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The structural study of small heme-containing proteins, such as myoglobin, in the apo-form lacking heme has been extensively described, but the characterization and stability of the giant Glossoscolex paulistus hemoglobin (HbGp), in the absence of heme groups, has not been studied. Spectroscopic data show efficient extraction of the heme groups from the hemoglobin, with relatively small secondary and tertiary structural changes in apo-HbGp noticed compared to oxy-HbGp. Electrophoresis shows a partial precipitation of the trimer abc (significantly lower intensity of the corresponding band in the gel), due to extraction of heme groups, and the predominance of the intense monomeric d band, as well as of two linker bands. AUC and DLS data agree with SDS-PAGE in showing that the apo-HbGp undergoes dissociation into the d and abc subunits. Subunits d and abc are characterized by sedimentation coefficients and percentage contributions of 2.0 and 3.0 S and 76 and 24%, respectively. DLS data suggest that the apo-HbGp is unstable, and two populations are present in solution: one with a diameter around 6.0 nm, identified with the dissociated species, and a second one with diameter 100-180 nm, due to aggregated protein. Finally, the presence of urea promotes the exposure of the fluorescent probes, extrinsic ANS and intrinsic protein tryptophans to the aqueous solvent due to the unfolding process. An understanding of the effect of heme extraction on the stability of hemoproteins is important for biotechnological approaches such as the introduction of non-native prosthetic groups and development of artificial enzymes with designed properties.

Glossoscolex paulistus hemoglobin structure is composed of 144 globin chains and 36 polypeptide chains lacking the heme group, with a total molecular mass of 3600 kDa. The current study focuses on the oxy-HbGp oligomeric stability, as a function of the storage time, at pH 7.0, using dynamic light scattering, analytical ultracentrifugation (AUC), optical absorption and size exclusion chromatography (SEC). HbGp stored in Tris-HCl buffer, pH 7.0, at 4 °C, for two years remains in the native form, while 4–6 years HbGp stocks present typical hemichrome species absorption spectra. AUC and SEC analyses show that the contribution of HbGp-subunits, such as, dodecamer (abcd)3, tetramer abcd, trimer abc and monomer d, increases with the protein aging due to the lower stability of the HbGp with the time. The dissociation and the oxidation of the iron noted for the older protein solutions indicate that HbGp storage for periods of time longer than two years changes its ability to carry oxygen. Despite the reduction of HbGp stability and oxygen carrying capacity with aging, the protein stability is still larger as compared to mammalian hemoglobins. Thus, the extracellular hemoglobins are quite stable and resistant to the auto-oxidation process, making them of interest for biotechnological applications.

In this work, isothermal titration and differential scanning calorimetric methods, in combination with pyrene fluorescence emission and dynamic light scattering have been used to investigate the interaction of dodecyltrimethylammonium bromide (DTAB) with the giant extracellular Glossoscolex paulistus hemoglobin (HbGp) in the oxy-form, at pH values around the isoelectric point (pI ≈ 5.5). Our ITC results have shown that the interaction of DTAB with the hemoglobin is more intense at pH 7.0, with a smaller cac (critical aggregation concentration) value. The increase of protein concentration does not influence the cac value of the interaction, at both pH values. Therefore, the beginning of the DTAB-oxy-HbGp premicellar aggregates formation, in the cac region, is not affected by the increase of protein concentration. HSDSC studies show higher Tm values at pH 5.0, in the absence and presence of DTAB, when compared with pH 7.0. Furthermore, at pH 7.0, an aggregation process is observed with DTAB in the range from 0.75 to 1.5 mmol/L, noticed by the exothermic peak, and similar to that observed for pure oxy-HbGp, at pH 5.0, and in the presence of DTAB. DLS melting curves show a decrease on the hemoglobin thermal stability for the oxy-HbGp-DTAB mixtures and formation of larger aggregates, at pH 7.0. Our present data, together with previous results, support the observation that the protein structural changes, at pH 7.0, occur at smaller DTAB concentrations, as compared with pH 5.0, due to the acidic pI of protein that favors the oxy-HbGp-cationic surfactant interaction at neutral pH.

Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been monitored in the presence of denaturant agents. 8-Anilino-1-naphtalene-sulfonic acid (ANS) was used, and spectroscopic and hydrodynamic studies were developed. Dodecyltrimethylammonium bromide (DTAB) induces an increase in ANS fluorescence emission intensity, with maximum emission wavelength blue-shifted from 517 to 493 nm. Two transitions are noticed, at 2.50 and 9.50 mmol/L of DTAB, assigned to ANS interaction with pre-micellar aggregates and micelles, respectively. In oxy-HbGp, ANS binds to protein sites less exposed to solvent, as compared to DTAB micelles. In DTAB–HbGp–ANS ternary system, at pH 7.0, protein aggregation, oligomeric dissociation and unfolding were observed, while, at pH 5.0, aggregation is absent. DTAB induced unfolding process displays two transitions, one due to oligomeric dissociation and the second one, probably, to the denaturation of dissociated subunits. Moreover, guanidine hydrochloride and urea concentrations above 1.5 and 4.0 mol/L, respectively, induce the full HbGp denaturation, with reduction of ANS-bound oxy-HbGp hydrophobic patches, as noticed by fluorescence quenching up to 1.0 and 5.0 mol/L of denaturants. Our results show clearly the differences in probe sensitivity to the surfactant, in the presence and absence of protein, and new insights into the denaturant effects on HbGp unfolding.

The giant extracellular hemoglobin from earthworm Glossoscolex paulistus (HbGp) reacts with hydrogen peroxide, displaying peroxidase activity in the presence of guaiacol. The formation of ferryl-HbGp (compound II) from the peroxidase cycle was studied in the present work. The hypervalent ferryl-HbGp species was formed directly by the reaction of oxy-HbGp and hydrogen peroxide. The oxy-HbGp heme groups (144) under different excess of H2O2, relative to heme, showed an influence in the total amount of ferryl-HbGp at the end of the reaction. The ferryl-HbGp was formed with second order rate constant of 27.1 ± 0.5 M− 1 s− 1, at pH 7.0 and 25 °C. The increase of the pH value to 8.0 induces both faster formation and decay of ferryl-HbGp, together with oligomeric dissociation induced by the presence of H2O2, as observed by DLS. This effect of dissociation increases the heme exposure and decreases the ferryl-HbGp stability, affecting the rate constant as a parallel reaction. At pH 7.0, high excess of H2O2, above 1:5 oxy-HbGp heme: H2O2, produces the aggregation of the protein. Our results show for the first time, for an extracellular giant hemoglobin, the possible effects of oxidative stress induced by hydrogen peroxide.

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.

Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been followed, in the presence of urea, using fluorescein isothiocyanate (FITC). Binding of FITC to HbGp results in a significant quenching of probe fluorescence. Tryptophan emission decays present four characteristic lifetimes: two in the sub-nanosecond/picosecond, and two in the nanosecond time ranges. Tryptophan decays for pure HbGp and HbGp-FITC systems are similar. In the absence of denaturant, and up to 2.5mol/L of urea, the shorter lifetimes predominate. At 3.5 and 6.0mol/L of urea, the longer lifetimes increase significantly their contribution. Urea-induced unfolding process is characterized by protein oligomeric dissociation and denaturation of dissociated subunits. FITC emission decays for FITC-HbGp system are also multi-exponential with three lifetimes: two in the sub-nanosecond and one in the nanosecond range with a value similar to free probe in buffer. Increase of urea concentration leads to increase of the longer lifetime contribution, implying the removal of the quenching observed for the native HbGp-FITC system. Anisotropy decays are characterized by two rotational correlation times associated to re-orientational motions of the probe relative to protein. Our results suggest that FITC bound to HbGp is useful to monitor denaturant effects on the protein.

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

Our aim was to investigate the interaction of the cationic meso-tetrakis (4-N-methylpyridyl) porphyrin, a photosensitizer used for photodynamic therapy, in its free base form (TMPyP) and complexed with Zn(II) (ZnTMPyP), with large unilamellar vesicles (LUVs), as a model for the gram-negative bacterial cell wall. Mixtures of the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) phospholipids, at different molar percentages, were used as LUVs. A significant increase of porphyrin affinity at higher POPG molar concentrations was observed from the binding constant values, K b, estimated by optical absorption and steady-state fluorescence. Besides, as demonstrated by time-resolved fluorescence, this affinity increase is also followed by a higher fraction of vesicle-bound porphyrin in the LUVs. Moreover, based on the K b values, we have observed a higher affinity of the ZnTMPyP to the POPG containing LUVs as compared to the TMPyP. Steady-state fluorescence quenching and zeta potential studies revealed that both porphyrins are possibly located at the LUVs Stern layer region. Therefore, the electrostatic attraction between the positively charged porphyrin peripheral groups and the negatively charged outer surface of the LUVs plays an important role in porphyrin association and localization. Our results have improved the understanding of the successful application of cationic porphyrins on the photo-inactivation of gram-negative bacteria. Since a higher accumulation of the ZnTMPyP in the bacterial cell wall would be expected, this porphyrin could be a more efficient therapeutic drug for this treatment.

The interaction of the cationic meso-tetrakis 4-N-methylpyridyl porphyrin (TMPyP) with large unilamellar vesicles (LUVs) was investigated in the present study. LUVs were formed by mixtures of the zwitterionic 1,2-dipalmitoyl-sn-glycero-phosphatidylcholine (DPPC) and anionic 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) phospholipids, at different DPPG molar percentages. All investigations were carried out above (50 °C) and below (25 °C) the main phase transition temperature of the LUVs (∼41 °C). The binding constant values, Kb, estimated from the time-resolved fluorescence study, showed a significant increase of the porphyrin affinity at higher mol% DPPG. This affinity is markedly increased when the LUVs are in the liquid crystalline state. For both situations, the increase of the Kb value was also followed by a higher porphyrin fraction bound to the LUVs. The displacement of the vesicle-bound porphyrins toward the aqueous medium, upon titration with the salt potassium chloride (KCl), was also studied. Altogether, our steady-state and frequency-domain fluorescence quenching data results indicate that the TMPyP is preferentially located at the LUVs Stern layer. This is supported by the zeta potential studies, where a partial neutralization of the LUVs surface charge, upon porphyrin titration, was observed. Dynamic light scattering (DLS) results showed that, for some phospholipid systems, this partial neutralization leads to the LUVs flocculation.

The interaction of bovine serum albumin (BSA) with the ionic surfactants sodium dodecylsulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) was studied by electron paramagnetic resonance (EPR) spectroscopy of spin label covalently bound to the single free thiol group of the protein. EPR spectra simulation allows to monitor the protein dynamics at the labeling site and to estimate the changes in standard Gibbs free energy, enthalpy and entropy for transferring the nitroxide side chain from the more motionally restricted to the less restricted component. Whereas SDS and CTAC showed similar increases in the dynamics of the protein backbone for all measured concentrations, HPS presented a smaller effect at concentrations above 1.5mM. At 10mM of surfactants and 0.15 mM BSA, the standard Gibbs free energy change was consistent with protein backbone conformations more expanded and exposed to the solvent as compared to the native protein, but with a less pronounced effect for HPS. In the presence of the surfactants, the enthalpy change, related to the energy required to dissociate the nitroxide side chain from the protein, was greater, suggesting a lower water activity. The nitroxide side chain also detected a higher viscosity environment in the vicinity of the paramagnetic probe induced by the addition of the surfactants. The results suggest that the surfactant-BSA interaction, at higher surfactant concentration, is affected by the affinities of the surfactant to its own micelles and micelle-like aggregates. Complementary DLS data suggests that the temperature induced changes monitored by the nitroxide probe reflects local changes in the vicinity of the single thiol group of Cys-34 BSA residue.

Structural and Electronic Properties of Dipyridamole and Derivatives R. S. Borges1 ∗, M. Castilho2, M. Tabak2, A. B. F. da Silva2, and C. N. Alves3 1Faculdade de Farmacia, Instituto de Ciencias da Saude, Universidade Federal do Para, 66075-110, Belem–PA, Brazil 2Instituto de Quimica de Sao Carlos, Universidade de Sao Paulo, CP 780, 13560-970, Sao Carlos–SP, Brazil 3Faculdade de Quimica, Instituto de Ciencias Exatas e Naturais, Universidade Federal do Para, CP 11101, 66075-110, Belem–PA, Brazil

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)3L3 (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. s020,w 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, Vbar, 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.

The equilibrium of meso-tetrakis(4-N-methylpyridiniumyl)porphyrin (TMPyP) in aqueous solution in the presence of surfactants was studied by optical spectroscopic techniques and SAXS (small angle X-ray scattering). Anionic SDS (sodium dodecyl sulfate), zwitterionic HPS (N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) and nonionic TRITON X-100 (t-octylphenoxypolyethoxyethanol), surfactants were used. TMPyP is characterized by a protonation equilibrium with a pKa around 1.0, associated with the diacid-free base transition, and a second pKa around 12.0 related with the transition between the free base and the monoanion form. Three independent species were observed for TMPyP at pH 6.0 as a function of SDS concentration: free TMPyP, TMPyP-SDS aggregates and porphyrin monomer bound to micelles. For HPS and TRITON X-100, the equilibrium of TMPyP as a function of pH is quite similar to that obtained in pure aqueous solution: no aggregation was observed, suggesting that electrostatic contribution is the major factor in the interaction between TMPyP and surfactants. SAXS data analysis demonstrated a prolate ellipsoidal shape for SDS micelles; no significant changes in shape and size were observed for SDS-TMPyP co-micelles. Moreover, the ionization coefficient, α, decreases with the increase of the porphyrin concentration, suggesting the "screening" of the anionic charge of SDS by the cationic porphyrin. These results are consistent with optical absorption, fluorescence and RLS (resonance light scattering) spectroscopies data, allowing to conclude that neutral surfactants present a smaller interaction with the cationic porphyrin as compared with an ionic surfactant. Therefore, the interaction of TMPyP with the ionic and nonionic surfactants is predominantly due to the electrostatic contribution.

