Dynamic light scattering and optical absorption spectroscopy study of pH and temperature stabilities of the extracellular hemoglobin of Glossoscolex paulistus

The extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted of subunits containing heme groups, monomers and trimers, and nonheme structures, called linkers, and the whole protein has a minimum molecular mass near 3.1 x 10(6) Da. This and other proteins of the same family are useful model systems for developing blood substitutes due to their extracellular nature, large size, and resistance to oxidation. HbGp samples were studied by dynamic light scattering (DLS). In the pH range 6.0-8.0, HbGp is stable and has a monodisperse size distribution with a z-average hydrodynamic diameter (D-h) of 27 +/- 1 nm. A more alkaline pH induced an irreversible dissociation process, resulting in a smaller D-h of 10 +/- 1 nm. The decrease in D-h suggests a complete hemoglobin dissociation. Gel filtration chromatography was used to show unequivocally the oligomeric dissociation observed at alkaline pH. At pH 9.0, the dissociation kinetics is slow, taking a minimum of 24 h to be completed. Dissociation rate constants progressively increase at higher pH, becoming, at pH 10.5, not detectable by DILS. Protein temperature stability was also pH-dependent. Melting curves for HbGp showed oligomeric dissociation and protein denaturation as a function of pH. Dissociation temperatures were lower at higher pH. Kinetic studies were also performed using ultraviolet-visible absorption at the Soret band. Optical absorption monitors the hemoglobin autoxidation while DLS gives information regarding particle size changes in the process of protein dissociation. Absorption was analyzed at different pH values in the range 9.0-9.8 and at two temperatures, 25 degrees C and 38 degrees C. At 25 degrees C, for pH 9.0 and 9.3, the kinetics monitored by ultraviolet-visible absorption presents a monoexponential behavior, whereas for pH 9.6 and 9.8, a biexponential behavior was observed, consistent with heme heterogeneity at more alkaline pH. The kinetics at 38 degrees C is faster than that at 25 degrees C and is biexponential in the whole pH range. DLS dissociation rates are faster than the autoxidation dissociation rates at 25 degrees C. Autoxiclation and dissociation processes are intimately related, so that oligomeric protein dissociation promotes the increase of autoxidation rate and vice versa. The effect of dissociation is to change the kinetic character of the autoxidation of hemes from monoexponential to biexponential, whereas the reverse change is not as effective. This work shows that DLS can be used to follow, quantitatively and in real time, the kinetics of changes in the oligomerization of biologic complex supramolecular systems. Such information is relevant for the development of mimetic systems to be used as blood substitutes.

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.

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.

Further characterization of the subunits of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) by SDS-PAGE electrophoresis and MALDI-TOF-MS

Further characterization of hemoglobin of Glossoscolex paulistus (HbGp) subunits was performed based on SDS-PAGE, size exclusion chromatography (SEC) and MALDI-TOF-MS analysis. SDS-PAGE has shown a total of four linker chains, two quite intense and two of lower intensity. HbGp fractions (I-VI), obtained by size exclusion chromatography (SEC), from oligomeric dissociation at alkaline pH 9.6, were monitored. Fraction I is identical to the whole protein. The monomeric chains c, obtained from the trimer abc reduction, present four isoforms with MM 17,336 Da, 17,414 Da, 17,546 Da and 17,620 Da. Furthermore, the trimer subunit presents two isoforms, T 1 and T 2, with MM 51,200 ± 60 and 51,985 ± 50 Da, respectively. Based on SDS-PAGE, the linker chains seem to be distributed along the different fractions of the SEC chromatogram, appearing along the peaks corresponding to fractions I-V. The fraction IV contains, predominantly, trimers with some linkers contamination. The strong interaction of linker chains L with the trimers abc, makes it difficult to obtain these subunits in pure form. The monomer d in fraction VI appears to be quite pure, in agreement with previous studies. © 2011 Elsevier Ltd. All rights reserved.

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with ionic surfactants: A MALDI-TOF-MS study

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by approximately 144 subunits containing heme groups with molecular masses in the range of 16-19 kDa forming a monomer (d) and a trimer (abc), and around 36 non-heme structures, named linkers (L). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) analysis was performed recently, to obtain directly information on the molecular masses of the different subunits from HbGp in the oxy-form. This technique demonstrated structural similarity between HbGp and the widely studied hemoglobin of Lumbricus terrestris (HbLt). Indeed, two major isoforms (d(1) and d(2)) of identical proportions with masses of 16,355+/-25 and 16,428+/-24 Da, respectively, and two minor isoforms (d(3) and d(4)) with masses around 16.6 kDa were detected for monomer d of HbGp. In the present work, the effects of anionic sodium dodecyl sulfate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) on the oligomeric structure of HbGp have been studied by MALDI-TOF-MS in order to evaluate the interaction between ionic surfactants and HbGp. The data obtained with this technique show an effective interaction of cationic surfactant CTAC with the two isoforms of monomer d, d(1) and d(2), both in the whole protein as well as in the pure isolated monomer. The results show that up to 10 molecules of CTAC are bound to each isoform of the monomer. Differently, the mass spectra obtained for SDS-HbGp system showed that the addition of the anionic surfactant SDS does not originate any mass increment of the monomeric subunits, indicating that SDS-HbGp interaction is, probably, significantly less effective as compared to CTAC-HbGp one. The acid pI of the protein around 5.5 is, probably, responsible for this behavior. The results of this work suggest also some interaction of both surfactants with linker chains as well as with trimers, as judged from observed mass increments. Our data are consistent with a recent spectroscopic study showing a strong interaction between CTAC and HbGp at physiological pH [P.S.Santiago, et al, Biochim. Biophys. Acta. 1770 (2007) 506-517.]. (C) 2007 Elsevier B.V. All rights reserved.

