Giant Extracellular Hemoglobin Research Articles

Structure-Function Relationships of Oxygen Transport Proteins in Marine Invertebrates Enduring Higher Temperatures and Deoxygenation.

AbstractPredictions for climate change-to lesser and greater extents-reveal a common scenario in which marine waters are characterized by a deadly trio of stressors: higher temperatures, lower oxygen levels, and acidification. Ectothermic taxa that inhabit coastal waters, such as shellfish, are vulnerable to rapid and prolonged environmental disturbances, such as heatwaves, pollution-induced eutrophication, and dysoxia. Oxygen transport capacity of the hemolymph (blood equivalent) is considered the proximal driver of thermotolerance and respiration in many invertebrates. Moreover, maintaining homeostasis under environmental duress is inextricably linked to the activities of the hemolymph-based oxygen transport or binding proteins. Several protein groups fulfill this role in marine invertebrates: copper-based extracellular hemocyanins, iron-based intracellular hemoglobins and hemerythrins, and giant extracellular hemoglobins. In this brief text, we revisit the distribution and multifunctional properties of oxygen transport proteins, notably hemocyanins, in the context of climate change, and the consequent physiological reprogramming of marine invertebrates.

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 × 10‒7mol 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.

Initial biophysical characterization of Amynthas gracilis giant extracellular hemoglobin (HbAg).

The aim of the present work was the biophysical characterization of the Amynthas gracilis hemoglobin (HbAg). The oxy-HbAg optical absorption data, with Soret and Q bands centered at 415, 540 and 575nm, were stable and unchanged at pH 7.0. An increase in pH promotes decrease in the intensity in the optical absorption bands, suggesting an oligomeric dissociation and partial oxidation. Identical stability at pH 7.0 was observed in DLS results that presented a hydrodynamic diameter of 28nm, characteristic of the whole oligomer. DLS shows that HbAg undergoes oligomeric dissociation and an aggregation/denaturation process that corroborates spectroscopic data. Our results showed that the monomer d presents four isoforms with molecular mass (MM) ranging from 16,244 to 16,855Da; the trimer subunit presents two isoforms, (abc)1 and (abc)2, with MM of 51,415 ± 20Da and 51,610 ± 14Da, respectively, and a less intense species, at 67,793Da, assigned to the tetramer abcd. Monomeric chains a, obtained from reduction of the disulfide-bonded trimer abc, present four isoforms with MM 17,015Da, 17,061Da, 17,138Da and 17,259Da. DLS and LSI revealed an isoeletric point (pI) of oxy-HbAg of 6.0 ± 0.3 and 5.5, respectively. Data analysis by IEF-SDS-PAGE revealed that the pI of oxy-HbAg is 6.11, correlating with DLS and LSI data. These studies indicate that oxy-HbAg is very stable, at pH 7.0, and has differing properties from orthologous giant hemoglobins.

Interaction of Glossoscolex paulistus extracellular hemoglobin with hydrogen peroxide: Formation and decay of ferryl-HbGp

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±0.5M−1s−1, at pH7.0 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 pH7.0, 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.

Interaction of cationic dodecyl-trimethyl-ammonium bromide with oxy-HbGp by isothermal titration and differential scanning calorimetric studies: Effect of proximity of isoelectric point.

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.

Purification of diverse hemoglobins by metal salt precipitation

Although donated blood is the preferred material for transfusion, its limited availability and stringent storage requirements have motivated the development of blood substitutes. The giant extracellular hemoglobin (aka erythrocruorin) of the earthworm Lumbricus terrestris (LtEc) has shown promise as a blood substitute, but an efficient purification method for LtEc must be developed to meet the potential large demand for blood substitutes. In this work, an optimized purification process that uses divalent and trivalent metal salts to selectively precipitate human, earthworm, and bloodworm hemoglobin (HbA, LtEc, and GdHb, respectively) from crude solutions was developed. Although several metal ions were able to selectively precipitate LtEc, Zn2+ and Ni2+ provided the lowest heme oxidation and highest overall yield of LtEc. In contrast, Zn2+ was the only metal ion that completely precipitated HbA and GdHb. Polyacrylamide gel electrophoresis (PAGE) analysis shows that metal precipitation removes several impurities to provide highly pure hemoglobin samples. Heme oxidation levels were relatively low for Zn2+-purified HbA and LtEc (2.4±1.3% and 5.3±2.1%, respectively), but slightly higher for Ni2+-purified LtEc (8.4±1.2%). The oxygen affinity and cooperativity of the precipitated samples are also identical to samples purified with tangential flow filtration (TFF) alone, indicating the metal precipitation does not significantly affect the function of the hemoglobins. Overall, these results show that hemoglobins from several different species can be highly purified using a combination of metal (Zn2+) precipitation and tangential flow filtration.

