Structure and Function of Adsorbed Hemoglobin on Silica Nanoparticles: Relationship between the Adsorption Process and the Oxygen Binding Properties

Abstract

The connection between the mechanisms of protein adsorption on nanoparticles and the structural and functional properties of the adsorbed protein often remains unclear. We investigate porcine hemoglobin adsorption on silica nanoparticles, and we analyze the structural and functional modifications of adsorbed hemoglobin by UV-vis spectrophotometry, circular dichroism, and oxygen binding measurement. The structural analysis of adsorbed hemoglobin on silica nanoparticles reveals a significant loss of secondary structure and a preservation of the heme electronic structure. However, adsorbed hemoglobin retains its quaternary structure and exhibits an enhanced oxygen affinity with cooperative binding. Moreover, the structural and functional modifications are fully reversible after complete desorption from silica nanoparticles at pH 8.7. The tunable adsorption and desorption of hemoglobin on SNPs with pH change, and the full control of hemoglobin activity by pH, temperature, and the addition of inorganic phosphate effectors opens the way to an interesting system whereby protein adsorption on nanoparticles can allow for full control over hemoglobin oxygen binding activity. Our results suggest that adsorption of hemoglobin on silica nanoparticles leads to a new structural, functional, and dynamic state with full reversibility in a way that significantly differs from protein denaturation.

Manipulating hemoglobin oxygenation using silica nanoparticles: a novel prospect for artificial oxygen carriers

“…17 However, Hb adsorption on SNPs is driven by multivalent interactions 16,18 and leads to stable and irreversible binding in physiological conditions. 11 Adsorption did not induce any oxidation of HbA, as indicated by spectrophotometry. 11 The oxygen binding curves of HbA hemolysate with SNPs at 37°C show a significant increase of the oxygen affinity of adsorbed HbA ( Figure 1B).…”

“…11 Adsorption did not induce any oxidation of HbA, as indicated by spectrophotometry. 11 The oxygen binding curves of HbA hemolysate with SNPs at 37°C show a significant increase of the oxygen affinity of adsorbed HbA ( Figure 1B). Moreover, the lower oxygen affinity of adsorbed HbA at pH 6 (P 50 5 10.7 6 0.5 mm Hg; supplemental Figure 1) compared with adsorbed HbA at pH 7.4 (P 50 5 7.2 6 0.5 mm Hg) indicates that adsorbed HbA retains its proton exchange capacity after adsorption on SNPs, which in turn controls its affinity (Bohr effect 19 ).…”

“…As DCLHb cannot dissociate into dimers (n 5 2.1 6 0.1), 21 this result further confirms that the modification of the oxygenation properties of adsorbed HbA is not a result of the breakdown of the tetramer but, rather, a result of specific changes of hemoglobin structure and dynamics on the silica surface. 11,18 Interestingly, the Hill plot shows that the highaffinity constant K 1 5 0.68 mm Hg 21 is the same for free and adsorbed HbA, whereas the low-affinity constant is higher for adsorbed HbA (K 2 5 0.08 mm Hg 21 ) compared with free HbA (K 3 5 0.02 mm Hg 21 ) ( Figure 1C). 22 This means that the change in the oxygenation properties is the result of a difference in the initial oxygen binding step (K 2 , K 3 ) between free HbA and adsorbed HbA, whereas the final binding step (K 1 ) is identical.…”

“…Despite differences in the adsorption of pig HbA, human HbA, and mutant HbS, the effect of SNPs on the oxygenation properties of hemoglobin are virtually identical. 11 This could indicate that local structural modifications may not be the key to explain the effect of SNPs on the oxygenation properties of hemoglobin but, rather, large structural or dynamic changes that globally affect adsorbed hemoglobin. Using incoherent neutron scattering experiments, we observed in a previous study that myoglobin adsorption on SNPs results in a large decrease of the protein dynamics.…”

“…11 The adsorption process leads to a modification of the protein structure associated with a remarkable retaining of its oxygen-binding properties and cooperativity. In this report, we investigated whether SNPs could enable one to tune the oxygenation properties of human native and mutant hemoglobin.…”