A DFT analysis of electronic, reactivity, and NLO responses of a reactive orange dye: the role of Hartree-Fock exchange corrections.

An experimental and theoretical study based on DFT/TD-DFT approximations is presented to understand the nature of electronic excitations, reactivity, and nonlinear optical (NLO) properties of reactive orange 16 dye (RO16), an azo chromophore widely used in textile and pharmacological industries. The results show that the solvent has a considerable influence on the electronic properties of the material. According to experimental results, the absorption spectrum is formed by four intense transitions, which have been identified as [Formula: see text] states using TD-DFT calculations. However, the TD-DFT results reveal a weak [Formula: see text] in the low-lying spectral region. Continuum models of solvation indicate that these states suffer from bathochromic (ca. 15 nm) and hypsochromic shifts (ca. 4 nm), respectively. However, the expected blue shift for the absorption [Formula: see text] is only described using long-range or dispersion-corrected DFT methods. RO16 is classified as a strong electrophilic system, with electrophilicity ω > 1.5 eV. Concerning the nucleophilicity parameter (N), from vacuum to solvent, the environment is active and changes the nucleophilic status from strong to moderate nucleophile (2.0 ≤ N ≤ 3.0 eV). The results also suggest that all electrical constants are strongly dependent on long-range and Hartree-Fock exchange contributions, and the absence of these interactions gives results far from reality. In particular, the results for the NLO response show that the chromophore presents a potential application in this field with a low refractive index and first hyperpolarizability ca. 214 times bigger than the value usually reported for urea (ß = 0.34 × 10- 30 esu), which is a standard NLO material. Concerning the solvent effects, the results indicate that the polarizability increases [Formula: see text] esu from gas to solvent while the first hyperpolarizability is calculated as [Formula: see text] esu, ca. 180%, regarding the vacuum. The results suggest RO16 is a potential compound in NLO applications. Graphical Abstract The frontier molecular orbitals, and the inverse relation between the energy-gap (Egap) and the first hyperpolarizability (ß).

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.

Characterization of the apo-form of extracellular hemoglobin of Glossoscolex paulistus (HbGp) and its stability in the presence of urea.

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.

A glassy carbon electrode modified with silver nanoparticles and functionalized multi-walled carbon nanotubes for voltammetric determination of the illicit growth promoter dienestrol in animal urine.

An electroanalytical method for determining dienestrol (DNL) in bovine urine samples is described. A glassy carbon electrode (GCE) modified with silver nanoparticles and functionalized multi-walled carbon nanotubes was used as working sensor. The modified GCE displays substantial analytical improvements including an amplified signal, fast electron transfer kinetics, and resistance to fouling. The irreversible oxidation signal of DNL is pH-dependent. Best reactivity is found at pH 3.0, where a typical anodic peak is recorded at 0.8 V (vs. Ag/AgCl). Square-wave voltammetry revealed a 8.4 nM detection limit (1.9 µg L-1), good repeatability and reproducibility (RSDs <5.0%), and good accuracy (93.2-99.4% recovery from spiked samples). The modified electrode is highly stable even in the presence of ions (Na+ and K+), urea and uric acid. The electrochemical sensor fulfills all requisites to be used as forensic device in surveillance of illegal livestock practices. Graphical abstract Schematic presentation of the construction of a glassy carbon electrode modified with silver nanoparticles and functionalized multi-walled carbon nanotubes. This sensor exhibited a remarkable performance for voltammetric detection of the illicit growth promoter dienestrol in animal urine.

Oligomeric stability of Glossoscolex paulistus hemoglobin as a function of the storage time.

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.

Glossoscolex paulistus hemoglobin with fluorescein isothiocyanate: Steady-state and time-resolved fluorescence.

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.

Ionic surfactants-Glossoscolex paulistus hemoglobin interactions: Characterization of species in the solution.

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.

Metals content of Glossoscolex paulistus extracellular hemoglobin: Its peroxidase activity and the importance of these ions in the protein stability.

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.

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.

Guanidine hydrochloride and urea effects upon thermal stability of Glossoscolex paulistus hemoglobin (HbGp).

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.

Denaturant effects on HbGp hemoglobin as monitored by 8-anilino-1-naphtalene-sulfonic acid (ANS) probe.

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.

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.

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.