On the molecular mass of the extracellular hemoglobin of Glossoscolex paulistus: Analytical ultracentrifugation reexamination

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by Subunits containing heme groups with molecular masses (M) in the range of 15 to 19 kDa, monomers of 16 kDa (d), and trimers of 51 to 52 kDa (abc) linked by nonheme structures named linkers of 24 to 32 kDa (L). HbGp is homologous to Lumbricus terrestris hemoglobin (HbLt). Several reports propose M of HbLt in the range of 3.6 to 4.4 MDa. Based on subunits M determined by mass spectrometry and assuming HbGp stoichiometry of 12(abcd)(3)L(3) (Vinogradov model) plus 144 heme groups, a Value of M for HbGp oligomer of 3560 kDa can be predicted. This Value is nearly 500 kDa higher than the unique HbGp M Value reported in the literature. In the current work, sedimentation velocity analytical ultracentrifugation (AUC) experiments were performed to obtain M for HbGp in oxy and cyano-met forms. s(20,w)(0), values of 58.1 +/- 0.2 S and 59.6 +/- 0.2 S, respectively, for the two oxidation forms were obtained. The ratio between sedimentation and diffusion coefficients supplied values for M of approximately 3600 100 and 3700 100 kDa for oxy and cyano-met HbGp forms, respectively. An independent determination of the partial specific volume, V(bar), for HbGp was performed based on density measurements, providing a value of 0.764 +/- 0.008, in excellent agreement with the estimates from SEDFIT software. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Therefore, HbGp possesses M very close to that of HbLt, suggesting an oligomeric assembly in agreement with the Vinogradov model. (c) 2008 Elsevier Inc. All rights reserved.

Structural studies of prephenate dehydratase from Mycobacterium tuberculosis H37Rv by SAXS, ultracentrifugation, and computational analysis

Tuberculosis (TB) is one of the most common infectious diseases known to man and responsible for millions of human deaths in the world. The increasing incidence of TB in developing countries, the proliferation of multidrug resistant strains, and the absence of resources for treatment have highlighted the need of developing new drugs against TB. The shikimate pathway leads to the biosynthesis of chorismate, a precursor of aromatic amino acids. This pathway is absent from mammals and shown to be essential for the survival of Mycobacterium tuberculosis, the causative agent of TB. Accordingly, enzymes of aromatic amino acid biosynthesis pathway represent promising targets for structure-based drug design. The first reaction in phenylalanine biosynthesis involves the conversion of chorismate to prephenate, catalyzed by chorismate mutase. The second reaction is catalyzed by prephenate dehydratase (PDT) and involves decarboxylation and dehydratation of prephenate to form phenylpyruvate, the precursor of phenylalanine. Here, we describe utilization of different techniques to infer the structure of M. tuberculosis PDT (MtbPDT) in solution. Small angle X-ray scattering and ultracentrifugation analysis showed that the protein oligomeric state is a tetramer and MtbPDT is a flat disk protein. Bioinformatics tools were used to infer the structure of MtbPDT A molecular model for MtbPDT is presented and molecular dynamics simulations indicate that MtbPDT i.s stable. Experimental and molecular modeling results were in agreement and provide evidence for a tetrameric state of MtbPDT in solution.

Thermodynamic characterization of the palm tree Roystonea regia peroxidase stability

The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N ->(k) D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization. (c) 2008 Elsevier Masson SAS. All rights reserved.

Human regulatory protein Ki-1/57 has characteristics of an intrinsically unstructured protein

The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 monoclonal antibody Ki-1; in Hodgkin lymphoma cells. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in peripheral blood leukocytes after mitogenic activation suggested its possible role in cell signaling. Ki-1/57 interacts with several other regulatory proteins involved in cellular signaling, transcriptional regulation and RNA metabolism, suggesting it may have pleiotropic functions. In a previous spectroscopic analysis, we observed a low content of secondary structure for Ki-1/57 constructs. Here, Circular dichroism experiments, in vitro RNA binding analysis, and limited proteolysis assays of recombinant Ki-1/57(122-413) and proteolysis assays of endogenous full length protein from human HEK293 cells suggested that Ki-1/57 has characteristics of an intrinsically unstructured protein. Small-angle X-ray scattering (SAXS) experiments were performed with the C-terminal fragment Ki-1/57(122-413). These results indicated an elongated shape and a partially unstructured conformation of the molecule in solution, confirming the characteristics of an intrinsically unstructured protein. Experimental curves together with ab initio modeling approaches revealed an extended and flexible molecule in solution. An elongated shape was also observed by analytical gel filtration. Furthermore, sedimentation velocity analysis suggested that Ki-1/57 is a highly asymmetric protein. These findings may explain the functional plasticity of Ki-1/57, as suggested by the wide array of proteins with which it is capable of interacting in yeast two-hybrid interaction assays.

