álcool Desidrogenase/química , Álcool Desidrogenase/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cristalografia por Raios X/métodos , Ligantes , Proteínas Ligantes de Maltose , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Espalhamento de Radiação , Soluções , Transferrina/química , Transferrina/metabolismo , Raios XRESUMO
The 18ID undulator beamline of the Biophysics Collaborative Access Team at the Advanced Photon Source, Argonne, IL, USA, is a high-performance instrument designed for, and dedicated to, the study of partially ordered and disordered biological materials using the techniques of small-angle X-ray scattering, fiber diffraction, and X-ray absorption spectroscopy. The beamline and associated instrumentation are described in detail and examples of the representative experimental results are presented.
Assuntos
Academias e Institutos , Biopolímeros/química , Cristalografia por Raios X/instrumentação , Software , Espectrometria por Raios X/instrumentação , Síncrotrons/instrumentação , Difração de Raios X/instrumentação , Biopolímeros/análise , Cristalografia por Raios X/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Illinois , Conformação Molecular , Pesquisa/instrumentação , Espectrometria por Raios X/métodos , Interface Usuário-Computador , Difração de Raios X/métodosRESUMO
BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.
Assuntos
Colágeno Tipo I/química , Tendões/química , Simulação por Computador , Cristalografia por Raios X/métodos , Modelos Moleculares , Propriedades de Superfície , SíncrotronsRESUMO
The Frank-Starling relationship of the heart has, as its molecular basis, an increase in the activation of myofibrils by calcium as the sarcomere length increases. It has been suggested that this phenomenon may be due to myofilaments moving closer together at longer lengths, thereby enhancing the probability of favorable acto-myosin interaction, resulting in increased calcium sensitivity. Accordingly, we have developed an apparatus so as to obtain accurate measurements of myocardial interfilament spacing (by synchrotron X-ray diffraction) as a function of sarcomere length (by video microscopy) over the working range of the heart, using skinned as well as intact rat trabeculas as model systems. In both these systems, lattice spacing decreased significantly as sarcomere length was increased. Furthermore, lattice spacing in the intact muscle was significantly smaller than that in the skinned muscle at all sarcomere lengths studied. These observations are consistent with the hypothesis that lattice spacing underlies length-dependent activation in the myocardium.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Contração Miocárdica/fisiologia , Miocárdio/ultraestrutura , Sarcômeros/ultraestrutura , Difração de Raios X/métodos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Feminino , Técnicas In Vitro , Masculino , Miocárdio/metabolismo , Ratos , Reprodutibilidade dos Testes , Difração de Raios X/instrumentaçãoRESUMO
X-ray methods based on synchrotron technology have the promise of providing time-resolved structural data based on the high flux and brightness of the X-ray beams. One of the most closely examined problems in this area of time-resolved structure determination has been the examination of intermediates in ligand binding to myoglobin. Recent crystallographic experiments using synchrotron radiation have identified the protein tertiary and heme structural changes that occur upon photolysis of the myoglobin--carbon monoxide complex at cryogenic temperatures [Schlichting, I., Berendzen, J., Phillips, G., & Sweet, R. (1994) Nature 371, 808--812]. However, the precision of protein crystallographic data (approximately 0.2 A) is insufficient to provide precise metrical details of the iron--ligand bond lengths. Since bond length changes on this scale can trigger reactivity changes of several orders of magnitude, such detail is critical to a full understanding of metalloprotein structure--function relationships. Extended X-ray absorption fine structure (EXAFS) spectroscopy has the potential for analyzing bond distances to a precision of 0.02 A but is hampered by its relative insensitivity to the geometry of the backscattering atoms. Thus, it is often unable to provide a unique solution to the structure without ancillary structural information. We have developed a suite of computer programs that incorporate this ancillary structural information and compute the expected experimental spectra for a wide ranging series of Cartesian coordinate sets (global mapping). The programs systematically increment the distance of the metal to various coordinating ligands (along with their associated higher shells). Then, utilizing the ab initio EXAFS code FEFF 6.01, simulated spectra are generated and compared to the actual experimental spectra, and the differences are computed. Finally, the results for hundreds of simulations can be displayed (and compared) in a single plot. The power of this approach is demonstrated in the examination of high signal to noise EXAFS data from a photolyzed solution sample of the myoglobin--carbon monoxide complex at 10 K. Evaluation of these data using our global mapping procedures placed the iron to pyrrole nitrogen average distances close to the value for deoxymyoglobin (2.05 +/- 0.01 A), while the distance from iron to the proximal histidine nitrogen is seen to be 2.20 +/- 0.04 A. It is also shown that one cannot uniquely position the CO ligand on the basis of the EXAFS data alone, as a number of reasonable minima (from the perspective of the EXAFS) are observed. This provides a reasonable explanation for the multiplicity of solutions that have been previously reported. The results presented here are seen to be in complete agreement with the crystallographic results of Schlichting et al. (1994) within the respective errors of the two techniques; however, the extended X-ray absorption fine structure data allow the iron--ligand bond lengths to be precisely defined. An examination of the available spectroscopic data, including EXAFS, shows that the crystallographic results of Schlichting et al. (1994) are highly relevant to the physiological solution state and must be taken into account in any attempt to understand the incomplete relaxation process of the heme iron for the Mb*CO photoproduct at low temperature.
