Modulatory Effects of Voxelotor and 5-HMF on Hemoglobin Affinity, Heme-Heme Cooperativity and Bohr Effect - Prospecting the Use of These Compounds in Cases of Hypoxemia, Such As in the Acute Respiratory Distress Syndrome (ARDS)
Authors: Lopes Diogo Grangani , Jorge Thaís Traina , Costa Fernando Ferreira , Jorge Susan Elisabeth 
Published: December 2024

The affinity of hemoglobin (Hb) for oxygen can be modulated by a variety of factors such as O2 pressure (PO2), H+/CO2 (Bohr Effect) and the presence of 2,3-bisphosphoglycerate (2,3-BPG). Pathological conditions that cause hypoxemia, such as in the Acute Respiratory Distress Syndrome (ARDS - acute lung inflammation) are characterized by decreased oxygen tension and reduced pH, difficulting Hb-O2 binding. Compounds that increase Hb-O2 affinity may be an alternative for lung diseases by improving the local oxygen uptake by hemoglobin. This study aims to investigate the effects of 5-hydroxymethylfurfural (5-HMF) and Voxelotor on native HbA, focusing on P50 values and Hill constants across a pH 6.5, 7.0 and 7.5 range to evaluate O2 affinity, heme-heme cooperativity and Bohr effect. The experiments were performed using HbA+HbA2 isolated from the whole blood of 3 healthy adults, whom consented to participate in this study and signed the informed consent form. Hb was purified by exclusion chromatography in Sephadex 25G columns (Sigma-Aldrich; Missouri, USA) - in 50 mM Hepes buffer (Sigma-Aldrich; Missouri, USA), and by ion exchange chromatography (Amberlite MB3; Sigma-Aldrich; Missouri, USA) to remove residual molecules and salts. Oxygen Dissociation Curves (ODC) were performed by spectrophotometry (DU 800, Beckman Coulter-Fullerton, EUA), temperature of 25°C, 70uM/Hb concentration and stoichiometry Hb:compounds of 1:1.5 [Hb:5-HMF or Voxelotor]. The experiments were also performed in the presence of Inositol Hexaphosphate (IHP, 1mM- Sigma-Aldrich) as a mimetic of 2,3-BPG. Oxygen affinity was determined by obtaining P50 (partial pressure of O2 required for the saturation of 50% of total Hb). Heme-heme cooperativity was determined by calculating the Hill constant (n), on which n=1 indicates non-cooperative interaction and n=2 indicates a cooperative heme-heme interaction. Voxelotor significantly increased Hb affinity at pH 6.5 (p=0.004), pH 7.0 (p=0.0104) in comparison to stripped Hb A (control) and in all pHs in comparison to 5-HMF (p=0.0448, p=0.0109 and p=0.0047, respectively). In contrast, P50 of 5-HMF-treated samples did not differ from control. In the presence of IHP, significant P50 differences between Voxelotor-treated samples were found across pHs 6.5, 7.0, and 7.5 (p=0.0038, p=0.043, p=0.0007), demonstrating a significant shift on the protein allosterism and a positive Bohr effect. Additionally, a significant difference between 5-HMF and Voxelotor was noted at pH 7.0 (p=0.0275). In regard to the heme-heme cooperativity, Voxelotor displayed n values below 1 for stripped HbA, indicating non-cooperative binding event, similarly to myoglobins. Significant differences in Hill constant values were observed between Voxelotor and control at pHs 6.5 (p=0.0114) and 7.0 (p=0.0331), and between Voxelotor and 5-HMF at all pHs (p=0.0047, p=0.026 and p=0.0248, respectively). In the presence of IHP, no significant differences in heme-heme cooperativity were found among the groups, and n values were approximately to 2, indicating that Hb-O2 binding remained cooperative and sensible to organic phosphates ligation, such as IHP. Voxelotor was designed to increase HbS affinity for oxygen by favoring Relaxed conformation (R state) and preventing its polymerization. Despite being designed for Sickle Cell Disease treatment, this compound may also offer new and undescribed effects that could be explored for other purposes. Our study suggests that Voxelotor might increase O2 affinity of HbA by stabilizing R conformation, reducing drastically the heme-heme cooperativity in the stripped state, demonstrating a very stable ligation between HbA and Voxelotor. However, in the presence of organic phosphates, as IHP, oxygen affinity of HbA remained increased while heme-heme cooperativity was reestablished and Bohr Effect was significantly present. In conclusion, Voxelotor significantly enhances Hb-O2 affinity at different pHs, particularly in acidic and alkaline environments, modulating Hb allosterism according to the presence of H+ and phosphates. These findings indicate some unexplored properties of Voxelotor into Hb-O2 binding dynamics which should be further investigated and which might be an alternative for pathological conditions of low pulmonary uptake in association with acidosis, such as in ARDS.