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.
Instituto de Química de São Carlos, Departamento de Química e Física Molecular, Universidade de São Paulo, São Carlos, SP, Brazil.
Fernanda Rosa
FR
Alves
Francisco Adriano O
FA
Carvalho
José Wilson P
JW
Carvalho
Marcel
M
Tabak
D000818:Animals; D002151:Calorimetry; D000067493:Dynamic Light Scattering; D005110:Extracellular Space / Q000737:chemistry*; D006454:Hemoglobins / Q000378:metabolism*; D057446:Hydrodynamics; D009835:Oligochaeta / Q000737:chemistry*; D055503:Protein Multimerization; D011721:Pyrenes / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature; D017186:Titrimetry
D006454:Hemoglobins; D011721:Pyrenes; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; C030984:pyrene
DLS; ITC; SDS; dissociation; oxy-HbGp; pyrene fluorescence emission