Spectroscopic studies of the met form of the extracellular hemoglobin from Glossoscolex paulistus

Agustinho, Sylvana Cardoso Miguel; Tinto, M H; Imasato, Hidetake; Tominaga, Tânia Toyomi; Perussi, Janice Rodrigues; Tabak, Marcel

doi:10.1016/s0167-4838(96)00127-6

Cited by 36 publications

(45 citation statements)

References 23 publications

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“…All the hexagonal bilayer structures exhibit a relative angular displacement of ϳ15°between the two hexagonal layers, and Lamy et al (1) have proposed that the relative movement of the latter may be involved in the globin-linker-globin interactions responsible for the full cooperativity in the native Hbs. The fact that the oxygenation does not lead to changes in shape of the whole Hb but the oxidation of the hemes renders the protein more susceptible to surfactant-induced dissociation is a strong evidence for changes in the subunit interactions upon oxidation of the heme iron, in agreement with our previous fluorescence studies (9,10). It would be of great interest to monitor the effects of surfactants on the oxygen binding and cooperativity of HbGp to understand more fully all the structural changes and modifications of subunit interactions involved in protein function and described in the present SAXS studies.…”

Section: Discussionsupporting

confidence: 89%

“…Changes in the fluorescence for the intact oligomer as well as for the monomer, trimer, and the (abcd) 2 fractions can be compared, showing quantum yields consistent with data from other hemoproteins, where a significant quenching due to energy transfer from tryptophans to hemes occurs (9,10). The oxidation of the iron with the production of the met-form of hemoglobin leads to a complete dissociation of the oligomer at pH 9 into its constituent small molecular mass subunits (linkers, trimers, and monomers).…”

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confidence: 66%

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Small Angle X-ray Scattering (SAXS) Study of the Extracellular Hemoglobin of Glossoscolex paulistus

Gelamo

Itri

Tabak

2004

Journal of Biological Chemistry

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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 Å 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 (I(0)) decreases a factor of 2.5, indicating protein dissociation. At 20 mM surfactant, further I(0) decrease is observed, with a reduction of radius of gyration to approximately 30 Å 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 Å shows that the giant hemoglobin dissociation into small subunits also occurs at alkaline pH. From the I(0) 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 Å 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.Annelid extracellular hemoglobins are giant molecules that have a characteristic hexagonal bilayer appearence in electron micrographs, high cooperativity of oxygen binding, and low iron and heme contents (1-4). The hemoglobin of the earthworm Lumbricus terrestris (HbLt) 1 is the most extensively studied annelid hemoglobin. It consists of about 180 polypeptide chains in an arrangement of two heme-containing subunits (monomer M and disulfide-bonded trimer T) and non-globin linker subunits with molecular masses in the range 24 -32 kDa. A model of quaternary structure (bracelet model) explains the properties and subunit structure of HbLt: three copies of monomer subunit and three copies of the trimer subunit form a dodecamer subunit, (abcd) 3 of about 200 kDa. Twelve such complexes of heme-containing chains are linked together by 36 heme-deficient linker chains to provide the total mass of about 3500 kDa. The 1/12 subunit of the whole protein is associated then to (abc) 3 d 3 L 3 , where L stands for the linker chains. Since 1996 a significant effort has been devoted by several laboratories to elucidate in more detail the arrangements between the subunits from the structural point of view to better understand the oxygenation of this giant hemoglobin. The three most important contributions in this research are associated with the mass spectrometry determination of the molecular masses of all subunits (3), to the three-dimensional reconstructions at a low resolution of 35 Å obtained by cryoelectron microscopy (1) and which were able to give results o...

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Section: Discussionsupporting

confidence: 89%

supporting

confidence: 66%

Small Angle X-ray Scattering (SAXS) Study of the Extracellular Hemoglobin of Glossoscolex paulistus

Gelamo

Itri

Tabak

2004

Journal of Biological Chemistry

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“…In present work, the level of cohesion in the whole hemoglobin is probably less effective when compared with the ferrous whole hemoglobin. Consequently, ferric whole hemoglobin (met-HbGp) must present a higher tendency to dissociation, in accordance with previous works involving acid and alkaline mediums (Perussi et al, 1990;Agustinho et al, 1996). In this context, it is important to notice the very interesting article of Arndt et al (2003), that discuss the correlation between pI, protein cohesion and oligomeric stability.…”

Section: Discussionsupporting

confidence: 83%

“…It is important to notice that previous works have demonstrated the occurrence of a process, at least partially irreversible, of oligomeric dissociation after pH changes (Moreira et al, 2008a;Santiago et al, 2008Santiago et al, , 2007Ribelatto et al, 2006Ribelatto et al, , 2005Agustinho et al, 1996Agustinho et al, , 1997. Likewise, it has been demonstrated that in alkaline (Moreira et al, 2008b) as well as in acid medium , the heme ferric transitions caused by more drastic pH transitions propitiate a process of secondary structure loss, which is detected by Circular Dichroism (CD), that is intrinsically related to the modifications that occur in the first sphere coordination of the ferric center.…”

Section: Discussionmentioning

confidence: 96%

“…Fluorescence studies of HbGp have shown that the dissociation of its oxy-form at alkaline pH 9.0 is not complete, resulting in (abcd) 2 fraction as the main product, while the met-form shows complete dissociation into trimers, Linkers and monomers (Perussi et al, 1990;Agustinho et al, 1996). This difference between the iron oxidation states is an example of the oligomeric implications associated to the properties of the first sphere of coordination in giant hemoglobins.…”

Section: Introductionmentioning

confidence: 99%

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

Moreira¹,

Poli²,

Lyon³

et al. 2008

Comparative Biochemistry and Physiology Part B: Biochemistry an

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

Alves

Carvalho

et al. 2014

Biopolymers

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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.

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Spectroscopic studies of the met form of the extracellular hemoglobin from Glossoscolex paulistus

Cited by 36 publications

References 23 publications

Small Angle X-ray Scattering (SAXS) Study of the Extracellular Hemoglobin of Glossoscolex paulistus

Small Angle X-ray Scattering (SAXS) Study of the Extracellular Hemoglobin of Glossoscolex paulistus

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

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

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