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Made available in DSpace on 2018-11-26T17:06:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-09-01 Oxy-HbRa thermal stability was evaluated by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) at pH 5.0, 7.0, 8.0, and 9.0. DLS results show that oxy-HbRa, at pH 7.0 and 5.0, remains stable up to 56 degrees C, undergoing denaturation/aggregation in acidic media above 60 degrees C, followed by partial sedimentation of aggregates. At alkaline pH values 8.0 and 9.0, oxy-HbRa oligomeric dissociation is observed above 30 degrees C, before denaturation. SAXS data show that oxy-HbRa, at 20 degrees C, is in its native form, displaying radius of gyration (R-g) and particle maximum dimension (D-max) of 108 +/- 1 and 300 +/- 10 angstrom, respectively. Oxy-HbRa, at pH 7.0, undergoes denaturation/aggregation at 60 degrees C. At pH 5.0-6.0, HbRa thermal denaturation/aggregation start earlier, at 50 degrees C, accompanied by an increase of R-g and D-max values. However, an overlap of oligomeric dissociation and denaturation in the system is observed upon temperature increase, with an increase in R-g and D-max. Analysis of experimental p(r) curves as a linear combination of theoretical curves obtained for HbGp fragments from the crystal structure shows an increasing contribution of dodecamer (abcd)(3) and tetramer (abcd) in solution, as a function of pH values (8.0 and 9.0) and temperature. Finally, our data show, for the first time, that oxy-HbRa, in neutral and acidic media, does not undergo oligomeric dissociation before denaturation, while in alkaline media the oligomeric dissociation process is an important step in the thermal denaturation. Inst Quim Sao Carlos, Sao Carlos, SP, Brazil Univ Estado Mato Grosso, Barra Do Bugres, MT, Brazil Univ Estadual Paulista, Registro, SP, Brazil Univ Estadual Paulista, Registro, SP, Brazil

The electrochemical oxidation of dipyridamole (DIP) has been studied in acidified aqueous solutions at platinum electrodes employing cyclic voltammetry and controlled-potential electrolysis. The progress of the anodic oxidation as a function of time was monitored by cyclic voltammetry with platinum ultramicroelectrodes, absorption and fluorescence optical spectroscopies, the resulting integrated charge being indicative of a two electron process. The cyclic voltammograms registered for low scan speeds are characterized by a single irreversible diffusion controlled anodic wave, the related cathodic wave being also observable for scan speeds higher than 1 V s-1. Oxidation reaction stoichiommetric parameters were obtained through Tafel slopes resulting in unitary reaction orders for DIP and H+. A oxidação eletroquímica do dipiridamol (DIP) foi estudada em soluções aquosas acidificadas, empregando-se as técnicas de voltametria cíclica e eletrólise a potencial controlado com eletrodos de platina. Os experimentos de eletrólise foram monitorados por voltametria cíclica com ultramicroeletrodo de platina, espectroscopia de absorção óptica e fluorescência, e a integração da corrente durante a eletrólise resultou em uma carga correspondente a 2 elétrons por molécula de DIP. Os voltamogramas cíclicos registrados para o DIP a baixas velocidades de varredura se caracterizam por um único pico de oxidação em processo controlado por difusão, com o aparecimento do pico de redução correspondente a velocidades de varredura maiores que 1 V s-1. A estequiometria da reação de oxidação obtida a partir de curvas de polarização de estado estacionário resultou em ordens de reação unitária em relação ao DIP e íons H+.

Giant extracellular hemoglobins, also known as erythrocruorins, have been investigated as a model of extreme complexity in oxygen-binding heme proteins [1,2]. They are characterized by a very high molecular mass (MM) of several megadalton (MDa), and their oligomeric structure together with the crowded and protected heme environment are two of the main factors responsible for the high redox stability [3,4]. Superoxide dimustase (SOD)-like intrinsic activity, observed for hemoglobins of Lumbricus terrestris (HbLt) and of Arenicola marina (HbAm), is another important factor [5,6]. These hemoglobins present a highly cooperative oxygen binding and a peculiar behavior associated to their oligomeric dissociation into smaller subunits and possible rearrangement back into the native oligomeric structure [7,8].

