Biorecognition of hydrogen peroxide using a novel electrochemical platform designed with Glossoscolex paulistus giant hemoglobin.

The giant extracellular hemoglobin of the annelid Glossoscolex paulistus (HbGp; 3.6 MDa) is a valuable and underexplored supramolecular hemoprotein system for the biorecognition of reactive oxygen species. In this work, an efficient and simple electrochemical platform was designed for analyzing H2O2, using HbGp covalently immobilized on Nafion®-modified glassy carbon electrode, named as HbGp/Nafion/GCE. Voltammetric and spectroscopic studies revealed the importance of prior modification of the electrodic support with the conducting polymer to obtain satisfactory hemoglobin electroactivity, as well as a biocompatible microenvironment for its immobilization. In terms of biological activity, it was observed a greater reactivity of the biomolecule in acidic medium, enabling the detection of the analyte by a quasi-reversible mechanism, whose kinetics was limited by analyte diffusion. In the presence of H2O2, the native structure of hemoglobin (oxy-HbGp (Fe2+)) oxidizes to ferryl-HbGp (Fe4+) and this redox reaction can be monitored on HbGp/Nafion/GCE with a detection limit of 8.5 × 10‒7  mol L-1. In addition to high sensitivity, the electrochemical biosensor also provided reproducible, consistent, and accurate measurements. The electroanalytical method showed an appropriate performance to quantify different levels of H2O2 in milk samples, proving the potential of HbGp/Nafion/GCE for this purpose.

Evaluation of the antigenotoxic and antioxidant activity induced by Croton antisyphiliticus.

This study aimed to investigate antigenotoxicity and antioxidant potential of extract, fractions and vitexin from C. antisyphiliticus. Methanolic extract was fractionated through solvents of increasing polarity. The composition of extracts and fractions were evaluated through phytochemical screening. Micronucleus test was performed in mice to evaluate the antigenotoxicity. Antioxidant activity was measured using the assay 1,1-diphenyl-2-picrylhydrazyl (DPPH), iron ion chelating, thiobarbituric acid assay and nitric oxide scavenging. Treatment with extract, fractions and vitexin did not produce an increase in Micronucleus mean values. However, Micronucleus (MN) mean values decreased in relation to control. methanolic extract presented antioxidant potential for DPPH (81%), iron ion chelating (77.8%), Thiobarbituric Acid (TBARS) (32.49%) and Nitric Oxide (NO) (80.97%). Ethyl acetate fraction showed the highest antioxidant activity (65.46%). The vitexin showed a Inhibitory Concentration (IC50) of DPPH value smaller in relation to control. Vitexin flavonoid was detected by High Performance Liquid Chromatography (HPLC), infrared spectrometry and nuclear magnetic resonance. It can be inferred that methanolic extract, fraction ethyl acetate and vitexin isolated from C. antisyphiliticus is endowed with antigenotoxic and antioxidant potential.

Thermal stability of extracellular hemoglobin of Rhinodrilus alatus (HbRa): DLS and SAXS studies.

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 °C, undergoing denaturation/aggregation in acidic media above 60 °C, followed by partial sedimentation of aggregates. At alkaline pH values 8.0 and 9.0, oxy-HbRa oligomeric dissociation is observed above 30 °C, before denaturation. SAXS data show that oxy-HbRa, at 20 °C, is in its native form, displaying radius of gyration (R g) and particle maximum dimension (D max) of 108 ± 1 and 300 ± 10 Å, respectively. Oxy-HbRa, at pH 7.0, undergoes denaturation/aggregation at 60 °C. At pH 5.0-6.0, HbRa thermal denaturation/aggregation start earlier, at 50 °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.

Cetyltrimethylammonium chloride (CTAC) effect on the thermal stability of oxy-HbGp: dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies.