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 1 mM while at pH 9.0 the surfactant effect is more intense above 1 mM. 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 (p I ) influence of the giant extracellular hemoglobins on their structure–activity relationship.

Glossoscolex paulistus hemoglobin (HbGp) is an oligomeric multisubunit protein with molecular mass of 3600kDa. In the current study, the interaction of sodium dodecyl sulfate (SDS) and cetyl trimethylammonium chloride (CTAC) surfactants with the monomer d and the whole oxy-HbGp, at pH 7.0, was investigated. For pure monomer d solution, SDS promotes the dimerization of subunit d, and the monomeric and dimeric forms have sedimentation coefficient values, s20,w, around 2.1-2.4 S and 2.9-3.2 S, respectively. Analytical ultracentrifugation (AUC) and isothermal titration calorimetry (ITC) data suggest that up to 26 DS- anions are bound to the monomer. In the presence of CTAC, only the monomeric form is observed in solution for subunit d. For the oxy-HbGp, SDS induces the dissociation into smaller subunits, such as, monomer d, trimer abc, and tetramer abcd, and unfolding without promoting the protein aggregation. On the other hand, lower CTAC concentration promotes protein aggregation, mainly of trimer, while higher concentration induces the unfolding of dissociated species. Our study provides strong evidence that surfactant effects upon the HbGp-subunits are different, and depend on the surfactant: protein concentration ratio and the charges of surfactant headgroups.

In this work we investigate the presence of divalent cations bound to the Glossoscolex paulistus (HbGp) hemoglobin and their effect over the protein stability and the peroxidase (POD) activity. Atomic absorption studies show that the HbGp iron content is consistent with the presence of 144 ions per protein. Moreover, using iron as a reference, the content of calcium was estimated as 30±4 ions per protein, independently of the EDTA pre-treatment or not prior to the acidic treatment performed in the protein digestion. The zinc content was 14±2 ions in the absence of EDTA pre-treatment, and 3±1 ions per protein in the presence of EDTA pre-treatment, implying the presence of one zinc ion per protomer (1/12 of the whole molecule). Finally, the copper concentration is negligible. Different from the vertebrate hemoglobins, where the effectors are usually organic anions, the hexagonal bilayer hemoglobins have as effectors inorganic cations that increase the oxygen affinity and stabilize the structure. Previous studies have suggested that the presence of divalent cations, such as copper and zinc, is related to the different types of antioxidant enzymatic activities as the superoxide dismutase (SOD) activity shown by giant hemoglobin from Lumbricus terrestris (HbLt). Recently, studies on HbGp crystal structure have confirmed the presence of Zn(2+) and Ca(2+) binding sites. The Ca(2+) sites are similar as observed in the HbLt crystal structure. Otherwise, the Zn(2+) sites have no relation with those observed in Cu/Zn SODs. Our peroxidase assays with guaiacol confirm the POD activity and the effect of the zinc ions for HbGp. Our present results on HbGp metal content and their stability effects is the first step to understand the role of these cations in HbGp function in the future.

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.

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein-surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein-surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein-surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein-surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.

In this work, isothermal titration and differential scanning calorimetric methods, in combination with pyrene fluorescence emission and dynamic light scattering have been used to investigate the interaction of dodecyltrimethylammonium bromide (DTAB) with the giant extracellular Glossoscolex paulistus hemoglobin (HbGp) in the oxy-form, at pH values around the isoelectric point (pI ≈ 5.5). Our ITC results have shown that the interaction of DTAB with the hemoglobin is more intense at pH 7.0, with a smaller cac (critical aggregation concentration) value. The increase of protein concentration does not influence the cac value of the interaction, at both pH values. Therefore, the beginning of the DTAB-oxy-HbGp premicellar aggregates formation, in the cac region, is not affected by the increase of protein concentration. HSDSC studies show higher Tm values at pH 5.0, in the absence and presence of DTAB, when compared with pH 7.0. Furthermore, at pH 7.0, an aggregation process is observed with DTAB in the range from 0.75 to 1.5 mmol/L, noticed by the exothermic peak, and similar to that observed for pure oxy-HbGp, at pH 5.0, and in the presence of DTAB. DLS melting curves show a decrease on the hemoglobin thermal stability for the oxy-HbGp-DTAB mixtures and formation of larger aggregates, at pH 7.0. Our present data, together with previous results, support the observation that the protein structural changes, at pH 7.0, occur at smaller DTAB concentrations, as compared with pH 5.0, due to the acidic pI of protein that favors the oxy-HbGp-cationic surfactant interaction at neutral pH.

The effect of the anionic surfactant sodium dodecyl sulfate (SDS) on the autoxidation process of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is addressed in the present work. The complex oligomeric assembly of hemoglobin subunits may influence the autoxidation rate and the exponential decay behavior. Kinetic studies were developed using UV–vis measurements at 415 nm. These spectroscopic measurements are analyzed at two pH values, 7.0 and 9.0, where the hemoglobin presents different oligomeric assembly. At pH 7.0 a high stability of the native form of the oxy-hemoglobin is observed, while at pH 9.0 an intense dissociation of the oligomer is promoted by alkalization. This difference is evident by comparison of the rate constants in the absence of surfactant: at pH 7.0 the kinetics presents a mono-exponential behavior with a rate constant of 0.27 × 10 −4 s −1 while at pH 9.0 a bi-exponential behavior was observed with rate constant increase to 7 × 10 −4 s −1 (fast process) and 1 × 10 −4 s −1 (slow process). In the autoxidation induced by SDS two factors affect significantly the process rate, namely, the oligomeric arrangement of the hemoglobin and the strength of the interaction between SDS and HbGp. At pH 7.0, for SDS concentrations up to 0.3 mM, a mono-exponential behavior was observed, showing rate constants around 0.4 × 10 −4 s −1 , which suggest that the hemoglobin still maintains the more compact structure observed at this pH for the native protein. In the SDS concentration range 0.75–1.0 mM, the mono-exponential process changes into a bi-exponential behavior with rate constants varying from 48 × 10 −4 up to 99 × 10 −4 s −1 for the fast process and from 1.7 × 10 −4 up to 3.7 × 10 −4 s −1 for the slow process, suggesting hemoglobin dissociation. At pH 9.0, a bi-exponential decay is observed for all studied SDS concentration range, presenting rate constants from 11.0 × 10 −4 up to 179 × 10 −4 s −1 for the fast process and from 1.0 × 10 −4 up to 8 × 10 4 s −1 for the slow process probably due to hemoglobin dissociation, which is already present in the absence of surfactant. At pH 7.0, the highly packed native protein structure should inhibit the autoxidation process, but the SDS/HbGp interaction is more intense as compared to pH 9.0, due to the acid p I value, promoting oligomeric dissociation. So, the autoxidation process is regulated at pH 7.0 by the interaction with SDS, which triggers oligomeric dissociation and increase of autoxidation rate. At pH 9.0, the autoxidation process should be very fast, probably due to the oligomeric dissociation, which is already present in the absence of surfactant. At alkaline pH, the interaction with SDS seems be weaker than at pH 7.0. This behavior at pH 7.0 can be observed through the higher autoxidation rate for the faster chains and it is associated to the acid p I of the giant extracellular hemoglobins.

