Abstract
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.
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Acknowledgments
The authors are grateful to the National Synchrotron Light Laboratory (LNLS), Campinas, Brazil, for making available the SAXS beam line used in these studies. Thanks are also due to the Brazilian agencies FAPESP, CNPq, and CAPES for partial financial support. F.A.O. Carvalho is a recipient of a post-doctoral grant from FAPESP (2013/09829-8). J.W.P. Carvalho is grateful for a post-doctoral grant from FAPESP (2013/09349-6) and M. Tabak is grateful to CNPq for a research grant. The authors are indebted to Prof. Dr. Carlos Ernesto Salmon Garrido, from Faculdade de Filosofia Ciências e Letras de Ribeirão Preto (FFCLRP), Universidade de São Paulo, for help with the analysis of linear combination of p(r) functions. The authors are also indebted to Dr. José Fernando Ruggiero Bachega for the help with the production of HbGp fragments from crystallographic structure used in the p(r) simulations as well as in Scheme 1. Thanks are also due to Mr. Ezer Biazin for efficient support in sample preparations.
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Carvalho, J.W.P., Carvalho, F.A.O., Santiago, P.S. et al. Thermal stability of extracellular hemoglobin of Rhinodrilus alatus (HbRa): DLS and SAXS studies. Eur Biophys J 45, 549–563 (2016). https://doi.org/10.1007/s00249-016-1121-6
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DOI: https://doi.org/10.1007/s00249-016-1121-6