Glossoscolex paulistus (HbGp) hemoglobin is an oligomeric protein, displaying a quaternary structure constituted by 144 globin and 36 non-globin chains (named linkers) with a total molecular mass of 3.6MDa. CTAC effects on the oxy-HbGp thermal stability were investigated, by DLS and SAXS, at pH 5.0, 7.0 and 9.0. DLS data show that the oxy-HbGp-CTAC interactions induce a significant decrease of the protein thermal stability, with the formation of larger aggregates, at pH 5.0 and 7.0. In the acidic pH, oxy-HbGp 0.5mg/mL, undergoes a partial oligomeric dissociation, on going from 0.2 to 0.6mmol/L of CTAC, accompanied by a decrease in the Dh values from 27±1 to 22±1nm. It is observed, for the first time, that in the absence and in the presence of CTAC, oxy-HbGp undergoes a partial oligomeric dissociation, with increase of temperature, before denaturation and aggregation at pH values 7.0 and 5.0. SAXS data show that oxy-HbGp undergoes denaturation at 60°C, in the presence of CTAC, pH 5.0. At neutral pH 7.0, the aggregation process starts at 20°C, with increase of Rg and Dmax parameters. At both pH values, 5.0 and 7.0, the denaturation and aggregation are accompanied by the sedimentation of the aggregates. At pH 9.0, oxy-HbGp is totally dissociated at 40°C, in the presence of 0.2mmol/L of CTAC, while in the presence of 0.4mmol/L of surfactant the aggregation process starts at 20°C, with the full denaturation of protein at higher temperature. Finally, our data show, for the first time, that the oligomeric dissociation is an important step in the thermal denaturation of oxy-HbGp, in the presence of CTAC, independently of both the pH and the protein concentration.

Characterization of Rhinodrilus alatus hemoglobin (HbRa) and its subunits: evidence for strong interaction with cationic surfactants DTAB and CTAC.

Rhinodrilus alatus is an annelid and its giant extracellular hemoglobin (HbRa) has a molecular mass (MM) of 3500kDa. In the current study, the characterization of MM values of the HbRa subunits, and the effects of surfactants and alkaline pH upon HbRa stability were monitored. Electrophoresis, MALDI-TOF-MS and AUC show that the MM values of HbRa subunits are very close, but not identical to the Glossoscolex paulistus hemoglobin (HbGp). The monomer d is found to exist in, at least, two isoforms: the main one, d1, displays a MM of 16,166±16Da, and the second one, d2, is less intense with MM of 16,490±20Da. For the trimer abc and tetramer abcd, single contributions around 51,470Da and 67,690Da were observed, respectively. Finally, the monomers a, b, and c, present MM values of 17,133, 17,290 and 15,506Da, respectively. Both CTAC and DTAB interact strongly with HbRa, and up to seven surfactant molecules are bound to the protein. On the other hand, spectroscopic studies show that HbRa is more stable at alkaline pH, as compared to HbGp. Thus, our data suggest that alkaline medium, up to pH10.0, induces the oligomeric dissociation, without promoting the subunits unfolding and heme iron oxidation. Our results suggest that the MM of the annelid hemoglobin subunits is conserved to a great extent in the evolution process of these species.

Sodium dodecyl sulfate (SDS) effect on the thermal stability of oxy-HbGp: dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies.

Glossoscolex paulistus (HbGp) hemoglobin is an oligomeric protein, presenting a quaternary structure constituted by 144 globin and 36 non-globin chains (named linkers) with a total molecular mass of 3.6 MDa. SDS effects on the oxy-HbGp thermal stability were studied, by DLS and SAXS, at pH 5.0, 7.0 and 9.0. DLS and SAXS data show that the SDS-oxy-HbGp interactions induce a significant decrease of the protein thermal stability, with the formation of larger aggregates, at pH 5.0. At pH 7.0, oxy-HbGp undergoes complete oligomeric dissociation, with increase of temperature, in the presence of SDS. Besides, oxy-HbGp 3.0mg/mL, pH 7.0, in the presence of SDS, has the oligomeric dissociation process reduced as compared to 0.5mg/mL of protein. At pH 9.0, oxy-HbGp starts to dissociate at 20 °C, and the protein is totally dissociated at 50 °C. The thermal dissociation kinetic data show that oxy-HbGp oligomeric dissociation at pH 7.0, in the presence of SDS, is strongly dependent on the protein concentration. At 0.5mg/mL of protein, the oligomeric dissociation is complete and fast at 40 and 42 °C, with kinetic constants of (2.1 ± 0.2) × 10(-4) and (5.5 ± 0.4) × 10(-4) s(-1), respectively, at 0.6 mmol/L SDS. However, at 3.0mg/mL, the oligomeric dissociation process starts at 46 °C, and only partial dissociation, accompanied by aggregates formation is observed. Moreover, our data show, for the first time, that, for 3.0mg/mL of protein, the oligomeric dissociation, denaturation and aggregation phenomena occur simultaneously, in the presence of SDS. Our present results on the surfactant-HbGp interactions and the protein thermal unfolding process correspond to a step forward in the understanding of SDS effects.

rBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures.

rBPI21 belongs to the antimicrobial peptide and protein (AMP) family. It has high affinity for lipopolysaccharide (LPS), acting mainly against Gram-negative bacteria. This work intends to elucidate the mechanism of action of rBPI21 at the membrane level. Using isothermal titration calorimetry, we observed that rBPI21 interaction occurs only with negatively charged membranes (mimicking bacterial membranes) and is entropically driven. Differential scanning calorimetry shows that membrane interaction with rBPI21 is followed by an increase of rigidity on negatively charged membrane, which is corroborated by small angle X-ray scattering (SAXS). Additionally, SAXS data reveal that rBPI21 promotes the multilamellarization of negatively charged membranes. The results support the proposed model for rBPI21 action: first it may interact with LPS at the bacterial surface. This entropic interaction could cause the release of ions that maintain the packed structure of LPS, ensuring peptide penetration. Then, rBPI21 may interact with the negatively charged leaflets of the outer and inner membranes, promoting the interaction between the two bacterial membranes, ultimately leading to cell death.

Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: a dissociation model.

The urea effect on the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) stability was studied by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). AUC data show that the sedimentation coefficient distributions curves c (S), at 1.0 mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0 mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204 kDa for the dodecamer (abcd)(3), 5.5S and 69 kDa for the tetramer (abcd), 4.1S and 52 kDa for the trimer (abc) and 2.0 S and 17 kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0 mol/L of denaturant, for oxy-HbGp, and above 6.0 mol/L for cyanomet-HbGp, an increase in the maximum dimension and gyration radius is observed, due to the unfolding process. According to AUC and SAXS data the HbGp unfolding is described by two phases: the first one, at low urea concentration, below 4.0 mol/L, characterizes the oligomeric dissociation, while the second one, at higher urea concentration, is associated to the unfolding of dissociated species. Our results are complementary to a recent report based on spectroscopic observations.

Thermal denaturation and aggregation of hemoglobin of Glossoscolex paulistus in acid and neutral media.

The thermal denaturation and aggregation of the HbGp, in the oxy- and cyanomet-forms, was investigated by DSC, AUC, DLS, optical absorption and CD, in the pH range from 5.0 to 7.0. Oxy-HbGp has a denaturation process partially reversible and dependent on the temperature. DSC melting curve is characterized by a single peak with T(c) value of 333.4 ± 0.2K for oxy-HbGp, while two peaks with T(c) values of 332.2 ± 0.1 and 338.4 ± 0.2K are observed for cyanomet-HbGp, at pH 7.0. In acidic pH oxy- and cyanomet-HbGp are more stable showing higher T(c) values and aggregation. AUC data show that, HbGp, at pH 7.0, upon denaturation, remains undissociated at 323 K, presenting oligomeric dissociation at 333 (12 ± 3% of tetramer and 88 ± 5% of whole HbGp) and 343 K (70 ± 5% of monomer and 30 ± 2% of trimer). DLS data show that the lag period before aggregation is dependent on the temperature and HbGp concentration. Optical absorption and CD results show that the increase of temperature leads to the oxy-HbGp oxidation and aggregation, above 331 K, in acidic pH. CD data, for HbGp, present a greater thermal stability in acid medium than at neutral pH, with similar T(c) values for both oxidation forms. Our data are consistent with previous studies and represents an advance in understanding the thermal stability of oligomeric HbGp structure.

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies.