Thermal decomposition of dipyridamole was followed by analysis of the residue by spectroscopic methods. The loss of mass observed in thermogravimetric (TG) experiments in N2 atmosphere occurs in essentially three steps. The first step, corresponding to 35% of mass loss, was monitored in an isothermal process, and the solid residue was analyzed by proton and carbon NMR, optical absorption, and fluorescence emission. Heating at 305°C leads to new products with optical absorption bands shifted to lower wavelengths relative to dipyridamole. The broad emission band is also shifted to lower wavelengths. NMR analysis demonstrates that the piperidine groups are probably one of the sites of modification because the corresponding resonance peaks are not present in proton or carbon spectra. Preliminary high‐pressure chromatography shows that two main compounds appear at significantly higher polarity as compared with dipyridamole. Isothermal decomposition leaves the pyrimido‐pyrimidine central ring essential...

Interactions of cationic FeTMPyP with ionic and nonionic micelles have been studied by optical absorption, resonance light scattering (RLS) and 1 H NMR spectroscopies. The equilibrium behavior of FeTMPyP as a function of pH is described by several species in aqueous solution. The presence of phosphate anions leads to the existence of additional species in the acid p H region, probably due to the coordination of phosphates to the iron. FeTMPyP solution as a function of pH in the presence of anionic SDS showed a simplified equilibrium in acidic pH region, favoring the transition to the dimeric species. Titration of FeTMPyP as a function of SDS surfactant concentration showed the presence of three different porphyrin species: free metalloporphyrin monomers (or dimers depending on pH), metalloporphyrin monomers (or dimers) bound to the micelles, and nonmicellar metalloporphyrin/surfactant aggregates. In the case of zwitterionic LPC and HPS, and nonionic TRITON X-100 the nonmicellar metalloporphyrin/surfactant aggregates were not observed. Binding constants were calculated from optical absorption data and have values of 2 × 103 M −1 for SDS being much smaller for HPS (58 M −1), LPC and TRITON X-100. Comparison with our previous data for anionic FeTPPS 4 shows that both the electrostatic factor and hydrophobic forces are relevant in the porphyrin-surfactant interaction: for FeTPPS 4 binding constants to cationic CTAC and zwitterionic HPS are of the same order of magnitude, 1-3 × 104 M −1; for FeTMPyP the delocalization of the positive charges from the periphery substituents into the macrocycle ring leads to reduction of both electrostatic attraction to the micelle as well as hydrophobic character of the porphyrin ring, leading to a 10-fold reduction of binding to the micelles of opposite charge to the porphyrin. NMR data indicated that FeTMPyP is bound to the micelles as an equilibrium of two forms of monomer at pH 2.0, and at pH 9.0 the bound aggregated form (possibly dimers) is observed predominantly with some amount of a monomeric form.

pH effects on the oligomeric structure of giant Glossoscolex paulistus extracellular hemoglobin in the oxyand met-forms have been studied as well as effects of the addition of anionic sodium dodecyl sulfate surfactant. A radius of gyration of 110 A is observed for a macromolecule. At 2 mm surfactant, the radius of gyration diminishes slightly for the oxy-form. However, the extrapolated initial scattering intensity (I0) decreases a factor of 2.5, indicating protein dissociation. At 20 mm surfactant, further I0 decrease is observed, with a reduction of radius of gyration to approximately 30 A consistent with dissociation into smaller subunits. At pH 9.0, the scattering curves are similar to that obtained for the protein in the presence of 20 mm surfactant at pH 7.0. A radius of gyration of approximately 35 A shows that the giant hemoglobin dissociation into small subunits also occurs at alkaline pH. From the I0 value, one can suggest that the tetramer is the main scatter at pH 9.0. At pH 7.0, the met-form dissociates to a larger extent at 2 mm surfactant as compared with the oxy-form, and the main scatters seem to be the 1/12 subunit. At pH 9.0, for the oxy-form, the addition of surfactant does not modify the scattering curve and a radius of gyration approximately 30 A is obtained, while for the met-form some kind of aggregation is observed. Our results give support to conclude that the iron oxidation state is an important factor modulating the oligomeric dissociation.

The aggregate morphology of meso-tetrakis(4-sulfonatophenyl) porphyrin (TPPS(4)) in aqueous solution is investigated by using small angle x-ray scattering (SAXS) technique. Measurements were performed at pH 4.0 and 9.0 to monitor the pH influence on the structural parameters of the aggregates. Radii of gyration were obtained from distance distribution functions p(r) analysis. The experimental data of TPPS(4) at pH 4.0 showed well-defined oscillations characteristic of large aggregates in contrast to the SAXS curve of 5 mM TPPS(4) at pH 9.0, where both a significant decrease in the intensity and the disappearance of the oscillation peaks suggest the dissociation of the aggregate. A 340-A long "hollow" cylinder with shell thickness of 20 A, compatible to the porphyrin molecule dimension, represents well the scattering curve of the aggregates at pH 4.0. According to the fitting parameters, 26 porphyrin molecules self-associate into a ringlike configuration in the plane of the cylinder cross-section. The total number of porphyrin molecules in the whole aggregate was also estimated as approximately 3000. The model compatible to SAXS data of a hollow cylinder with J-aggregation in the cross-section and H-aggregation (columnar stacking) between the cylinder layers is consistent with optical absorption spectroscopic data both in the literature and obtained in this work.