Characterization of Amynthas Gracilis Hemoglobin (HbAg) and its Subunits by AUC and MALDI-TOF-MS

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

Evaluating the Capacity to Generate and Preserve Nitric Oxide Bioactivity in Highly Purified Earthworm Erythrocruorin: A GIANT POLYMERIC HEMOGLOBIN WITH POTENTIAL BLOOD SUBSTITUTE PROPERTIES

The giant extracellular hemoglobin (erythrocruorin) from the earth worm (Lumbricus terrestris) has shown promise as a potential hemoglobin-based oxygen carrier (HBOC) in in vivo animal studies. An important beneficial characteristic of this hemoglobin (LtHb) is the large number of heme-based oxygen transport sites that helps overcome issues of osmotic stress when attempting to provide enough material for efficient oxygen delivery. A potentially important additional property is the capacity of the HBOC either to generate nitric oxide (NO) or to preserve NO bioactivity to compensate for decreased levels of NO in the circulation. The present study compares the NO-generating and NO bioactivity-preserving capability of LtHb with that of human adult hemoglobin (HbA) through several reactions including the nitrite reductase, reductive nitrosylation, and still controversial nitrite anhydrase reactions. An assignment of a heme-bound dinitrogen trioxide as the stable intermediate associated with the nitrite anhydrase reaction in both LtHb and HbA is supported based on functional and EPR spectroscopic studies. The role of the redox potential as a factor contributing to the NO-generating activity of these two proteins is evaluated. The results show that LtHb undergoes the same reactions as HbA and that the reduced efficacy for these reactions for LtHb relative to HbA is consistent with the much higher redox potential of LtHb. Evidence of functional heterogeneity in LtHb is explained in terms of the large difference in the redox potential of the isolated subunits.

Glossoscolex paulistus extracellular hemoglobin (HbGp) oligomeric dissociation upon interaction with sodium dodecyl sulfate: Isothermal titration calorimetry (ITC).

Annelid erythrocruorins are respiratory proteins with high cooperativity and low autoxidation rates. The giant extracellular hemoglobin of the earthworm, Glossoscolex paulistus (HbGp), has a molecular mass of 3.6 MDa. In this work, isothermal titration calorimetry (ITC), together with DLS and fluorescence emission have been used to investigate the interaction of SDS with the HbGp in the oxy-form, at pH 7.0. Our ITC and DLS results show that addition of SDS induces oxy-HbGp oligomeric dissociation, while a small amount of protein aggregation is observed only by DLS. Moreover, the oligomeric dissociation process is favored at lower protein concentrations. The temperature effect does not influence significantly the interaction of SDS with the hemoglobin, due to the similarities presented by the critical aggregation concentration (cac) and critical micelle concentration (cmc') for the mixtures. The increase of oxy-HbGp concentration leads to a slight variation of the cac values for the SDS-oxy-HbGp mixture, attributed mainly to the noncooperative electrostatic binding of surfactant to protein. However, the cmc' values increase considerably, associated to a more cooperative hydrophobic binding. Complementary pyrene fluorescence emission studies show formation of pre-micellar structures of the mixture already at lower SDS concentrations. This study opens the possibility of the evaluation of the surfactant effect on the hemoglobin stability by ITC, which is made for the first time with this extracellular hemoglobin.