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). DLS melting curves were measured for met-HbGp at different concentrations. SAXS temperature studies were performed for oxy-, cyanomet- and met-HbGp forms, at several pH values. At pH 5.0 and 6.0, the scattering curves are identical from 20 to 60 °C, and Rg is 108 Å, independent of the oxidation form. At pH 7.0, protein denaturation and aggregation occurs above 55 °C and 60 °C, for oxy and met-HbGp, respectively. Cyanomet-HbGp, at pH 7.0, is stable up to 60 °C. At alkaline pH (8.0-9.0) and higher temperature, an irreversible dissociation process is observed, with a decrease of Rg, Dmax and I(0). Analysis by p(r), obtained from GNOM, and OLIGOMER, was used to fit the SAXS experimental scattering curves by a combination of theoretical curves obtained for HbLt fragments from the crystal structure. Our results show clearly the increasing contribution of smaller molecular weight fragments, as a function of increasing pH and temperature, as well as, the order of thermal stabilities: cyanomet->oxy->met-HbGp.

On the stability of the extracellular hemoglobin of Glossoscolex paulistus, in two iron oxidation states, in the presence of urea.

The stability of the Glossoscolex paulistus hemoglobin (HbGp), in two iron oxidation states (and three forms), as monitored by optical absorption, fluorescence emission and circular dichroism (CD) spectroscopies, in the presence of the chaotropic agent urea, is studied. HbGp oligomeric dissociation, denaturation and iron oxidation are observed. CD data show that the cyanomet-HbGp is more stable than the oxy-form. Oxy- and cyanomet-HbGp show good fits on the basis of a two state model with critical urea concentrations at 220-222 nm of 5.1±0.2 and 6.1±0.1 mol/L, respectively. The three-state model was able to reveal a subtle second transition at lower urea concentration (1.0-2.0 mol/L) associated to partial oligomeric dissociation. The intermediate state for oxy- and cyanomet-HbGp is very similar to the native state. For met-HbGp, a different equilibrium, in the presence of urea, is observed. A sharp transition at 1.95±0.05 mol/L of denaturant is observed, associated to oligomeric dissociation and hemichrome formation. In this case, analysis by a three-state model reveals the great similarity between the intermediate and the unfolded states. Analysis of spectroscopic data, by two-state and three-state models, reveals consistency of obtained thermodynamic parameters for HbGp urea denaturation.

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 (s20,w0) 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 ß-mercaptoethanol, were also studied. Our results indicate that for samples of pure subunit d, besides the monomeric species with s20,w0 of 2.0 S, formation of dimer of subunit d is observed with s20,w0 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.

Thermal stability of extracellular hemoglobin of Glossoscolex paulistus: determination of activation parameters by optical spectroscopic and differential scanning calorimetric studies.

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 (E(a)) 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.

Isoelectric point determination for Glossoscolex paulistus extracellular hemoglobin: oligomeric stability in acidic pH and relevance to protein-surfactant interactions.

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.

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(0)(20,w) values of 58.1+/-0.2S and 59.6+/-0.2S, 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.

On the localization of water-soluble porphyrins in micellar systems evaluated by static and time-resolved frequency-domain fluorescence techniques.

Fluorescence quenching of meso-tetrakis-4-sulfonatophenyl (TPPS(4)) and meso-tetrakis-4-N-methylpyridil (TMPyP) porphyrins is studied in aqueous solution and upon addition of micelles of sodium dodecylsulfate (SDS), cetyltrimethylammonium chloride (CTAC), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and t-octylphenoxypolyethoxyethanol (Triton X-100). Potassium iodide (KI) was used as quencher. Steady-state Stern-Volmer plots were best fitted by a quadratic equation, including dynamic (K(D)) and static (K(S)) quenching. K(S) was significantly smaller than K(D). Frequency-domain fluorescence lifetimes allowed estimating bimolecular quenching constants, k(q). At 25 degrees C, in aqueous solution, TMPyP shows k(q) values a factor of 2-3 higher than the diffusional limit. TPPS(4) shows collisional quenching with pH dependent k(q) values. For TMPyP quenching results are consistent with reported binding constants: a significant reduction of quenching takes place for SDS, a moderate reduction is observed for HPS and almost no change is seen for Triton X-100. Similar data were obtained at 50 degrees C. For CTAC-TPPS(4) system an enhancement of quenching was observed as compared to pure buffer. This is probably associated to accumulation of iodide at the cationic micellar interface. The attraction between CTAC headgroups and I(-), and repulsion between SDS and I(-), enhances and reduces the fluorescence quenching, respectively, of porphyrins located at the micellar interface. The small quenching of TPPS(4) in Triton X-100 is consistent with strong binding as reported in the literature.

