Glossoscolex paulistus Extracellular Hemoglobin (HbGp) Oligomeric Dissociation upon Interaction with Sodium Dodecyl Sulfate: Isothermal Titration Calorimetry (ITC) Fernanda Rosa Alves, 1 Francisco Adriano O. Carvalho, 1 Jos  e Wilson P. Carvalho, 1,2 Marcel Tabak 1 1 Instituto de Qu  ımica de S~ ao Carlos, Departamento de Qu  ımica e F  ısica Molecular, Universidade de S~ ao Paulo, S~ ao Carlos, SP, Brazil 2 Universidade do Estado de Mato Grosso, Campus Rene Barbour, Barra do Bugres, MT, Brazil Received 19 March 2014; revised 6 May 2014; accepted 13 May 2014 Published online 19 May 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22506 ABSTRACT: 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 dissoci- ation process is favored at lower protein concentrations. The temperature effect does not influence significantly the interaction of SDS with the hemoglobin, due to the simi- larities presented by the critical aggregation concentration (cac) and critical micelle concentration (cmc 0 ) 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 0 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. V C 2014 Wiley Periodicals, Inc. Biopolymers 101: 1065–1076, 2014. Keywords: oxy-HbGp; SDS; ITC; DLS; dissociation; pyrene fluorescence emission This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by email- ing the Biopolymers editorial office at biopolymers@wiley.com INTRODUCTION T he extracellular hemoglobin of Glossoscolex paulistus (HbGp) has a hexagonal bilayer structure, with a molecular mass of 3.6 MDa, and a high oligomeric sta- bility. 1 Its oligomeric structure is composed by 144 heme-containing subunits, and 36 additional chains, lacking the heme group, named linkers, similar to the ortholo- gous Lumbricus terrestris hemoglobin (HbLt). 2 Extracellular hemoglobins have been studied in recent years as a new prom- ising blood substitute. 3 Studies on the interaction of globular proteins with surfac- tants, particularly with SDS, have been performed to Correspondence to: Fernanda Rosa Alves; e-mail: frosaalves@gmail.com Contract grant sponsor: FAPESP, Brazil Contract grant number: 2013/09349-6 and 2011/09863-6 Contract grant sponsor: CNPq, Brazil Contract grant sponsor: CAPES, Brazil Contract grant sponsor: FAPESP Contract grant number: 2013/09829-8 Contract grant sponsor: CNPq V C 2014 Wiley Periodicals, Inc. Biopolymers Volume 101 / Number 10 1065