© 2010 Elsevier B.V.All rights reserved, Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS), optical absorption spectroscopy (UV–VIS) and differential scanning calorimetry (DSC). At pH 7.0, cyanomet-HbGp is very stable, no oligomeric dissociation is observed, while denaturation occurs at 56 °C, 4 °C higher as compared to oxy-HbGp. The oligomeric dissociation of HbGp occurs simultaneously with some protein aggregation. Kinetic studies for oxy-HbGp using UV–VIS and DLS allowed to obtain activation energy (Ea) values of 278–262 kJ/mol (DLS) and 333 kJ/mol (UV–VIS). Complimentary DSC studies indicate that the denaturation is irreversible, giving endotherms strongly dependent upon the heating scan rates, suggesting a kinetically controlled process. Dependence on protein concentration suggests that the two components in the endotherms are due to oligomeric dissociation effect upon denaturation. Activation energies are in the range 200–560 kJ/mol. The mid-point transition temperatures were in the range 50–65 °C. Cyanomet-HbGp shows higher mid-point temperatures as well as activation energies, consistent with its higher stability. DSC data are reported for the first time for an extracellular hemoglobin., The authors are grateful to the Brazilian agencies FAPESP, CNPq and CAPES and to the Portuguese FCT-MCTES for partial financial support.

© 2010 American Chemical Society, The extracellular hemoglobin from Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 MDa. It has a high oligomeric stability at pH 7.0 and low autoxidation rates, as compared to vertebrate hemoglobins. In this work, fluorescence and light scattering experiments were performed with the three oxidation forms of HbGp exposed to acidic pH. Our focus is on the HbGp stability at acidic pH and also on the determination of the isoelectric point (pI) of the protein. Our results show that the protein in the cyanomet form is more stable than in the other two forms, in the whole pH range. Our zeta-potential data are consistent with light scattering results. Average values of pI obtained by different techniques were 5.6±0.5, 5.4±0.2 and 5.2±0.5 for the oxy, met, and cyanomet forms. Dynamic light scattering (DLS) experiments have shown that, at pH 6.0, the aggregation (oligomeric) state of oxy-, met- and cyanomet-HbGp remains the same as that at pH 7.0. The interaction between the oxy-HbGp and ionic surfactants at pH 5.0 and 6.0 was also monitored in the present study. At pH 5.0, below the protein pI, the effects of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium chloride (CTAC) are inverted when compared to pH 7.0. For CTAC, in acid pH 5.0, no precipitation is observed, while for SDS an intense light scattering appears due to a precipitation process. HbGp interacts strongly with the cationic surfactant at pH 7.0 and with the anionic one at pH 5.0. This effect is due to the predominance, in the protein surface, of residues presenting opposite charges to the surfactant headgroups. This information can be relevant for the development of extracellular hemoglobin-based artificial blood substitutes., The authors are grateful to Brazilian agencies FAPESP, CNPq, and CAPES and to the Portuguese FCTMCTES for partial financial support.M.M.D. also acknowledges the Ph.D. fellowship SFRH/BD/41750/2007 from FCT-MCTES.

To obtain a detailed understanding of both the general structure and function of specific members of the class of hemoproteins, comparative studies using homologous proteins from a wide variety of organisms are very useful. Myoglobins from a number of invertebrate species have been characterized both structurally and functionally [ 11. Myoglobin of AppZysia limacina was isolated, characterized [2,3] and several structural and functional properties have been studied [4-71. Applysia brasiliana is common on the eastern coast of Brazil. myoglobin was dialysed against the appropriate buffer to remove free spin label. The buffers used were Tris 0.05 M with adjusted pH values. EPR spectra were taken with a Varian X band E-9 spectrometer equipped with a system for temperature control.

Glossoscolex paulistus (HbGp) extracellular hemoglobin is a giant oligomeric protein. It is constituted by 144 heme containing subunits and non-heme structures (linkers), with a total molecular mass of 3.6MDa. AUC and DLS studies were developed for three HbGp forms, oxy-, met- and cyanomet-, at several pH values, in order to characterize the species in solution upon oligomeric dissociation. Isolated SEC fractions, trimer and dodecamer, are less stable as compared to the whole oxy-HbGp. The monomer d displays a large thermal stability up to 59°C. Hydrodynamic properties of the isolated subunits are very similar to those described for them in the whole protein, in the presence of urea or at pH 10.0. The degree of HbGp oligomeric dissociation, in alkaline pH, depends significantly on the iron oxidation state. Also on the ligand coordinated to the heme iron. Thus, at pH 8.0, the oxy-HbGp is partially dissociated, while the met-form is fully dissociated. The cyanomet-HbGp remains undissociated. Our present results show that the effect of pH on the HbGp oligomeric stability is similar to that associated to the urea-induced unfolding. Simultaneous use of AUC and DLS allowed the characterization of the species in the SEC fractions of isolated HbGp subunits.