In vivo biodistribution and oxygenation potential of a new generation of oxygen carrier

Natural giant extracellular hemoglobins (Hbs) from polychaete annelids are currently actively investigated as promising oxygen carriers. Their powerful oxygenating ability and their safety have been demonstrated in preclinical studies, motivating their development for therapeutic and industrial applications. HEMARINA-M101 (M101) is derived from the marine invertebrate Arenicola marina. It is formulated as a manufactured product designated HEMOXYCarrier® (HEMARINA SA, France). The aim of the present study was to unveil the fate of M101 after a single intravenous (i.v.) injection in mice. For this purpose, M101 was tagged with a far-red fluorescent dye. Repeated non-invasive fluorescent imaging revealed a rapid diffusion of M101 in the whole body of animals, reaching all the examined organs such as brain, liver, lungs and ovaries. Functional M101 was circulating in bloodstream for several hours, without inducing any obvious side-effects. Last, a single i.v. injection of M101 in mice bearing human-derived subcutaneous tumors demonstrated the ability of this Hb to reduce hypoxia in poorly vascularized tissues, thus supporting the biological relevance of M101 oxygen release to vertebrate tissues. Altogether, these results further encourage the development of M101 as an oxygen carrying therapeutic.

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 1.0mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204kDa for the dodecamer (abcd)3, 5.5S and 69kDa for the tetramer (abcd), 4.1S and 52kDa for the trimer (abc) and 2.0 S and 17kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0mol/L of denaturant, for oxy-HbGp, and above 6.0mol/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 4.0mol/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.

Microcirculation and NO-CO Studies of a Natural Extracellular Hemoglobin Developed for an Oxygen Therapeutic Carrier

Extracellular soluble hemoglobins (Hbs) have long been studied for their possible use as safe and effective alternatives to blood transfusion. While remarkable progress has been made in the use of cell-free Hb as artificial oxygen carrier, significant problems remain, including susceptibility to oxidative inactivation and propensity to induce vasoconstriction. Hemarina-M101 is a natural giant extracellular hemoglobin (3600 kDa) derived from marine invertebrate (polychaete annelid). Hemarina-M101 is a biopolymer composed of 156 globins and 44 non-globin linker chains and formulated in a product called HEMOXYCarrier®. Prior work has shown Hemarina-M101 to possess unique anti-oxidant activity and a high oxygen affinity. Topload experiment with this product into rats did not revealed any effect on heart rate (HR) and mean arterial pressure (MAP). A pilot study with the hamster dorsal skinfold window chamber model showed absence of microvascular vasoconstriction and no significant impact on mean arterial blood pressure. In vitro nitric oxide (NO) and carbon monoxide (CO) reaction kinetics measurements show that Hemarina-M101 has different binding rates as compared to human Hb. These results revealed for the first time that the presence of this marine hemoglobin appears to have no vasoactivity at the microvascular level in comparison to others hemoglobin based oxygen carriers (HBOCs) developed so far and merits further investigation.

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

Further characterization of hemoglobin of Glossoscolex paulistus (HbGp) subunits was performed based on SDS-PAGE, size exclusion chromatography (SEC) and MALDI-TOF-MS analysis. SDS-PAGE has shown a total of four linker chains, two quite intense and two of lower intensity. HbGp fractions (I–VI), obtained by size exclusion chromatography (SEC), from oligomeric dissociation at alkaline pH 9.6, were monitored. Fraction I is identical to the whole protein. The monomeric chains c, obtained from the trimer abc reduction, present four isoforms with MM 17,336Da, 17,414Da, 17,546Da and 17,620Da. Furthermore, the trimer subunit presents two isoforms, T1 and T2, with MM 51,200±60 and 51,985±50Da, respectively. Based on SDS-PAGE, the linker chains seem to be distributed along the different fractions of the SEC chromatogram, appearing along the peaks corresponding to fractions I–V. The fraction IV contains, predominantly, trimers with some linkers contamination. The strong interaction of linker chains L with the trimers abc, makes it difficult to obtain these subunits in pure form. The monomer d in fraction VI appears to be quite pure, in agreement with previous studies.