In this work small angle X-ray scattering (SAXS) studies on the interaction of the phenothiazine trifluoperazine (TFP, 2-10 mM), a cationic drug, with micelles of the zwitterionic surfactant 3-(N-hexadecyl-N,N-dimethylammonium) propane sulfonate (HPS, 30 mM) and the anionic surfactant sodium dodecyl sulfate (SDS, 40 mM) at pH 4.0, 7.0, and 9.0 are reported. The data were analyzed through the modeling of the micellar form factor and interference function, as well as by means of the distance distribution function p(r). For anionic micelles (SDS), the results evidence a micellar shape transformation from prolate ellipsoid to cylinder accompanied by micellar growth and surface charge screening as the molar ratio TFP:SDS increases in the complex for all values of pH. Small ellipsoids with axial ratio nu=1.5+/-0.1 (long dimension of 60 A) grow and reassemble into cylinder-like aggregates upon 5 mM drug incorporation (1 TFP:8 SDS monomers) with a decrease of the micelle surface charge. At 10 mM TFP:40 mM SDS cylindrical micelles are totally screened with an axial ratio nu approximately 4 (long dimension approximately 140 A at pH 7.0 and 9.0). However, at pH 4.0, where the drug is partially diprotonated, 10 mM TFP incorporation gives rise to a huge increase in micellar size, resulting in micelles at least 400 A long, without altering the intramicellar core. For zwitterionic micelles (HPS), the results have shown that the aggregates also resemble small prolate ellipsoids with averaged axial ratio approximately nu=1.6+/-0.1. Under TFP addition, both the paraffinic radius and the micellar size show a slight decrease, giving evidence that the micellar hydrophobic core may be affected by phenothiazine incorporation rather than that observed for the SDS/TFP comicelle. Therefore, our results demonstrate that the axial ratio and shape evolution of the surfactant:TFP complex are both dependent on surfactant surface-charge and drug:surfactant molar ratio. The results are compared with those recently obtained for another phenothiazine drug, chlorpromazine (CPZ), in SDS and HPS micelles (Caetano, Gelamo, Tabak, and Itri, J. Colloid Interface Science 248 (2002) 149).

The equilibrium behavior of cationic iron(III) meso-tetrakis(4-N-methyl-pyridiniumyl) porphyrin, Fe(III)TMPyP, in aqueous solution was studied as a function of pH by optical absorption, EPR and (1)H NMR spectroscopies. The presence of several Fe(III)TMPyP species in solution was unequivocally demonstrated: monomeric porphyrin species (a monoaqueous five-coordinated complex, a diaaqueous six-coordinated complex and a monoaqueous-hydroxo six-coordinated complex), a micro-oxo dimer and a bis-hydroxo complex. The addition of salt to the porphyrin solution leads to a simplification of the equilibrium as a function of pH. In this case, only three species were observed in solution: a monomeric porphyrin species, a micro-oxo dimer and a bis-hydroxo complex. Optical absorption, EPR and (1)H NMR spectra contributed to the characterization of these species. Four critical pH values (pK) for Fe(III)TMPyP were obtained in pure buffer and only three pK values were observed in the presence of NaCl. The addition of salt favors the presence of the dimeric species in solution and simplifies the equilibrium in the acidic pH range.

The sequences of all seven polypeptide chains from the giant haemoglobin of the free-living earthwormGlossoscolex paulistus(HbGp) are reported together with the three-dimensional structure of the 3.6 MDa complex which they form. The refinement of the full particle, which has been solved at 3.2 Å resolution, the highest resolution reported to date for a hexagonal bilayer haemoglobin composed of 12 protomers, is reported. This has allowed a more detailed description of the contacts between subunits which are essential for particle stability. Interpretation of features in the electron-density maps suggests the presence of metal-binding sites (probably Zn2+and Ca2+) and glycosylation sites, some of which have not been reported previously. The former appear to be important for the integrity of the particle. The crystal structure of the isolateddchain (d-HbGp) at 2.1 Å resolution shows different interchain contacts betweendmonomers compared with those observed in the full particle. Instead of forming trimers, as seen in the complex, the isolateddchains associate to form dimers across a crystallographic twofold axis. These observations eliminate the possibility that trimers form spontaneously in solution as intermediates during the formation of the dodecameric globin cap and contribute to understanding of the possible ways in which the particle self-assembles.

Electrooxidation of dipyridamole (DIP) and several of its derivatives, RA14, RA47, RA143 and RA25, was studied in acetonitrile. It is observed that all the derivatives (except RA143) like DIP, exhibit a consecutive two-step one-electron electrochemical oxidation. The oxidation potentials for DIP, RA14 and RA47 are quite similar, while those for RA25 are higher. The derivative RA143 which lacks two of the nitrogens linked in positions 2 and 6 of the pyrimido-pyrimidine ring is not electroactive in the potential window used. The spectral changes following oxidation were monitored by UV–vis optical absorption and ESR, which in conjunction were able to shed additional light on the species involved in the complex equilibrium. In the first wave process cation radicals of DIP and RA25 are produced which decay to the final products with half-lives of 160 and 90 min. The decay for the cation radical of RA25 is faster than that of DIP, due to its lower stability in ACN solution. The ESR spectrum of RA25 cation radical is very similar to that of DIP in agreement with recent theoretical calculations that show that the electron is removed from the conjugated system based on the pyrimido-pyrimidine ring. The analysis of the spectra registered after the two electron oxidation of DIP and RA25 derivative shows that while, for DIP the reaction produces one additional intermediate, for RA25 it leads directly to the final product(s).

Small-angle X-ray scattering (SAXS) studies are reported on the interaction of chlorpromazine (CPZ) with micelles of anionic surfactant sodium dodecyl sulfate (SDS). Isotropic solutions of SDS (40 and 100 mM) at pH 4.0, 7.0, and 9.0 in the absence and presence of CPZ (2-25 mM) were investigated at the National Laboratory of Synchrotron Light (LNLS, Campinas, Brazil). The data were analyzed through the modeling of the micellar form factor and interference function. The results evidence a micellar shape transformation from prolate ellipsoid to cylinder accompanied by micellar growth and surface charge screening as the molar ratio CPZ : SDS increases in the complex. Small ellipsoids with axial ratio nu=1.5+/-0.1 at 40 mM SDS grow and reassemble into cylinder-like aggregates upon 5 mM drug incorporation (1 CPZ : 8 SDS monomers) with a decrease of the micelle surface charge. At 10 mM CPZ : 40 mM SDS cylindrical micelles are totally screened with an axial ratio nu approximately 2.5. The data also indicate the presence of small prolate ellipsoids (nu=1.7+/-0.1) in solutions of 100 mM SDS (no drug) and micellar growth (nu approximately 2.0 and 4.0) when 10 and 25 mM CPZ are added to the system. In the latter case, the aggregate is also better represented by a cylinder-like form. Therefore, our results demonstrate that the axial ratio and shape evolution of the surfactant : phenothiazine complex are both SDS concentration and drug : SDS molar ratio dependent. The drug location close to the SDS polar headgroup region without disrupting in a significant way both the paraffinic hydrophobic core and the polar shell thickness is inferred. SAXS data made it possible to obtain the shapes and dimensions of CPZ/SDS aggregates.

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern-Volmer quenching constants of (1.1+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) for SDS, HPS and CTAC, respectively, which are associated to the 'effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA-myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.

