Trehalose Glass-Facilitated Thermal Reduction of Metmyoglobin and Methemoglobin

Abstract

The reduction of ferric derivatives of hemeproteins in solution typically requires moderate to strong reducing agents. Reducing sugars are not adequate to reduce ferric myoglobins or hemoglobins under solution conditions favorable to protein stability. We find that embedding aquo-met derivatives of horse myoglobin and human adult hemoglobin in a glucose-doped glassy matrix derived from trehalose facilitates an efficient thermally initiated reduction that yields a five-coordinate high-spin ferrous heme. The trehalose glass plays a central role by stabilizing the reduction-prone bis-histidine heme (hemichrome) intermediate under the high-temperature conditions that favor the open reducing form of glucose. Due to glass-imposed limitations on conformational reorganization, this process has clear applications in biophysics where it can be used to generate nonequilibrium ferrous derivatives having the initial conformation of the aquo-met derivative. Since the glassy matrix can be redissolved to release the embedded protein, this technique is not only a basis for a relatively benign method of reducing hemoglobin-based blood substitutes that have undergone autoxidation during storage but may also be a way to reactivate stored proteins that have undergone oxidation.

Sugar-derived Glasses Support Thermal and Photo-initiated Electron Transfer Processes over Macroscopic Distances

“…Although the spectra show no indication of reduction, they do demonstrate the progressive formation of the oxidized six-coordinate derivative of the two proteins known as the hemichrome (18). Hemichrome formation, often associated with significant osmotic stress (13,19), is the result of the imidazole side chain of the distal histidine replacing water as the sixth ligand. Dissolving the glass in aqueous buffer resulted in the full recovery of the standard spectra of the initial aquomet derivatives.…”

“…Redox reactions, although influenced by protein dynamics, can still occur even when the proteins are immobilized. In a preliminary study (13) it was shown that doping trehalose glasses containing either methemoglobin or metmyoglobin with glucose (a reducing sugar) resulted in samples that undergo facile thermal reduction. In the present work we demonstrate not only that such processes are likely to be general but that these glass-facilitated redox reactions can occur over surprisingly large macroscopic distances.…”

Proteins in Saccharides Matrices and the Trehalose Peculiarity: Biochemical and Biophysical Properties

“…To this aim, a series of studies on the properties of electron transfer in sugar based glassy matrices have been performed. In particular, by doping trehalose based solid matrices with the reducing sugar glucose, it was possible to reduce metmyoglobin to deoxymyoglobin (or met-Hb to deoxy-Hb) by thermal inducing the electron transfer from the doping glucose molecules to the iron Fe 3+ ions contained in the active site of the embedded proteins [238]. The same process could be obtained using two distinct trehalose sandwich layers, one containing met-Hb without any glucose, the other doped with glucose (or other reducing monosaccharides such as fructose or tagatose) but free of any protein; if the two glassy sandwiches are put in contact and heated at 75 °C for 45 min, full reduction of met-Hb to deoxy-Hb is obtained [239].…”

Comparison between two different hemichromes of hemoglobins (HbA and HbS) induced by n-dodecyl trimethylammonium bromide: Chemometric study

“…This surface activity difference is almost certainly the origin of the greater susceptibility of HbS to precipitation, presumably as a result of surface denaturation [8][9][10]. Now, we have understood hemoglobin structure, the mechanisms of its oxygen transport function and its electron transfer on electrode surface [11][12][13][14][15], but the research on its interaction with other molecules are almost limited to a few biological molecules, such as bacterial endotoxin [16,17], hydroxyurea [18] and trehalose [19], with the conclusion that some biological molecules can convert oxyhemoglobin (oxyHb) to methemoglobin (metHb) and hemichrome, while some others may slow or reverse the auto-oxidation process of Hb [16,19]. Since hemichrome accumulation in red cells is typical of some blood diseases [20,21] and aging of the erythrocyte [22], the interrelated study of the conversion between metHb and hemichrome will be worthwhile not only in theory but also in practice.…”