Assuntos
Monóxido de Carbono/química , Músculo Esquelético/química , Mioglobina/química , Animais , Monóxido de Carbono/metabolismo , Simulação por Computador , Cristalografia por Raios X , Heme/metabolismo , Histidina/química , Histidina/metabolismo , Cavalos , Ferro/química , Ferro/metabolismo , Mioglobina/metabolismo , Fotólise , Software , Espectrofotometria , Análise Espectral Raman , TemperaturaRESUMO
The design of the time-resolved x-ray diffraction experiments reported in this and an accompanying paper was based on direct measurements of enzyme phosphorylation using [gamma-32P]ATP that were employed to determine the extent to which the lanthanides La3+ and Tb3+ activate phosphorylation of the Ca2+ATPase and their effect on the kinetics of phosphoenzyme formation and decay. We found that, under the conditions of our experiments, the two lanthanides are capable of activating phosphorylation of the ATPase, resulting in substantial levels of phosphoenzyme formation and they slow the formation and dramatically extend the lifetime of the phosphorylated enzyme conformation, as compared with calcium activation. The results from the time-resolved, nonresonance x-ray diffraction work reported in this paper are consistent with the enzyme phosphorylation experiments; they indicate that the changes in the profile structure of the SR membrane induced by terbium-activated phosphorylation of the ATPase enzyme are persistent over the much longer lifetime of the phosphorylated enzyme and are qualitatively similar to the changes induced by calcium-activated phosphorylation, but smaller in magnitude. These results made possible the time-resolved, resonance x-ray diffraction studies reported in an accompanying paper utilizing the resonance x-ray scattering from terbium, replacing calcium, to determine not only the location of high-affinity metal-binding sites in the SR membrane profile, but also the redistribution of metal density among those sites upon phosphorylation of the Ca2+ATPase protein, as facilitated by the greatly extended lifetime of the phosphoenzyme.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Retículo Sarcoplasmático/enzimologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/efeitos da radiação , Animais , Fenômenos Biofísicos , Biofísica , ATPases Transportadoras de Cálcio/efeitos da radiação , Elétrons , Técnicas In Vitro , Membranas Intracelulares/química , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Cinética , Lantânio/farmacologia , Lipídeos de Membrana/química , Modelos Químicos , Estrutura Molecular , Fosforilação , Fotólise , Coelhos , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/efeitos dos fármacos , Térbio/farmacologia , Difração de Raios XRESUMO
Time-resolved, terbium resonance x-ray diffraction experiments have provided the locations of three different high-affinity metal-binding/transport sites on the Ca2+ATPase enzyme in the profile structure of the sarcoplasmic reticulum (SR) membrane. By considering these results in conjunction with the known, moderate-resolution profile structure of the SR membrane (derived from nonresonance x-ray and neutron diffraction studies), it was determined that the three metal-binding sites are located at the "headpiece/stalk" junction in the Ca2+ATPase profile structure, in the "transbilayer" portion of the enzyme profile near the center of the membrane phospholipid bilayer, and at the intravesicular surface of the membrane profile. All three metal-binding sites so identified are simultaneously occupied in the unphosphorylated enzyme conformation. Phosphorylation of the ATPase causes a redistribution of metal density among the sites, resulting in a net movement of metal density toward the intravesicular side of the membrane, i.e., in the direction of calcium active transport. We propose that this redistribution of metal density is caused by changes in the relative binding affinities of the three sites, mediated by local structural changes at the sites resulting from the large-scale (i.e., long-range) changes in the profile structure of the Ca2+ATPase induced by phosphorylation, as reported in an accompanying paper. The implications of these results for the mechanism of calcium active transport by the SR Ca2+ATPase are discussed briefly.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Metais/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Sítios de Ligação , Fenômenos Biofísicos , Biofísica , ATPases Transportadoras de Cálcio/química , Elétrons , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Transporte de Íons , Lantânio/metabolismo , Modelos Químicos , Fosforilação , Conformação Proteica , Coelhos , Térbio/metabolismo , Difração de Raios XRESUMO