Glossoscolex paulistus hemoglobin (HbGp) has a molecular mass of 3600kDa. It belongs to the hexagonal bilayer hemoglobin class, which consists of highly cooperative respiratory macromolecules found in mollusks and annelids. The present work focusses on oxy-HbGp thermal stability, in the presence of urea and guanidine hydrochloride (GuHCl), monitored by several techniques. Initially, dynamic light scattering data show that the presence of GuHCl induces the protein oligomeric dissociation, followed by a significant 11-fold increase in the hydrodynamic diameter (DH) values, due to the formation of protein aggregates in solution. In contrast, urea promotes the HbGp oligomeric dissociation, followed by unfolding process at high temperatures, without aggregation. Circular dichroism data show that unfolding critical temperature (Tc) of oxy-HbGp decreases from 57°C, at 0.0 mol/L of the denaturant, to 45°C, in the presence of 3.5 mol/L of urea, suggesting the reduction of HbGp oligomeric stability. Moreover, differential scanning calorimetry results show that at lower GuHCl concentrations, some thermal stabilization of the hemoglobin is observed, whereas at higher concentrations, the reduction of stability takes place. Besides, HbGp is more stable in the presence of urea when compared with the guanidine effect, as deduced from the differences in the concentration range of denaturants.

The stratum corneum (SC) protein dynamics in the sulfhydryl group regions was studied by electron paramagnetic resonance (EPR) spectroscopy of a covalently attached maleimide derivative spin label. A two-state model for the nitroxide described the coexistence of two spectral components in the EPR spectra. The so-called strongly immobilized component arises from a spin-label fraction with the nitroxide moiety hydrogen-bonded to protein (rigid structure) and the weakly immobilized component is provided by the spin labels with higher mobility (approximately 10 times greater) exposed to the aqueous environment. The relative populations between these two states are in thermodynamic equilibrium. The apparent energetic gain for the nitroxide to form a hydrogen bond with the backbone rather than to be dissolved in the local environment was approximately 10 kcal/mol in the temperature range of 2-30 degrees C and approximately 6 kcal/mol in the range of 30-70 degrees C. Urea treatment caused a drastic increase in the segmental motion of the polypeptide chains that was completely reversible by its removal. Our analyses also indicated that the urea induced unfolding of the SC proteins opening the thiol group cavities. This work can also be useful to improve the spectral analysis of site-directed spin-labeling, especially for a more quantitative description of the nitroxide side chain mobility.

PM3 calculations of charge distributions for dipyridamole (DIP) in the neutral, single- and double-ionized states allowed to estimate the first and second ionization potentials. Results are compared with electrochemical oxidation, a sequential two-step process. Single ionization produces a cation radical, the electron being removed from the nitrogen atoms in the substituent positions 2,4,6,8 with participation of the carbons in the pyrimido-pyrimidine ring. Protonation of one of the nitrogens is allowed energetically while a second protonation is forbidden due to the high energy required. Our calculations allow to explain some interesting experimental results related to electrochemical oxidation and protonation of the drug.

Bovine (BSA) and human (HSA) serum albumins are frequently used in biophysical and biochemical studies since they have a similar folding, a well known primary structure, and they have been associated with the binding of many different categories of small molecules. One important difference of BSA and HSA is the fact that bovine albumin has two tryptophan residues while human albumin has a unique tryptophan. In this work results are presented for the interaction of BSA and HSA with several ionic surfactants, namely, anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS), as monitored by fluorescence spectroscopy of intrinsic tryptophans and circular dichroism spectroscopy. On the interaction of all three surfactants with BSA, at low concentrations, a quenching of fluorescence takes place and Stern-Volmer analysis allowed to estimate their 'effective' association constants to the protein: for SDS, CTAC and HPS at pH 7.0 these constants are, respectively, (1.4+/-0.1) x 10(5) M(-1), (8.9+/-0.1) x 10(3) M(-1) and (1.4+/-0.1) x 10(4) M(-1). A blue shift of maximum emission is observed from 345 to 330 nm upon surfactant binding. Analysis of fluorescence emission spectra allowed to separate three species in solution which were associated to native protein, a surfactant protein complex and partially denatured protein. The binding at low surfactant concentrations follows a Hill plot model displaying positive cooperativity and a number of surfactant binding sites very close to the number of cationic or anionic residues present in the protein. Circular dichroism data corroborated the partial loss of secondary structure upon surfactant addition showing the high stability of serum albumin. The interaction of the surfactants with HSA showed an enhancement of fluorescence at low concentrations, opposite to the effect on BSA, consistent with the existence of a unique buried tryptophan residue in this protein with considerable static quenching in the native state. The effects of surfactants at low concentrations were very similar to those of myristic acid suggesting a non specific binding through hydrophobic interaction modulated by eletrostatic interactions. The changes in the vicinity of the tryptophan residues are discussed based on the recently published crystallographic structure of HSA myristate complex (S. Curry et al., Nat. Struct. Biol. 5 (1998) 827).

The anodic oxidation of dipyridamole (DIP) has been studied in micellar solutions of the anionic surfactant sodium dodecylsulfate (SDS), the cationic cetyltrimethylammonium bromide (CTAB) and the neutral t -octyl phenoxy polyethoxyethanol (Triton X-100). The oxidation reaction was studied by employing cyclic voltammetry at a glassy carbon, and rotating disc voltammetry which allowed the estimation of the diffusion coefficients, D , for DIP in the presence of the micelles. The values of D in the case of CTAB (0.42×10 −6 cm 2 s −1 ) and Triton X-100 (0.39×10 −6 cm 2 s −1 ) at pH 7.0 are consistent with DIP being transported to the electrode together with the micelle — so, for nonionic and cationic micelles the oxidation of DIP is diffusion controlled by drug micelle binding. In the case of SDS the values of D (0.16×10 −7 cm 2 s −1 at pH 7.0) are significantly lower than those expected for the pure SDS micelles. Furthermore, the interaction of SDS with the electrode surface could also contribute to preclude the efficient transport of DIP to the electrode surface, explaining the low diffusion constant found. In acid solution, at pH 3.0 or below, the oxidation of DIP is characterized by a single two-electron wave. Upon alkalinization to a more physiological pH range (pH 6–9) a two step oxidation behavior is found only in the presence of anionic SDS. Diffusion coefficients for both waves are very similar suggesting that both processes are governed by the same mechanism.

The lipid chain motions in stratum corneum (SC) membranes have been studied through electron paramagnetic resonance (EPR) spectroscopy of stearic acid spin-labeled at the 5th, 12th and 16th carbon atom positions of the acyl chain. Lipids have been extracted from SC with a series of chloroform/methanol mixtures, in order to compare the molecular dynamics and the thermotropic behavior in intact SC, lipid-depleted SC (containing covalently bound lipids of the corneocyte envelope) and dispersion of extracted SC lipids. The segmental motion of 5- and 12-doxylstearic acid (5- and 12-DSA) and the rotational correlation time of 16-doxylstearic acid (16-DSA) showed that the envelope lipids are more rigid and the extracted lipids are more fluid than the lipids of the intact SC over the range of temperature measured. The lower fluidity observed for the corneocyte envelope, that may be caused mainly due to lipid-protein interactions, suggests a major contribution of this lipid domain to the barrier function of SC. Changes in the activation energy for reorientational diffusion of the 16-DSA spin label showed apparent phase transitions around 54 degrees C, for the three SC samples. Some lipid reorganization may occur in SC above 54 degrees C, in agreement with results reported from studies with several other techniques. This reorganization is sensitive to the presence of the extractable intercellular lipids, being different in the lipid-depleted sample as compared to native SC and lipid dispersion. The results contribute to the understanding of alkyl chain packing and mobility in the SC membranes, which are involved in the mechanisms that control the permeability of different compounds through skin, suggesting an important involvement of the envelope in the skin barrier.