Carbohydrate gluing, an architectural mechanism in the supramolecular structure of an annelid giant hemoglobin.

We report a carbohydrate-dependent supramolecular architecture in the extracellular giant hemoglobin (Hb) from the marine worm Perinereis aibuhitensis; we call this architectural mechanism carbohydrate gluing. This study is an extension of our accidental discovery of deterioration in the form of the Hb caused by a high concentration of glucose. The giant Hbs of annelids are natural supramolecules consisting of about 200 polypeptide chains that associate to form a double-layered hexagonal structure. This Hb has 0.5% (wt) carbohydrates, including mannose, xylose, fucose, galactose, glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). Using carbohydrate-staining assays, in conjunction with two-dimensional polyacrylamide gel electrophoresis, we found that two types of linker chains (L1 and L2; the nomenclature of the Hb subunits followed that for another marine worm, Tylorrhynchus heterochaetus) contained carbohydrates with both GlcNAc and GalNAc. Furthermore, two types of globins (a and A) have only GlcNAc-containing carbohydrates, whereas the other types of globins (b and B) had no carbohydrates. Monosaccharides including mannose, fucose, glucose, galactose, GlcNAc, and GalNAc reversibly dissociated the intact form of the Hb, but the removal of carbohydrate with N-glycanase resulted in irreversible dissociation. These results show that carbohydrate acts noncovalently to glue together the components to yield the complete quaternary supramolecular structure of the giant Hb. We suggest that this carbohydrate gluing may be mediated through lectin-like carbohydrate-binding by the associated structural chains ("linkers").

Mutations in B3GALT6, which encodes a glycosaminoglycan linker region enzyme, cause a spectrum of skeletal and connective tissue disorders.

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes, A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality, For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties, The imidazole function was conjugated to these CS-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid), The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments, 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates, In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates, The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2′-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XbaI and by mass spectrometry of the PCR products.

Species Fractionation In Atomic Chains From Mechanically Stretched Alloys.

Bettini et al (2006 Nat. Nanotechnol. 1 182-5) reported the first experimental realization of linear atomic chains (LACs) composed of different atoms (Au and Ag). The different contents of Au and Ag were observed in the chains from what was found in the bulk alloys, which raises the question of what the wire composition is, if it is in equilibrium with a bulk alloy. In this work we address the thermodynamic driving force for species fractionation in LACs under tension, and we present the density-functional theory results for Ag-Au chain alloys. A pronounced stabilization of the wires with an alternating Ag-Au sequence is observed, which could be behind the experimentally observed Au enrichment in LACs from alloys with high Ag content.

Linearization of Chains and Sideward Movement

360

Interleukin-22 forms dimers that are recognized by two interleukin-22R1 receptor chains

Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.

Formation of atomic carbon chains from graphene nanoribbons

The formation of one-dimensional carbon chains from graphene nanoribbons is investigated using ab initio molecular dynamics. We show under what conditions it is possible to obtain a linear atomic chain via pulling of the graphene nanoribbons. The presence of dimers composed of two-coordinated carbon atoms at the edge of the ribbons is necessary for the formation of the linear chains, otherwise there is simply the full rupture of the structure. The presence of Stone-Wales defects close to these dimers may lead to the formation of longer chains. The local atomic configuration of the suspended atoms indicates the formation of single and triple bonds, which is a characteristic of polyynes.

Defect-induced effects on carrier migration through one-dimensional poly(para-phenylenevinylene) chains

Defects in one-dimensional (1D) systems can be intrinsically distinct from its three-dimensional counterparts, and polymer films are good candidates for showing both extremes that are difficult to individuate in the experimental data. We study theoretically the impact of simple hydrogen and oxygen defects on the electron transport properties of one-dimensional poly(para-phenylenevinylene) chains through a multiscale technique, starting from classical structural simulations for crystalline films to extensive ab initio calculations within density functional theory for the defects in single crystalline-constrained chains. The most disruptive effect on carrier transport comes from conjugation breaking imposed by the overcoordination of a carbon atom in the vinyl group independently from the chemical nature of the defect. The particular case of the [C=O] (keto-defect) shows in addition unexpected electron-hole separation, suggesting that the experimentally detected photoluminescence bleaching and photoconductivity enhancement could be due to exciton dissociation caused by the 1D characteristics of the defect.

Renyi entropy and parity oscillations of anisotropic spin-s Heisenberg chains in a magnetic field

Using the density matrix renormalization group, we investigate the Renyi entropy of the anisotropic spin-s Heisenberg chains in a z-magnetic field. We considered the half-odd-integer spin-s chains, with s = 1/2, 3/2, and 5/2, and periodic and open boundary conditions. In the case of the spin-1/2 chain we were able to obtain accurate estimates of the new parity exponents p(alpha)((p)) and p(alpha)((o)) that gives the power-law decay of the oscillations of the alpha-Renyi entropy for periodic and open boundary conditions, respectively. We confirm the relations of these exponents with the Luttinger parameter K, as proposed by Calabrese et al. [Phys. Rev. Lett. 104, 095701 (2010)]. Moreover, the predicted periodicity of the oscillating term was also observed for some nonzero values of the magnetization m. We show that for s > 1/2 the amplitudes of the oscillations are quite small and get accurate estimates of p(alpha)((p)) and p(alpha)((o)) become a challenge. Although our estimates of the new universal exponents p(alpha)((p)) and p(alpha)((o)) for the spin-3/2 chain are not so accurate, they are consistent with the theoretical predictions.