The mas oncogene codes for a seven transmembrane helix protein. The amino acid sequence 253–266, from the third extracellular loop and beginning of helix 7, was synthesized either blocked or carrying an amino acid spin label at the N-terminus. Peptide binding to bilayers and micelles was monitored by ESR, fluorescence and circular dichroism. Binding induced tighter lipid packing, and caused an increase of peptide secondary structure. While binding to bilayers occurred only when peptide and phospholipid bore opposite charges, in micelles the interaction took place irrespective of charge. The results suggest that changes in lipid packing could modulate conformational changes in receptor loops related to the triggering of signal transduction.

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 DLS. 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. Autoxidation 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.

Binding and localization of the vasodilator and antitumor drug coactivator dipyridamole (DIP) and one of its derivatives, RA25, to phospholipid vesicles of DMPC (dimyristoylphosphatidylcholine) and DPPC (dipalmitoylphosphatidylcholine) was studied using fluorescence spectroscopy as well as quenching of fluorescence. The analysis of fluorescence data indicates that neutral dipyridamole binds to the phospholipids in their liquid crystalline phase with an association constant of 950 M−1 and 1150 M−1 to DMPC and DPPC, respectively. Protonation of DIP leads to a 3-fold reduction of the association constant. For the gel phospholipid phase, the binding is smaller (a factor of 2), independently of pH, suggesting that the more flexible lipid packing in the liquid crystalline phase facilitates the binding of the drug. The association constant of RA25 neutral form is considerably lower than for DIP, being around 295 M−1. Fluorescence quenching with nitroxides TEMPO and stearic acid doxyl derivatives suggests the localization of DIP to be closer to the 5th carbon of alkyl chain. The quenching effect of 5-DSA below the lipid phase transition suggests that a strong static quenching may be operative. The quenching effect of 16-DSA is almost as great as that for 5-DSA below the phase transition, being even higher above the phase transition. This effect is probably due to the trans-gauche isomerization of the stearic acid nitroxide, making the encounter of its paramagnetic fragment with the DIP chromophore possible. Our data are consistent with DIP location close to the bilayer surface in the border of hydrophobic-polar heads interface which is similar to the data in micellar systems. In the case of RA25, the drug is in the outer part of the head group interface being much exposed to the aqueous phase and being significantly less accessible to the membrane nitroxide quenchers.

The giant extracellular hemoglobin (HbAg) of the annelid Amynthas gracilis has a molecular mass (MM) of 3400kDa. In the current work, the characterization of MM values of HbAg and its subunits is presented. Electrophoresis, MALDI-TOF-MS and AUC show that the MM values of HbAg subunits are very close, but not identical to those of Glossoscolex paulistus (HbGp) and Rhinodrilus alatus (HbRa) hemoglobins. Analytical ultracentrifugation (AUC) sedimentation velocity experiments were performed to obtain M for HbAg in oxy- form. value of 59.3 ± 0.2 S was obtained for native HbAg. From the ratio between sedimentation and diffusion coefficients values for M of approximately 3400 ± 100 kDa for oxy-HbAg was obtained. MALDI-TOF-MS data gave MM for HbAg subunits. Monomer d is found to exist in, at least, four isoforms with MM 16,244±3Da, 16,459±5Da, 16,667±5Da and 16,855±3Da, as noticed for HbGp, and not observed for HbRa. Furthermore, the trimer subunit presents two isoforms ((abc)1 and (abc)2) with MM 51,415±20Da and 51,610±14Da, respectively. This might indicate that the monomers a, b and c do have isoforms, as found for HbGp and not for HbRa. The monomeric chains a, obtained from the trimer abc reduction, present three isoforms with MM 17,015Da, 17,061Da and 17,138Da, differing from HbGp that presents four isoforms. A less intense species is observed at 67,717, and is due to the tetramer abcd contribution. Finally, AUC and MALDI-TOF-MS data are very close as compared to that obtained for HbGp and HbRa. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Finantial support: FAPESP and CNPq Brazilian agencies.