Hemoglobin Crystals Research Articles

Highly efficient recovery of noble metals using hemoglobin crystals through one-step process

Highly efficient recovery of noble metals using hemoglobin crystals through one-step process

Tissue Clearing and Its Application in the Musculoskeletal System.

The musculoskeletal system is an integral part of the human body. Currently, most skeletal muscle research is conducted through conventional histological sections due to technological limitations and the structure of skeletal muscles. For studying and observing bones and muscles, there is an urgent need for three-dimensional, objective imaging technologies. Optical tissue-clearing technologies seem to offer a novel and accessible approach to research of the musculoskeletal system. Using this approach, the components which cause refraction or prevent light from penetrating into the tissue are physically and chemically eliminated; then the liquid in the tissue is replaced with high-refractive-index chemicals. This innovative method, which allows three-dimensional reconstruction at the cellular and subcellular scale, significantly improves imaging depth and resolution. Nonetheless, this technology was not originally developed to image bones or muscles. When compared with brain and nerve organs which have attracted considerable attention in this field, the musculoskeletal system contains fewer lipids and has high levels of hemoglobin, collagen fibers, and inorganic hydroxyapatite crystals. Currently, three-dimensional imaging methods are widely used in the diagnosis and treatment of skeletal and muscular illnesses. In this regard, it is vitally important to review and evaluate the optical tissue-clearing technologies currently employed in the musculoskeletal system, so that researchers may make an informed decision. In the meantime, this study offers guidelines and recommendations for expanding the use of this technology in the musculoskeletal system.

HEMOGLOBIN C TRAIT PRESENTING WITH BILATERAL CENTRAL RETINAL VEIN OCCLUSION.

To report a case of bilateral sequential, central retinal vein occlusion (CRVO) in a man with newly diagnosed hemoglobin C trait. A 67-year-old man presented with a one-month history of declining visual acuity. He was diagnosed with left CRVO. Bilateral temporal retinal ischemia and arteriovenous anastomoses on fluorescein angiography suggestive of sickle cell retinopathy prompted a systemic work-up. Hemoglobin electrophoresis revealed an underlying hemoglobin C trait. Six-months after his initial presentation, the patient developed symptomatic right CRVO. The initial left CRVO was complicated by optic disc swelling and macular edema. Intravitreal antiangiogenic therapy was initiated and the macular edema resolved. The left eye subsequently developed an epiretinal membrane which was surgically removed. Macular edema in the right eye also resolved after intravitreal antiangiogenic therapy. This is the first reported case of bilateral CRVO in a case of hemoglobin C trait. It is possible that erythrocyte inflexibility, caused by hemoglobin C-induced dehydration and crystallization, acted concomitantly with hypertension to produce occlusive microangiopathy. This case highlights the need for further investigation in patients presenting with central retinal vein occlusion, especially when bilateral, or when retinal angiography reveals bilateral pathology.

Preparation of hemoglobin crystals of desired size

Preparation of hemoglobin crystals of desired size

Macromolecular crystallization: basics and advanced methodologies

For the first time, about 155 years ago and as a laboratory curiosity, German biologists observed the crystals of hemoglobin from worms and fishes. Since then, the crystallization of proteins, nucleic acids and big biological structures, like viruses, has been developed into a broad research field including several applications, for example in the drug discovery. This review is divided into four major sections. The first section addresses the specific physicochemical properties of biomolecular crystals accompanied by kinetics of supersaturation, nucleation and growth which are the three main steps required to achieve macromolecular crystals. Besides, various physical, chemical and biochemical parameters influencing the process of macromolecular crystallization are reviewed. The second part deals with classical approaches, such as vapor and batch diffusion methods, available to create macromolecular crystals. The third part overviews novel approaches including microgravity, cocrystallization, membrane-assisted crystallization and microfluidic array chips involved in more complicated techniques for growing macromolecular crystals and controlling their size and orientation. In the end, considering the very significant role of automation in providing biomolecules’ crystals in recent years, we provided a brief explanation about robotics and their importance in developing high-throughput crystallization. RDC-NMR and SAXS/WAXS hybrid methods with the aim of obtaining structural information of complex macromolecular assemblies are also discussed.