Autoxidation of giant extracellular hemoglobin of Glossoscolex paulistus: Molecular mechanism and oligomeric implications

Giant extracellular hemoglobins present high redox stability due to their supramolecular architecture, high number of polypeptide chains and great compaction of protein subunits. The oligomeric assembly and the changes in the polypeptidic structure can influence the autoxidation rate of the heme proteins, being that different nucleophiles can act in this process due to pH alterations. In the present work, we have studied the autoxidation rate of whole Glossoscolex paulistus (HbGp) giant extracellular hemoglobin, as well as the autoxidation rate of the isolated d monomer of HbGp studied regarding pH variations. The kinetic decay behavior is dependent on pH, presenting mono-exponential or bi-exponential character, depending on the oligomeric state of the protein. Thus, the oligomeric dissociation in specific pH values demonstrated a bi-exponential kinetic decay. A mono-exponential kinetic behavior was verified in the pH range of 5.9–7.3, which is assigned to the native whole protein. In alkaline medium, the presence of hydroxide ions leads the autoxidation of whole hemoglobin to a complex behavior, which is described by the combination of two first-order kinetics. The slow process occurs due to the d monomer autoxidation. At pH 7.0, the kinetic is mono-exponential, indicating a highly conserved oligomeric structure. In acid medium, the proton-catalyzed autoxidation occurs both on the whole hemoglobin and in the d monomer. It has been found that proximal and distal histidines develop determinant roles regarding the autoxidation rate, being that the distal histidine controls the contact of ligands with the ferrous center through a very interesting “swinging door” mechanism. Despite the significant sensitivity of the distal histidine to the presence of protons, water molecules and anions, the influence of chemical changes around the heme, such as pH changes, is much more effective in hemoproteins without this amino acid as distal residue. This fact denotes the ability of HbGp to adapt to environmental disturbances caused by the presence of the distal histidine, which is responsible for the great redox and oligomeric stabilities encountered in HbGp.

Hemoglobina extracelular gigante de Glossoscolex paulistus: um extraordinário sistema supramolecular hemoproteico

Giant extracellular hemoglobins are considered the summit of complexity in systems that carry oxygen, constituting an extraordinary model system to the study of hemoproteins. This class includes the hemoglobin of the annelid Glossoscolex paulistus that presents high cooperativity, great oligomeric and redox stabilities and ability of oligomeric reassociation. These properties have motivated evaluations about its utilization as prototype of artificial blood and biosensor. Kinetic studies involving autoxidation and detailed spectroscopic characterizations of its ferrous and ferric species have propitiated information about the structure-activity relationship of this macromolecule. The present review analyzes several biochemical issues, evaluating the state-of-art of this subject.

Molecular masses and sedimentation coefficients of extracellular hemoglobin of Glossoscolex paulistus: Alkaline oligomeric dissociation

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) has a molecular mass ( M) of 3600 ± 100 kDa and a standard sedimentation coefficient ( s 20 , w 0 ) of 58 S, estimated by analytical ultracentrifugation (AUC). In the present work, further AUC studies were developed for HbGp, at pH 10.0, which favors oligomeric dissociation into lower M species. The HbGp oligomer is formed by globin chains a , b , c and d plus the linker chains. The pure monomeric fraction, subunit d , and HbGp at pH 10.0, in the presence of β-mercaptoethanol, were also studied. Our results indicate that for samples of pure subunit d , besides the monomeric species with s 20 , w 0 of 2.0 S, formation of dimer of subunit d is observed with s 20 , w 0 of around 2.9 S. For the whole HbGp at pH 10.0 contributions from monomers, trimers and linkers are observed. No contribution from 58 S species was observed for the sample of oxy-HbGp at pH 10.0, showing its complete dissociation. For cyanomet-HbGp form a contribution of 17% is observed for the un-dissociated oligomer, consistent with data from other techniques that show the cyanomet-form is more stable as compared to oxy-HbGp. Masses of HbGp subunits, especially trimer abc and monomeric chains a , b , c and d , were also estimated from sedimentation equilibrium data, and are in agreement with the results from MALDI-TOF-MS.

Ligand changes in ferric species of the giant extracellular hemoglobin ofGlossoscolex paulistusas function of pH: correlations between redox, spectroscopic and oligomeric properties and general implications with different hemoproteins

The present review is focused on the relationship between oligomeric and heme properties of HbGp, emphasizing the characteristics that can be generalized to other hemoproteins. This study represents the state-of-the-art with respect to the approaches for investigating giant extracellular hemoglobins as well as the correlation between oligomeric assembly alterations and their consequent changes in the first coordination sphere. A wide introduction focused on the properties of this hemoglobin is developed. Indeed, this hemoprotein is considered an interesting prototype of blood substitute and biosensor due to its peculiar properties, such as resistance to autoxidation and oligomeric stability. Previous studies by our group employing UV-vis, EPR and CD spectroscopies have been revised in a complete approach, in agreement with recent and relevant data from the literature. In fact, a consistent and inter-related spectroscopic study is described propitiating a wide assignment of "fingerprint" peaks found in the techniques evaluated in this paper. This review furnishes physicochemical information regarding the identification of ferric heme species of hemoproteins and metallic complexes through their spectroscopic bands. This effort at the attribution of UV-vis, EPR and CD peaks is not restricted to HbGp, and includes a comparative analysis of several hemoproteins involving relevant implications regarding several types of iron-porphyrin systems.