Molecular masses and sedimentation coefficients of extracellular hemoglobin of Glossoscolex paulistus: Alkaline oligomeric dissociation

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) has a molecular mass (M) of 3600 +/- 100 kDa and a standard sedimentation coefficient (s(20.w)(0)) of 58 S. estimated by analytical ultracentrifugation (AUC). In the present work, further AUC studies were developed for HbGp, at pH 10.0, which favors oligomeric dissociation into lower M species. The HbGp oligomer is formed by globin chains a, b, c and d plus the linker chains. The pure monomeric fraction, subunit d, and HbGp at pH 10.0, in the presence of beta-mercaptoethanol, were also studied. Our results indicate that for samples of pure subunit d, besides the monomeric species with s(20.w)(0) of 2.0 S, formation of dimer of subunit d is observed with s(20.w)(0) of around 2.9 S. For the whole HbGp at pH 10.0 contributions from monomers, trimers and linkers are observed. No contribution from 58 S species was observed for the sample of oxy-HbGp at pH 10.0, showing its complete dissociation. For cyanomet-HbGp form a contribution of 17% is observed for the un-dissociated oligomer, consistent with data from other techniques that show the cyanomet-form is more stable as compared to oxy-HbGp. Masses of HbGp subunits, especially trimer abc and monomeric chains a, b, c and d, were also estimated from sedimentation equilibrium data, and are in agreement with the results from MALDI-TOF-MS. (C) 2010 Elsevier B.V. All rights reserved.

Stoichiometry and thermodynamics of the interaction between the C-terminus of human 90 kDa heat shock protein Hsp90 and the mitochondrial translocase of outer membrane Tom70

A large majority of the 1000-1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K(D) of 360 30 nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated. (C) 2011 Elsevier Inc. All rights reserved.

Analytical solutions of accreting black holes immersed in a Lambda CDM model

The evolution of the mass of a black hole embedded in a universe filled with dark energy and cold dark matter is calculated in a closed form within a test fluid model in a Schwarzschild metric, taking into account the cosmological evolution of both fluids. The result describes exactly how accretion asymptotically switches from the matter-dominated to the Lambda-dominated regime. For early epochs, the black hole mass increases due to dark matter accretion, and on later epochs the increase in mass stops as dark energy accretion takes over. Thus, the unphysical behaviour of previous analyses is improved in this simple exact model. (C) 2010 Elsevier B.V. All rights reserved.

Analytical quantification of low-density lipoprotein using europium tetracycline indicator

Low-density lipoprotein (LDL) is known as `bad` cholesterol. If too much LDL circulates in the blood it can be retained in the walls of the arteries, causing atherosclerosis. In this paper we showed an alternative method to quantify LDL using the europium tetracycline (EuTc) indicator. The optical properties of the EuTc complex were investigated in aqueous solutions containing LDL. An enhancement was observed of the europium luminescence in the solutions with LDL compared those without the lipoprotein. A method to quantify the amount of LDL in a sample, based on EuTc enhanced luminescence, is proposed. The enhancement mechanism is also discussed. Copyright (C) 2009 John Wiley & Sons, Ltd.

Investigation of the Europium Emission Spectra of the Europium-Oxytetracycline Complex in the Presence of Human Low-Density Lipoproteins

Low-Density Lipoprotein (LDL), often known as ""bad cholesterol"" is one of the responsible to increase the risk of coronary arterial diseases. For this reason, the cholesterol present in the LDL particle has become one of the main parameters to be quantified in routine clinical diagnosis. A number of tools are available to assess LDL particles and estimate the cholesterol concentration in the blood. The most common methods to quantify the LDL in the plasma are the density gradient ultracentrifugation and nuclear magnetic resonance (NMR). However, these techniques require special equipments and can take a long time to provide the results. In this paper, we report on the increase of the Europium emission in Europium-oxytetracycline complex aqueous solutions in the presence of LDL. This increase is proportional to the LDL concentration in the solution. This phenomenum can be used to develop a method to quantify the number of LDL particles in a sample. A comparison between the performances of the oxytetracycline and the tetracycline in the complexes is also made.