What can light scattering spectroscopy do for membrane-active peptide studies?

Highly charged peptides are important components of the immune system and belong to an important family of antibiotics. Although their therapeutic activity is known, most of the molecular level mechanisms are controversial. A wide variety of different approaches are usually applied to understand their mechanisms, but light scattering techniques are frequently overlooked. Yet, light scattering is a noninvasive technique that allows insights both on the peptide mechanism of action as well as on the development of new antibiotics. Dynamic light scattering (DLS) and static light scattering (SLS) are used to measure the aggregation process of lipid vesicles upon addition of peptides and molecular properties (shape, molecular weight). The high charge of these peptides allows electrostatic attraction toward charged lipid vesicles, which is studied by zeta potential (zeta-potential) measurements.

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 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.

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS): effects of oligomeric dissociation.

The present work focuses on the interaction between the zwitterionic surfactant N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp). Electronic optical absorption, fluorescence emission and circular dichroism spectroscopy techniques, together with Gel-filtration chromatography, were used in order to evaluate the oligomeric dissociation as well as the autoxidation of HbGp as a function of the interaction with HPS. A peculiar behavior was observed for the HPS-HbGp interaction: a complex ferric species formation equilibrium was promoted, as a consequence of the autoxidation and oligomeric dissociation processes. At pH 7.0, HPS is more effective up to 1mM while at pH 9.0 the surfactant effect is more intense above 1mM. Furthermore, the interaction of HPS with HbGp was clearly less intense than the interaction of this hemoglobin with cationic (CTAC) and anionic (SDS) surfactants. Probably, this lower interaction with HPS is due to two factors: (i) the lower electrostatic attraction between the HPS surfactant and the protein surface ionic sites when compared to the electrostatic interaction between HbGp and cationic and anionic surfactants, and (ii) the low cmc of HPS, which probably reduces the interaction of the surfactant in the monomeric form with the protein. The present work emphasizes the importance of the electrostatic contribution in the interaction between ionic surfactants and HbGp. Furthermore, in the whole HPS concentration range used in this study, no folding and autoxidation decrease induced by this surfactant were observed. This is quite different from the literature data on the interaction between surfactants and tetrameric hemoglobins, that supports the occurrence of this behavior for the intracellular hemoglobins at low surfactant concentration range. Spectroscopic data are discussed and compared with the literature in order to improve the understanding of hemoglobin-surfactant interaction as well as the acid isoelectric point (pI) influence of the giant extracellular hemoglobins on their structure-activity relationship.

Interaction of meso-tetrakis (4-sulfonatophenyl) porphyrin with cationic CTAC micelles investigated by small angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR).

Small angle X-ray scattering (SAXS) and electron paramagnetic resonance (EPR) have been used to investigate the interaction of the water-soluble meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS(4)) with cationic cethyltrimethylammonium chloride (CTAC) micelles. To evaluate if the porphyrin protonation state affects its interaction with the micelle, both SAXS and EPR measurements were performed at pH 4.0 and 9.0. The best-fit SAXS curves were obtained assuming for CTAC micelle a prolate ellipsoidal shape in the absence and upon incorporation of 2-10 mM TPPS(4). SAXS results show that the presence of porphyrin impacts on micellar hydrophobic core, leading to a micellar reassembling into smaller micelles. Lineshapes of EPR spectra of 5- and 16-doxyl stearic acids (5- and 16-DSA, respectively) bound to 100 mM CTAC micelles exhibited slight changes as a function of porphyrin concentration. Spectral simulations revealed an increase of mobility restriction for both spin probes, especially at higher porphyrin concentration, where a small reduction of environment polarity was also observed for 16-DSA. The spin labels monitored only slight differences between pH 4.0 and 9.0, in agreement with the SAXS results.