Ferromagnetism in 2D organic iron hemoglobin crystals based on nitrogenated conjugated micropore materials.

In this work we study a low-cost two-dimensional ferromagnetic semiconductor with possible applications in biomedicine, solar cells, spintronics, and energy and hydrogen storage. From first principle calculations we describe the unique electronic, transport, optical, and magnetic properties of a π-conjugated micropore polymer (CMP) with three iron atoms placed in the middle of an isolated pore locally resembling heme complexes. This material exhibits strong Fe-localized dz2 bands. The bandgap is direct and equal to 0.28 eV. The valence band is doubly degenerate at the Γ-point and for larger k-wavevectors the HOMO band becomes flat with low contribution to charge mobility. The absorption coefficient is roughly isotropic. The conductivity is also isotropic with the nonzero contribution in the energy range 0.3-8 eV. The xy-component of the imaginary part of the dielectric tensor determines the magneto-optical Faraday and Kerr rotation. Nonvanishing rotation is observed in the interval of 0.5-5.0 eV. This material is found to be a ferromagnet of an Ising type with long-range exchange interactions with a very high magnetic moment per unit cell, m = 6 μB. The exchange integral is calculated by two independent methods: (a) from the energy difference between ferromagnetic and antiferromagnetic states and (b) from a magnon dispersion curve. In the former case Jnn = 27 μeV. In the latter case the magnon dispersion is fitted by the Ising model with the nearest and next-nearest neighbor spin interactions. From these estimations we find that Jnn = 19.5 μeV and Jnnn = -3 μeV. Despite the different nature of the calculations, the exchange integrals are only within 28% difference.

Hematological and Renal Function Evaluation in Dogs with IMHA

Background: The IMHA is a common cause of anemia in dogs and characterized by direct destruction or phagocytosis of erythrocytes opsonized by IgG, IgM and/or complement. The diagnosis is based on the identification of erythrocytes destruction in the presence of anti-erythrocyte antibodies, producing spherocytes, auto-agglutination, Coomb’s test or flow cytometry test positive, in addition to anemia and clinical signs of hemolysis. The renal biochemical profile and urinalysis may reveal important changes due to the severity of the kidney demage. The aim of this study were to evaluate the incidence of hematological and renal abnormalities, and the prevalence of immunoglobulin’s classes involved in IMHA.Materials, Methods & Results: In a total of 87 anemic dogs were selected and tested by Coomb’s test, flow cytometry (FC), and auto-agglutination, along with CBC, reticulocyte count, renal profile (ureia and creatinine), hemoparasite search in peripheral blood smears, and Ehrlichia sp. and leptospirosis tests. The results were analyzed by t test or Mann-Whitney with 5% of significance. Therefore, 61 dogs (70.11%) were positive for IMHA by FC, 31 (35.63%) by Coomb’s test, and 24 (27.58%) by auto-agglutination. There was not a predominance of IgG or IgM involvement. The hematological and clinical changes in dogs with IMHA included macrocytic, hypochromic regenerative anemia, and reticulocytosis, as well as icterus, fever, auto-agglutination, hyperglobulinemia and bilirrubinuria. Spherocytosis was found in 9.8% of dogs with IMHA, and 29.5% of dogs had leukocytosis, 39.6% neutrophilia, and 72.1% thrombocytopenia. Mostly of cases of IHMA (74.6%) were attributed to infectious diseases and associated with Ehrlichia sp. (secondary IMHA), 21.4% of dogs with IMHA had azotemia, and 51.8% had increased urine protein creatinine ratio.Discussion: The FC was confirmed as a more sensitive technique for the diagnosis of IMHA compared to auto-agglutination and Coomb’s tests. The auto-agglutination test was more specific than the Coomb’s test, however the last one was more sensitive. The similar prevalence of IgG and IgM in IMHA did not indicate which class of immunoglobulin would be a better choice for diagnosis by the FC technique. Antibodies are produced against normal red cells (primary or idiopathic IMHA) or to red cells that are antigenical changed by the action of drugs, neoplasia or infectious diseases (leptospirosis, babesiosis, canine ehrlichiosis), known as secondary IMHA. This study alerts for the high prevalence of IMHA in dogs, in most cases characterized by a regenerative anemia associated with intense thrombocytopenia and secondary to Ehrlichia sp. in areas endemic to this infectious disease. In Brazil, the presence of endemic areas for various infectious diseases may contribute to the high prevalence of secondary IMHA. Kidney damage may occurs because tissue hypoxia increases the risk of progressive injury, due to acute hematocrit decrease (below 22%), leading to renal tubular necrosis, whereas the deposition of immune complexes, mainly in the renal parenchyma, may aggravate the renal injury, further complicating the clinical state of the animal. Direct renal injury caused by crystallization of free hemoglobin in the renal tubules may result in renal azotemia. Hypoxia and nephrotoxicity caused by hemoglobinemia reflects increased liver enzymes and azotemia, respectively. Significant proteinuria and the increase in urine protein creatinine ratio revealed evidence of renal injury in dogs with IMHA.