An analytical relation between entropy production and quantum Lyapunov exponents for Gaussian bipartite systems

We study and compare the information loss of a large class of Gaussian bipartite systems. It includes the usual Caldeira-Leggett-type model as well as Anosov models ( parametric oscillators, the inverted oscillator environment, etc), which exhibit instability, one of the most important characteristics of chaotic systems. We establish a rigorous connection between the quantum Lyapunov exponents and coherence loss, and show that in the case of unstable environments coherence loss is completely determined by the upper quantum Lyapunov exponent, a behavior which is more universal than that of the Caldeira-Leggett-type model.

Heteroscedastic controlled calibration model applied to analytical chemistry

In chemical analyses performed by laboratories, one faces the problem of determining the concentration of a chemical element in a sample. In practice, one deals with the problem using the so-called linear calibration model, which considers that the errors associated with the independent variables are negligible compared with the former variable. In this work, a new linear calibration model is proposed assuming that the independent variables are subject to heteroscedastic measurement errors. A simulation study is carried out in order to verify some properties of the estimators derived for the new model and it is also considered the usual calibration model to compare it with the new approach. Three applications are considered to verify the performance of the new approach. Copyright (C) 2010 John Wiley & Sons, Ltd.

Approaches for multicopper oxidases in the design of electrochemical sensors for analytical applications

We report an effective approach for the construction of a biomimetic sensor of multicopper oxidases by immobilizing a cyclic-tetrameric copper(II) species, containing the ligand (4-imidazolyl)ethylene-2-amino-1-ethylpyridine (apyhist), in the Nafion (R) membrane on a vitreous carbon electrode surface. This complex provides a tetranuclear arrangement of copper ions that allows an effective reduction of oxygen to water, in a catalytic cycle involving four electrons. The electrochemical reduction of oxygen was studied at pH 9.0 buffer solution by using cyclic voltammetry, chronoamperometry, rotating disk electrode voltammetry and scanning electrochemical microscopy techniques. The mediator shows good electrocatalytic ability for the reduction of O(2) at pH 9.0, with reduction of overpotential (350 mV) and increased current response in comparison with results obtained with a bare glassy carbon electrode. The heterogeneous rate constant (k(ME)`) for the reduction of O(2) at the modified electrode was determined by using a Koutecky-Levich plot. In addition, the charge transport rate through the coating and the apparent diffusion coefficient of O(2) into the modifier film were also evaluated. The overall process was found to be governed by the charge transport through the coating, occurring at the interface or at a finite layer at the electrode/coating interface. The proposed study opens up the way for the development of bioelectronic devices based on molecular recognition and self-organization. (C) 2010 Elsevier Ltd. All rights reserved.

Exploring Liquid Sequential Injection Chromatography To Teach Fundamentals of Separation Methods: A Very Fast Analytical Chemistry Experiment

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)

Development of an Analytical Methodology for Quantification of Strong Acid in Diesel Oil

Many factors can affect the quality of diesel oil, in particular the degradation processes that are directly related to some organosulfur compounds. During the degradation process, these compounds are oxidized into their corresponding sulfonic acids, generating a strong acid content during the process. p-Toluene sulfonic acid analysis was performed using the linear sweep voltammetry technique with a platinum ultramicroelectrode in aqueous solution containing 3 mol L(-1) potassium chloride. An extraction step was introduced prior to the voltammetric detection in order to avoid the adsorption of organic molecules, which inhibit the electrochemical response. The extraction step promoted the transference of sulfonic acid from the diesel oil to an aqueous phase. The method was accurate and reproducible, with detection and quantification limits of 5 ppm and 15 ppm, respectively. Recovery of sulfonic acid was around 90%.

Graphite-silicone rubber composite electrode: Preparation and possibilities of analytical application

Composite electrodes were prepared using graphite powder and silicone rubber in different compositions. The use of such hydrophopic materials interned to diminish the swallowing observed in other cases when the electrodes are used in aqueous solutions for a long time. The composite was characterized for the response reproducibility, ohmic resistance, thermal behavior and active area. The voltammetric response in relation to analytes with known voltammetric behavior was also evaluated, always in comparison with the glassy carbon. The 70% (graphite, w/w) composite electrode was used in the quantitative determination of hydroquinone (HQ) in a DPV procedure in which a detection limit of 5.1 x 10(-8) mol L-1 was observed. HQ was determined in a photographic developer sample with errors lower then 1% in relation to the label value. (c) 2007 Elsevier B.V. All rights reserved.