Optical absorption monitors the hemoglobin autoxidation while DLS gives information regarding particle size changes in the process of protein dissociation. Kinetic studies were also performed using ultraviolet-visible absorption at the Soret band. Dissociation temperatures were lower at higher pH. Melting curves for HbGp showed oligomeric dissociation and protein denaturation as a function of pH. Protein temperature stability was also pH-dependent. Dissociation rate constants progressively increase at higher pH, becoming, at pH 10.5, not detectable by DLS. At pH 9.0, the dissociation kinetics is slow, taking a minimum of 24 h to be completed. Gel filtration chromatography was used to show unequivocally the oligomeric dissociation observed at alkaline pH. The decrease in D h suggests a complete hemoglobin dissociation. A more alkaline pH induced an irreversible dissociation process, resulting in a smaller D h of 10 ± 1 nm. In the pH range 6.0–8.0, HbGp is stable and has a monodisperse size distribution with a z-average hydrodynamic diameter ( D h) of 27 ± 1 nm. HbGp samples were studied by dynamic light scattering (DLS). This and other proteins of the same family are useful model systems for developing blood substitutes due to their extracellular nature, large size, and resistance to oxidation. The extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted of subunits containing heme groups, monomers and trimers, and nonheme structures, called linkers, and the whole protein has a minimum molecular mass near 3.1 × 10 6 Da.
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