Size and Shape Controlled Crystallization of Hemoglobin for Advanced Crystallography

While high-throughput screening for protein crystallization conditions have rapidly evolved in the last few decades, it is also becoming increasingly necessary for the control of crystal size and shape as increasing diversity of protein crystallographic experiments. For example, X-ray crystallography (XRC) combined with photoexcitation and/or spectrophotometry requires optically thin but well diffracting crystals. By contrast, large-volume crystals are needed for weak signal experiments, such as neutron crystallography (NC) or recently developed X-ray fluorescent holography (XFH). In this article, we present, using hemoglobin as an example protein, some techniques for obtaining the crystals of controlled size, shape, and adequate quality. Furthermore, we describe a few case studies of applications of the optimized hemoglobin crystals for implementing the above mentioned crystallographic experiments, providing some hints and tips for the further progress of advanced protein crystallography.

Development of an X-ray fluorescence holographic measurement system for protein crystals.

Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α2β2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm(3)) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

New Structural Analysis Methods for Hemoglobin Crystals. Time-Resolved Cryogenic X-Ray Crystallography with Extended Pulsed-Laser Pumping and 3D Imaging by X-Ray Fluorescence Holography

Hemoglobin (Hb) has long served as a model for studying protein allostery in general. Static crystal structures of deoxy tense (T) state and oxy relaxed (R) state of Hb reveal multiple differences at both tertiary and quaternary levels. However, we still lack an adequate understanding of how these structural differences control ligand binding. Here we present two methods for Hb crystals to investigate the ligation-linked protein motion and the atomic details of the heme active site structure.The first method is time-resolved X-ray crystallography combined with high-repetition pulsed-laser pumping and cryogenic trapping. At cryogenic temperatures under 50 K, a photo-dissociated ligand molecule can move to only the limited range near the heme, and the protein moiety can undergo only small structural changes. By contrast, at 100-140 K under continuous pulsed-laser irradiation, we found that ligand migration and protein relaxation can be observed. We have succeeded in tracking the photo-dissociated ligand molecule that migrates along the pathway connecting inner cavities of Hb in a different direction in each subunit.The second method is atomic-resolution X-ray fluorescence holography (XFH), which utilizes fluorescing atoms as a wave source within a crystal sample. So far, XFH has generally been applied to inorganic materials to investigate the microscopic environments of trace metals. This is the first application of XFH for protein crystals. For Hb crystals, XFH can provide the three dimensional atomic images around the iron central atoms of the individual hemes. We have carried out XFH measurements at 100 K of CO-bound Hb crystals at BL6C, KEK, Japan. We will present our latest data and share with you the results and discussion of this work.