We have recently developed x-ray diffraction methods to derive the profile structure of ultrathin lipid multilayer films having one to five bilayers (e.g., Skita, V., W. Richardson, M. Filipkowski, A.F. Garito, and J.K. Blasie. 1987. J. Physique. 47:1849-1855). Furthermore, we have employed these techniques to determine the location of a monolayer of cytochrome c bound to the carboxyl group surface of various ultrathin lipid multilayer substrates via nonresonance x-ray diffraction (Pachence, J.M., and J.K. Blasie. 1987. Biophys. J. 52:735-747). Here an intense tunable source of x-rays (beam line X9-A at the National Synchrotron Light Source at the Brookhaven National Laboratory) was utilized to measure the resonance x-ray diffraction effect from the heme-Fe atoms within the cytochrome c molecular monolayer located on the carboxyl surface of a five monolayer arachidic acid film. Lamellar x-ray diffraction was recorded for energies above, below, and at the Fe K-absorption edge (E = 7,112 eV). An analysis of the resonance x-ray diffraction effect is presented, whereby the location of the heme-Fe atoms within the electron density profile of the cytochrome c/arachidic acid ultrathin multilayer film is indicated to +/- 3 A accuracy.
Assuntos
Grupo dos Citocromos c/metabolismo , Ácidos Eicosanoicos , Bicamadas Lipídicas , Silanos , Silício , Heme/análise , Ferro/análise , Conformação Molecular , Ligação Proteica , Conformação Proteica , Difração de Raios XAssuntos
Brucelose/veterinária , Infecções por Campylobacter/veterinária , Zoonoses , Animais , Brucelose/diagnóstico , Brucelose/microbiologia , Infecções por Campylobacter/diagnóstico , Infecções por Campylobacter/microbiologia , Campylobacter fetus , Doenças do Cão/diagnóstico , Doenças do Cão/microbiologia , Cães , Testes Sorológicos/veterináriaRESUMO
Photolysis and recombination of carboxymyoglobin at low temperatures have been studied by a variety of methods. This paper combines optical and structural studies of carboxymyoglobin photolysis and recombination in the temperature range 4-120 K. The absorbance changes indicate ablation of the characteristic optical transitions of carboxymyoglobin and formation of a photoproduct (Mb*CO) differing from deoxymyoglobin. When the X-ray absorption changes in the 7150-7200-eV region of the X-ray absorption spectrum are used as an indicator of structural change, the photoproduct at 4 K as measured with respect to the unphotolyzed sample is 60% of that observed for the chemically produced deoxy form. Saturation of the change is obtained with repetitive flashes totaling several thousand joules of energy from a xenon flash lamp by using a thin sample (1 mm) at 4 mM concentration as measured by both optical transmission and X-ray absorption criteria. The kinetics of the reaction show the change to occur at 10 K within the resolving time currently available (2 s) in the X-ray absorption measurements. The amplitude of the light-induced change decreases to half its maximal value at 40 K and to zero at 90 K. Steady illumination suggests at least two recombination processes. Analysis of the extended X-ray absorption fine structure (EXAFS) data on Mb*CO indicates small distance changes in the first shell of Fe-N and Fe-C that can be attributed to lengthening of the pyrrole nitrogen bonds and proximal histidine motion, together with a small displacement of the CO molecule on photolysis--a form here designated Mb*CO. This structure of the germinate state, Mb*CO, may elucidate the nature of elementary steps in chemical reactions and in tunneling processes.
Assuntos
Mioglobina/metabolismo , Microanálise por Sonda Eletrônica/instrumentação , Microanálise por Sonda Eletrônica/métodos , Congelamento , Cinética , Fotólise , Ligação ProteicaRESUMO
An on-line, real-time histogram display of heterogeneity of oxygen delivery to perfused and in situ organs is afforded by a flying-spot fluorometer that provides excitation for either oxidized flavoprotein of the mitochondrial space or reduced pyridine nucleotide of mitochondrial and cytosolic spaces. Emission from the two fluorochromes is acquired at 10(4) to 10(5) data points/s and histograms of the fluorescence intensity versus the number of occurrences of that intensity are displayed at 1--10 times per second. The histograms show alterations of the intensity and of the degree of heterogeneity of the redox states of perfused heart with model coronary occlusion, of perfused and in situ rat liver, and of rat and gerbil models of stroke. The percentage change of oxygen delivery to the intracellular space can be calculated from the areas under the histogram.