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+.

The characteristics of binding of two phenothiazine antipsychotic drugs, namely, chlorpromazine (CPZ) and trifluoperazine (TFP), to cationic cetyltrimethylammonium chloride (CTAC), zwitterionic N -hexadecyl- N , N -dimethyl-3-ammonio-1-propanesulfonate (HPS), neutral t -octylphenoxypolyethoxyethanol (TRITON X-100) and polyoxyethylene dodecyl ether (Brij-35) micelles were investigated using electronic absorption spectroscopy. Binding constants K b and p K a values of drugs in micelles were estimated using the red shifts of the maximum absorption upon alkalization or in the presence of detergents. The p K a of TFP seems to be shifted by 2.5–4.1 units to lower values in the presence of different surfactants as compared to the experimental value of p K a obtained in buffer which is around 7.0. Consideration of the second p K a around 4.0 reported in the literature for TFP leads to a better rationalization of p K a changes for this compound. The changes in p K a contributed by electrostatic effects are all positive, small for CTAC (+0.2), and greater for HPS (+0.9). For CPZ the p K a shift due to its interaction with micelles is in the 0.7–2.3 range, the direction of the shift depending on the charge of the polar head. The electrostatic contribution for the shift is great for CTAC (−0.8) and smaller for HPS (+0.2). This result suggests a more polar localization in the micelle of CPZ as compared to TFP. The values of binding constants K b for TFP and CPZ in different protonation states show that electrostatic interactions are essential in the affinity of the drugs to micelles bearing different charges on their headgroups (CTAC, HPS). Data for Brij-35 demonstrate that the additional charge on the TFP ring at pH 2.0 leads to a decrease of binding constant probably due to the repulsion of the phenothiazine ring from the protons accumulated at the polar head of the micelle at acidic pH values. For this micelle at pH 5.0 TFP has a K b 3-fold greater than that for CPZ while at pH 2.0 K b for TFP is 3-fold less than that for CPZ. So, for more hydrophobic TFP electrostatic interactions due to protonation of the ring are quite important even considering its deeper penetration into the micelle interior as compared to CPZ.

Interactions of the water-soluble tetra(4-sulfonatophenyl)porphyrin (TPPS4) with ionic micelles in aqueous solutions have been studied by optical absorption, fluorescence, resonance light-scattering (RLS), and 1H NMR spectroscopies. The presence of three different species of TPPS4 in cationic cetyltrimethylammonium chloride (CTAC) solution has been unequivocally demonstrated: free porphyrin monomers, monomers bound to micelles, and nonmicellar porphyrin/surfactant aggregates. This result is similar to our previous findings for TPPS4 interactions with biomacromolecules (serum albumin and DNA). The surfactant:porphyrin ratio for maximum aggregate formation is around 4:1−5:1 and 14:1 at pH 3.0 and pH 7.5, respectively. In the case of zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) the aggregates were not observed. Binding constants estimated from these data were of the order of 104 M-1 for CTAC and HPS. Our data show that solubilization of porphyrins within nonpolar regions of micel...

The KM+ lectin exhibits a novel and unusual circular dichroism (CD) spectrum that could be explained by a high proline content that would be inducing deformation of the beta-structure and/or unusual turns. KM+ was shown to be a very rigid lectin, which was very stable under a broad variety of conditions (urea, guanidine, hydrolysis, pH, etc.). Only incubation for 60 min at 333-338 K and extreme basic pH were able to induce conformational changes which could be observed by CD and fluorescence measurements. Data from CD are typical for protein denaturing associated with changes in the overall secondary structure. Data from high-performance size exclusion chromatography (SEC) showed that the denatured forms produced at pH 12.0 are eluted in clusters that co-elute with the native forms. A significant contribution from the tyrosines to the fluorescence emission upon denaturation was observed above 328 K. In fact at 328 K some broadening of the emission spectrum takes place followed by the appearance of a shoulder (approx. 305 nm) at 333 K and above. The sensitivity of tryptophan fluorescence to the addition of sugar suggests a close proximity of the tryptophan residues to the sugar binding site, K(a)=(2.9+/-0.6)x10(3) M(-1). The fraction of chromophore accessible to the quencher obtained is f(a)=0.43+/-0.08, suggesting that approximately 50% of the tryptophan residues are not accessible to quenching by d-mannose. KM+ thermal denaturation was found to be irreversible and was analyzed using a two-state model (N-->D). The results obtained for the activation energy and transition temperature from the equilibrium CD studies were: activation energy, E(a)=134+/-11 kJ/mol and transition temperature, T(m)=339+/-1 K, and from the fluorescence data: E(a)=179+/-18 kJ/mol and T(m)=337+/-1 K. Kinetic studies gave the following values: E(a)=108+/-18 kJ/mol and E(a)=167+/-12 kJ/mol for CD and fluorescence data, respectively.

The eletrochemical oxidation of dipyridamole DIP was studied by cyclic voltammetry with ultramicroelectrodes, electronic absorption and fluorescence spectroscopies, and ESR spectroscopy in acetonitrile and ethanol. Extensive electrolysis of DIP was performed in these solvents and the progress of the oxidation reaction as a function of time was monitored through cyclic voltammetry, absorption and fluorescence optical spectroscopies, taking advantage of the fact that DIP is a highly fluorescent compound. Voltammograms at high and low scanning rates showed a similar behavior in the different solvents characterized by two almost reversible voltammetric waves corresponding to the consecutive one-electron release. The corresponding E 1/2 were 120 and 400 mV in acetonitrile and 470 and 680 mV in ethanol. The release of one electron produces a radical cation which is readily detected by ESR spectroscopy. The oxidation is accompanied by the decrease of fluorescence intensity and significant changes in the optical absorption spectrum of DIP with the appearence of the characteristic absorption of the radical in the blue spectral region. The oxidation in ethanol seems to be affected by the solvent in the sense that it is not as easy as in acetonitrile and longer reaction times are required to complete the process. The relative ease of oxidation is in agreement with its reported anti-oxidant activity both in model systems and in physiological media.