Thermal-induced force release in oxyhemoglobin.

Oxygen is released to living tissues via conformational changes of hemoglobin from R-state (oxyhemoglobin) to T-state (desoxyhemoglobin). The detailed mechanism of this process is not yet fully understood. We have carried out micromechanical experiments on oxyhemoglobin crystals to determine the behavior of the Young’s modulus and the internal friction for temperatures between 20 °C and 70 °C. We have found that around 49 °C oxyhemoglobin crystal samples undergo a sudden and strong increase of their Young’s modulus, accompanied by a sudden decrease of the internal friction. This sudden mechanical change (and the ensuing force release) takes place in a partially unfolded state and precedes the full denaturation transition at higher temperatures. After this transformation, the hemoglobin crystals have the same mechanical properties as their initial state at room temperatures. We conjecture that it can be relevant for explaining the oxygen-releasing function of native oxyhemoglobin when the temperature is increased, e.g. due to active sport. The effect is specific for the quaternary structure of hemoglobin, and is absent for myoglobin with only one peptide sequence.

The first protein Structure Determinations

Max Perutz had been studying the structure of haemoglobin for 21 years using highly original and creative techniques, when I arrived in Cambridge in the summer of 1958. Most recently he had explored the use of isomorphous replacement by substituting specific amino acids with heavy metal compounds. Together with Ann Cullis, he had collected three dimensional diffraction data on native haemoglobin crystals and on three heavy atom derivatives. But now Max needed help in analyzing this data. I had had experience in determining three small aromatic hydrocarbon structures as a graduate student with J.M. Robertson at the University of Glasgow using only mechanical hand calculators, a slide rule and Beevers-Lipson strips. At the University of Minnesota, as a post doc with Bill Lipscomb, I had learned how to program one of the earliest commercial computers and solved a plant terpenoid structure in three dimensions. Thus, when I joined Max's lab, I was soon busy programming the bran new "home build" EDSAC2 computer to determine and refine the heavy atom parameters for phase determination by isomorphous replacement. When we calculated the 5.5Å resolution horse oxy haemoglobin electron density map in the summer of 1959 we were amazed to see that the two polypeptide chains of haemoglobin each had the same fold as that of myoglobin, another oxygen carrying protein determined by John Kendrew at 6Å resolution two years earlier at the Cavendish. John and his group were extending the structure of myoglobin to 2Å resolution. The high resolution map of myglobin was calculated using my Fourier program the week after the low resolution haemoglobin map. We then saw the atomic structure of α-helices as had been predicted by Linus Pauling. Seeing evolution for the first time at a molecular level has inspired my thinking and choice of research topics ever since. In the following few years, stimulated by these discoveries , David Blow (who had returned from the US later that summer after two years of post-doc studies) and I developed the single isomorphous replacement method, the use of anomalous dispersion for phase determination, phase combination procedures and, most importantly, molecular replacement.

Purification, Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P(50)) and 1.96 (n value), and a small Bohr effect (δH(+) = -0.29) at a pH of 6.9-8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O(2) affinity at P(50) of 2.5 mmHg which may assure efficient utilization of the lung O(2) reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10-13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl(2)·6H(2)O] solution at a pH of 7.0-8.5.

