The Cys 201 residue in one subunit and the Cys 317 residue of the paired subunit are at close distance and can easily form intermolecular disulfide bonds following moderate oxidative stress [36, 43] and following the binding of hemichromes [18, 35]

The Cys 201 residue in one subunit and the Cys 317 residue of the paired subunit are at close distance and can easily form intermolecular disulfide bonds following moderate oxidative stress [36, 43] and following the binding of hemichromes [18, 35]. sensor regulating its own phosphorylation and that hemichromes leading to the protracted phosphorylation of band 3 may trigger a cascade of events finally leading to hemolysis. 1. Introduction Due to their function in carrying oxygen and their high iron content, red blood cells (RBCs) are constantly exposed to oxidative stress [1]. In addition, RBCs may transiently experience oxidative stress when they are exposed to ROS crossing inflammatory tissues or interacting with oxidant contained in drugs or foods [2C4]. Moreover, a number of hemolytic disorders are also known to damage the RBC membrane increasing the production of free radicals originating from denatured hemoglobin species (hemichromes), invariably present in thalassemia, sickle cell disease [5C7] or with decreased ability of RBCs to deal with extracellular oxidants as in G6PD deficiency [8]. It is noteworthy that approximately 7% of world population is affected by those mutations which have been selected by malaria. It is well 2”-O-Galloylhyperin known that RBCs respond to oxidative stress with a metabolic response finalized to maximize the production of NADPH and to regenerate the stores of GSH and thioredoxin. In parallel, RBCs also respond by activating tyrosine kinases determining the tyrosine (Tyr) phosphorylation of band 3, the most abundant RBC membrane protein and the major linkage between the cytoskeleton and the lipid bilayer [9C12]. In RBCs, hyperphosphorylation of band 3 has been constantly reported in all the prooxidant hemolytic disorders [13C15] and in malaria [16, 17], but the mechanisms leading to its phosphorylation and its pathophysiological significance have been partially defined. We recently described that band 3 phosphorylation appears to be increased in intermediate thalassemia [18] and that this phenomenon is closely related to the formation of hemichromes. Band 3 phosphorylation and hemichromes formation have been also described in malaria infected RBC [19]. In both pathological situations, band 3 phosphorylation appears to play a permissive role in the release of membrane microparticles. Current knowledge appears to be still insufficient to explain the molecular details of the underlying mechanism, although some recent findings clearly indicate a role of band 3 phosphorylation in the regulation of metabolism mediated by the binding of deoxygenated hemoglobin (Hb) [20C22] and in the modification of the affinity between band 3 and ankyrin following oxidative stress [23]. The redox regulation of band 3 Tyr phosphorylation apparently involves different components. In a previous report, it has been demonstrated that oxidized band 3 is selectively phosphorylated [9]. Lyn is responsible for the phosphorylation of Tyr 359 and Syk is responsible for the phosphorylation of Tyr 8 and Tyr 21 [24C26]. Interestingly, all of those residues are located in the cytoplasmic domain of 2”-O-Galloylhyperin ETV7 band 3. Phosphatases (PTPs) have also been implicated in the phosphorylation of band 3 that follows oxidative stress [27C29] and inhibition of PTPs is due to the inhibitory Cys residue present in the catalytic site of some PTPs but the reactivity to H2O2 of the inhibitory Cys is 0.005-fold lower than GSH, indicating that, at its normal concentrations, GSH should very effectively protect PTPs from oxidative inhibition [30, 31]. Additional regulatory components could be also involved in the band 3 phosphorylation: Lyn kinase has been described to act as redox sensor [32]; Lyn activates Syk in different cell types and the role of 2”-O-Galloylhyperin Syk autophosphorylation remains to be elucidated [25, 26]. Moreover, all of those regulations have been mainly studied in immune cells and very little information is available on RBCs. In the present report we performed a series of experiments to gain more information on the mechanisms that are involved in the Tyr phosphorylation of band 3 following a reversible membrane protein oxidation triggered by diamide and H2O2 and by hemichromes which cause irreversible oxidation. 2. Materials and Methods 2.1. Treatment of Red Blood Cells Venous blood was.