On the molecular mass of the extracellular hemoglobin of Glossoscolex paulistus: Analytical ultracentrifugation reexamination

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by subunits containing heme groups with molecular masses ( M) in the range of 15 to 19 kDa, monomers of 16 kDa ( d), and trimers of 51 to 52 kDa ( abc) linked by nonheme structures named linkers of 24 to 32 kDa ( L). HbGp is homologous to Lumbricus terrestris hemoglobin (HbLt). Several reports propose M of HbLt in the range of 3.6 to 4.4 MDa. Based on subunits M determined by mass spectrometry and assuming HbGp stoichiometry of 12( abcd) 3 L 3 (Vinogradov model) plus 144 heme groups, a value of M for HbGp oligomer of 3560 kDa can be predicted. This value is nearly 500 kDa higher than the unique HbGp M value reported in the literature. In the current work, sedimentation velocity analytical ultracentrifugation (AUC) experiments were performed to obtain M for HbGp in oxy and cyano-met forms. s 0 20,w values of 58.1 ± 0.2 S and 59.6 ± 0.2 S, respectively, for the two oxidation forms were obtained. The ratio between sedimentation and diffusion coefficients supplied values for M of approximately 3600 ± 100 and 3700 ± 100 kDa for oxy and cyano-met HbGp forms, respectively. An independent determination of the partial specific volume, V bar, for HbGp was performed based on density measurements, providing a value of 0.764 ± 0.008, in excellent agreement with the estimates from SEDFIT software. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Therefore, HbGp possesses M very close to that of HbLt, suggesting an oligomeric assembly in agreement with the Vinogradov model.

Ferric species of the giant extracellular hemoglobin of Glossoscolex paulistus as function of pH: An EPR study on the irreversibility of the heme transitions

The present article is focused on the transitions of ferric heme species of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) induced by successive alterations in pH, involving alkaline and acid mediums. Electron paramagnetic resonance (EPR) is the spectroscopy used to evaluate the transitions that occur in the first coordination sphere of ferric ion as a consequence of ligand changes in a wide range of pH, since this tool is very sensitive to slight changes that occur in the heme pocket of paramagnetic species. This approach is adequate to obtain information regarding the reversibility/irreversibility that involves the heme transitions induced by pH, since the degree of reversibility is associated to the intensity of the changes that occur in the spatial configuration of the polypeptide chains, which is clearly associated to the first coordination sphere. The results demonstrate a significant degree of irreversibility of heme transitions, since the final species, which do not present any change after 6 h of its respective formations, are quite different of the initial species. The results denote that the more stable species are the bis-histidine (hemichrome) and pentacoordinate species, due to the properties of their ligands and to the mechanical influence of the respective subunits. EPR spectra allow to distinguish the types of hemichrome species, depending on the reciprocal orientation between the histidine axial ligands, in agreement with Walker's Classification [Walker, F.A., 1999. Magnetic spectroscopic (EPR, ESEEM, Mössbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins. Coord. Chem. Rev. 185–186, 471–534]. However, these transitions are not completed, i.e., the appearance of a determined species does not mean the total consumption of its precursor species, implying the coexistence of several types of species, depending on pH. Furthermore, it is possible to conclude that a “pure” EPR spectrum of aquomet ferric species is an important indicator of a high level of conservation referent to the “native” configuration of whole hemoglobin, which is only encountered at pH 7.0. The results allow to infer important physico-chemical properties as well as to evaluate aspects of the structure–activity relationship of this hemoprotein, furnishing information with respect to the denaturation mechanism induced by drastic changes in pH. These data are very useful since HbGp has been proposed as prototype of substitute of blood, thus requiring wide knowledge about its structural and chemical properties.