Capturing the Hemoglobin Allosteric Transition in a Single Crystal Form

Allostery in many oligomeric proteins has been postulated to occur via a ligand-binding-driven conformational transition from the tense (T) to relaxed (R) state, largely on the basis of the knowledge of the structure and function of hemoglobin, the most thoroughly studied of all allosteric proteins. However, a growing body of evidence suggests that hemoglobin possesses a variety of intermediates between the two end states. As such intermediate forms coexist with the end states in dynamic equilibrium and cannot be individually characterized by conventional techniques, very little is known about their properties and functions. Here we present complete structural and functional snapshots of nine equilibrium conformers of human hemoglobin in the half-liganded and fully liganded states by using a novel combination of X-ray diffraction analysis and microspectrophotometric O2 equilibrium measurements on three isomorphous crystals, each capturing three distinct equilibrium conformers. Notably, the conformational set of this crystal form varies according to shifts in the allosteric equilibrium, reflecting the differences in hemoglobin ligation state and crystallization solution conditions. We find that nine snapshot structures cover the complete conformational space of hemoglobin, ranging from T to R2 (the second relaxed quaternary structure) through R, with various relaxed intermediate forms between R and R2. Moreover, we find a previously unidentified intermediate conformer, between T and R, with an intermediate O2 affinity, sought by many research groups over a period of decades. These findings reveal a comprehensive picture of the equilibrium conformers and transition pathway for human hemoglobin.

Purification, crystallization and preliminary crystallographic studies of haemoglobin from mongoose (Helogale parvula) in two different crystal forms induced by pH variation.

Haemoglobin (Hb) is a respiratory pigment; it is a tetrameric protein that ferries oxygen from the lungs to tissues and transports carbon dioxide on the return journey. The oxygen affinity of haemoglobin is regulated by the concentration of oxygen surrounding it and several efforts have revealed the shapes of Hb in different states and with different functions. However, study of the molecular basis of Hbs from low-oxygen-affinity species is critically needed in order to increase the understanding of the mechanism behind oxygen adaptation. The present study reports the preliminary crystallographic study of low-oxygen-affinity haemoglobin from mongoose, a burrowing mammal. Haemoglobin from mongoose was purified by anion-exchange chromatography, crystallized using the hanging-drop vapour-diffusion method and diffraction data sets were collected from monoclinic (2.3 Å resolution) and orthorhombic (2.9 Å resolution) crystal forms obtained by pH variation. The monoclinic and orthorhombic asymmetric units contained half and a whole biological molecule, respectively.

Haemolytic anaemia and acute pancreatitis associated with zinc toxicosis in a dog

We describe a case of zinc toxicity in a 14-month-old, female, neutered, Cavalier King Charles spaniel with a 48-hour history of haematochezia, icterus and collapse. Regenerative anaemia with a packed-cell...

Selective X-ray-induced NO photodissociation in haemoglobin crystals: evidence from a Raman-assisted crystallographic study

Despite their high physiological relevance, haemoglobin crystal structures with NO bound to haem constitute less than 1% of the total ligated haemoglobins (Hbs) deposited in the Protein Data Bank. The major difficulty in obtaining NO-ligated Hbs is most likely to be related to the oxidative denitrosylation caused by the high reactivity of the nitrosylated species with O(2). Here, using Raman-assisted X-ray crystallography, it is shown that under X-ray exposure (at four different radiation doses) crystals of nitrosylated haemoglobin from Trematomus bernacchii undergo a transition, mainly in the β chains, that generates a pentacoordinate species owing to photodissociation of the Fe-NO bond. These data provide a physical explanation for the low number of nitrosylated Hb structures available in the literature.

Haemolytic anaemia and acute pancreatitis associated with zinc toxicosis in a dog

We describe a case of zinc toxicity in a 14-month-old, female, neutered, Cavalier King Charles spaniel with a 48-hour history of haematochezia, icterus and collapse. Regenerative anaemia with a packed-cell...

Structures and Oxygen Affinities of Crystalline Human Hemoglobin C (β6 Glu→Lys) in the R and R2 Quaternary Structures

Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (β6 Glu→Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 Å resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K(4)) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2-3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.

1I1324 P32 2種類のリラックス状態ヘモグロビン結晶の機能比較(ヘム蛋白質 1,第49回日本生物物理学会年会)

1I1324 P32 2種類のリラックス状態ヘモグロビン結晶の機能比較(ヘム蛋白質 1,第49回日本生物物理学会年会)