Interaction of the coronary vasodilator dipyridamole (DIP) with vesicles of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and mixtures of them has been studied by fluorescence spectroscopy of the drug. Association constants were determined both below and above the phase transition temperature for the lipids, at different pHs (4.0 and 7.0). These constants at pH 7.0 were 633 M -1 (30 °C) and 1.15 × 10 3 M -1 (50 °C) for pure DPPC and 727 M -1 (30 °C) and 1.48 × 10 3 M -1 (50 °C) for pure DPPG. At pH 4.0 the association constants showed different behavior; K b values decreased by a factor of 3 for DPPC but increased for DPPG due to the electrostatic interaction of the protonated drug with the phospholipid headgroup. In the mixed system formed by DPPC and DPPG (11% and 20%) the resulting variations in the mixture compositions have a marked effect on drug-vesicle interaction; a decrease of the association constants was observed consistent with a more tightly packed bilayer. Steady-state anisotropy binding data demonstrated the validity of the two-state binding model. Fluorescence quenching experiments with 5-doxylstearic acid (5-DSA) were also performed at pH 4 and pH 7 and with different compositions of lipids. The results support the interfacial location of the drug (close to the fifth carbon of the alkyl chain), suggesting also a strong dependence of binding on lipid packing and the presence of charges at the membrane interface. Fluorescence anisotropy decay experiments were also performed for dipyridamole (DIP) in pure DPPC and DPPG at different pHs and temperatures. The results show clearly that the initial anisotropy r 0 of DIP in model systems (glycerol, sucrose) is quite high, above 0.3, attaining values of 0.20-0.24 in DPPG and being smaller in DPPC. These values are consistent with steady-state anisotropy values at saturating lipid concentrations. The anisotropy decays are best described as a single rotational correlation time which varies in the range 1.5-5.0 ns together with a limiting anisotropy value which also varies between 0.03 and 0.08. Data for anisotropy are in agreement with the binding data, also suggesting the location of the drug at the membrane interface.

Interactions of the water soluble meso-tetrakis(4-N-methyl-pyridiniumyl) porphyrin (TMPyP) in its free base, Mn(III) and Fe(III) forms with DNA in aqueous solutions have been studied by optical absorption, fluorescence and resonance light-scattering (RLS) spectroscopies. Optical absorption and fluorescence measurements have demonstrated the presence of three different species of TMPyP free base and its Mn(III) form in DNA solutions. These species correspond to free porphyrin monomers, monomers bound to DNA and porphyrin aggregates formed on the surface of DNA molecules. This assignment correlates very well with the RLS data. Aggregation reduces the fluorescence of the TMPyP free base. Fe(III)TMPyP also forms aggregates, however, more than three species exist in the presence of DNA due to the equilibria between its free and bound monomers and μ-oxo dimers. The degree of aggregation of Mn(III) and Fe(III) forms of TMPyP is higher than that of its free base. One of the possible explanations of this fact lies in the competition between intercalation and aggregation of TMPyP free base in the process of its binding to DNA; the intercalation of porphyrin should reduce its degree of aggregation. For the Mn(III) and Fe(III) TMPyP forms this competition does not exist as they do not intercalate.

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.

Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus in the met form presents an unique band at pH 7.0 and two bands at pH 9.0 as a result of alkaline dissociation. SDS-PAGE of the intact protein obtained at pH 7.0 is very similar to that for the oxyhemoglobin. Chromatography at pH 9.0 indicates complete dissociation of the oligomeric protein into two low molecular weight fractions corresponding to the trimers and monomers, showing that the oxidized extracellular hemoglobin is less stable than the oxyhemoglobin with respect to alkaline dissociation. Fluorescence quantum yields of different fractions obtained in the chromatography, as well as extinction coefficients at 280 nm and 415 nm, were estimated and compared to human methemoglobin. The fluorescence data are consistent with the high content of aromatic residues in G. paulistus hemoglobin. The increase in the fluorescence quantum yield upon both alkalinization and dissociation was correlated with the reduction of intramolecular quenching but the exposure of tryptophan residues to the solvent did not changed significantly as occurs for the oxy form. The intact native protein has a quantum yield of 0.36% at pH 7.0, increasing to 1.89% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 1.1% at pH 7.0 increasing to 1.43% at pH 9.0. The maximum emission wavelength of the intact protein (330 nm) is consistent with tryptophan residues being relatively buried; they become more fluorescent upon dissociation into smaller subunits but not more exposed since the wavelength of maximum emission is essentially unchanged at pH 9.0. In the monomer, the tryptophan residues also remain buried inside the protein molecule at pH 9.0 (328 nm). The dependencies of fluorescence quantum yields on the pH show in a clear way the hemichrome transitions observed by optical absorption spectroscopy indicating that the formation of two types of hemichromes accompany the distinct increase in fluorescence quantum yield. One type of hemichrome is irreversibly formed around pH 7.5–8.0 and a second reversible hemichrome is formed above pH 9.5–10.0. They are associated with the bis-imidazole low spin hemichrome and with a high spin hemichrome where the weakening of the iron bond to proximal histidine takes place. Addition of cyanide to the metHb solution produces the cyanomet form of the protein which is considerably more stable towards alkaline dissociation and presents a smaller change in quantum yield as a function of pH. Circular dichroism suggests that the formation of hemichromes is not accompanied by significant protein denaturation.

The interaction of dipyridamole (DIP) with reversed micelles (RM) of cetyltrimethylammonium chloride (CTAC) in CDCl3 at different water contents was studied. The position and T1 relaxation of the water peak upon addition of extra water revealed three concentration ranges of CTAC:10 mM (impurity water is mainly dispersed in CDCl3),50-100 mM (water mainly inside the RM), and intermediate range. The resonances of CTAC protons in the polar layer broadened and displaced by up to 0.07 ppm as a function of CTAC concentration and extra water. At 10 mM CTAC, the addition of 40 mM DIP shifted the head group signals to high field by about 0.1 ppm. At high and intermediate CTAC concentrations, four nitroxide spin probes, hydrophobic 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), hydrophilic 4-amine-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPAMINE), and lipophilic 5- and 16-doxyl stearic acids (5- and 16-DSA), underwent partial immobilization. The rotational correlation time of TEMPAMINE (rather than TEMPO, 5-, and 16-DSA) in RM moderately increased upon addition of 1.5-2.0 mM DIP. At an excess of CTAC, only one DIP peak at 3.88 ppm remained measurable, and its selective T1 fell from 0.34 to 0.12 s. The association constant for DIP and CTAC was between 10 and 35 M-1. Thus, DIP incorporates into the polar region of RM influencing packing and dynamics of surfactant head groups. In contrast, in aqueous CTAC micelles, the preferential localization of DIP substituents is inside the nonpolar micelle core, and the binding constant is two orders of magnitude above that for RM.

The interaction of the water soluble meso -tetrakis( p -sulfonato-phenyl)porphyrin (TPPS 4 ) and meso -tetrakis(4- N -methyl-pyridiniumyl)porphyrin (TMPyP) with bovine serum albumin (BSA) in aqueous solutions has been studied by optical absorption, fluorescence and resonance light scattering spectroscopies. The formation of two types of aggregates due to this interaction has been demonstrated: aggregates of the TPPS 4 on the BSA molecule surface and aggregates of BSA molecules around the TPPS 4 molecule. The reduction of integral fluorescence intensity of TPPS 4 due to the porphyrin aggregation and its increase due to the BSA aggregation have been demonstrated. The influence of the porphyrin charge on the aggregation process has been also clearly observed and explained on the basis of known BSA binding properties.