A combination of dynamic light scattering and polarized resonance Raman scattering applied in the study of Arenicola Marina extracellular hemoglobin

Abstract

Arenicola Marina extracellular hemoglobin (Hbl Hb) is considered to be a promising candidate as a blood substitute. To entangle some of the properties of extracellular giant hexagonal bilayer hemoglobin (Hbl Hb) of Arenicola Marina, we combined polarized resonance Raman scattering (532 nm excitation) with dynamic light scattering (DLS) (632.8 nm). An analysis of the depolarization ratio of selected a(2g) skeletal modes of the heme in native Hbl Hb and porcine Hb, shows that the distortion of the heme group away from its ideal fourfold symmetry is much smaller for heme groups bound in the Hbl Hb than for heme groups bound in porcine Hb. Using DLS, the average hydrodynamic diameter (<d(h)>) of Hbl Hb was measured at pH = 5, 7, 8, 9, and 10. At pH = 5 to 7, the Hbl Hb was found in its native form with <d(h)> equal to 24.2 nm, while at pH = 8 and 9, a dissociation process starts to take place resulting in <d(h)> = 9 nm. At pH = 10, only large aggregates of fragmented Hbl Hb with <d(h)> larger than 1000 nm was detected, however, a comparison of the DLS results with the polarized resonance Raman scattering (RRS) revealed that the coupling between the fragments did not involve direct interaction between the heme groups, but also that the local heme environment seems to be comparable in the aggregates and in the native Hbl Hb. By comparing the unpolarized RRS results obtained for erythrocytes (RBC) with those for Hbl Hb, led us to the important conclusion that Hbl Hb is much easier photolyzed than porcine RBC.

Thermal stability of extracellular hemoglobin of Rhinodrilus alatus (HbRa): DLS and SAXS studies

“…Arenicola marina hemoglobin at 20 °C also presents similar D H values to oxy-HbRa in the pH range of 5.0-7.0. However, at pH 8.0, it is partially dissociated with a hydrodynamic diameter of 15.5 nm, and, at pH 9.0, HbAm is completely dissociated, displaying a D H of 9 nm (Jernshøj et al 2013). Thus, our current study indicates that oxy-HbRa has a pH stability similar to that of oxy-HbGp, being more stable than HbAm.…”

“…HbLt and HbGp are the most extensively studied giant hemoglobins (Bachega et al 2011;Carvalho et al 2012Carvalho et al , 2013Carvalho et al , 2015Krebs et al 1998;Oliveira et al 2007;Poli et al 2005;Royer et al 2005Royer et al , 2006Rousselot et al 2006;Santiago et al 2008Santiago et al , 2010a. Another widely studied giant protein is Arenicola marina hemoglobin (HbAm), orthologous of HbGp and HbLt, presenting quite similar hierarchic structural organization and biophysical properties (Zal and Rousselot 2014;Jernshøj et al 2013). These hemoglobins present high cooperativity in oxygen binding and high resistance to the oxidation process as compared to human hemoglobin (Poli et al 2005;Santiago et al 2008).…”

Direct comparison of oligochaete erythrocruorins as potential blood substitutes

“…Therefore, it appears that EvEc, EeEc, and EhEc may have dissociated during tangential flow filtration (as was visibly observed during purification of AgEc) or it could reflect a structural instability of these Ecs at pH 7.4. Indeed, many other Ecs have been shown to dissociate at alkaline pH …”

Metals content of Glossoscolex paulistus extracellular hemoglobin: Its peroxidase activity and the importance of these ions in the protein stability

“…Another strong evidence for the absence of amorphous aggregates at pH 8.0 is the low polydispersity index of 0.18 at high temperatures, as compared to 0.27-0.50 at pH 7.0. Previous studies by several techniques have observed that the oligomeric structure of the giant extracellular hemoglobins, such as, Glossoscolex paulistus and Arenicola marina, dissociate in the alkaline pH, and smaller subunits are noted in the solution [46,52]. The extent of dissociation depends on the protein concentration and the oxidation ion state of heme group.…”