Anion transport across erythrocyte membrane and reversibility of band 3 protein sulphydrylic groups oxidation in canine leishmaniasis

CO2 transport through erythrocytes membrane is mediated by band 3, an integral membrane protein allowing a rapid Cl/HCO3- exchange, whose cytoplasmic domain binds ankyrin and cytoskeletal proteins. Since canine leishmaniasis is associated with degradation of membrane proteins, the aim of the present study is to verify whether structural modification, i.e. oxidation of band 3 protein sulphydrylic groups and, hence, unbalanced anion transport, in erythrocytes of infected animals may be restored by an antioxidant treatment. To accomplish this aim, sulphate kinetic influx quantification by turbidimetric method, as an index for anion transport capability, and determination of sulphydryl groups by Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB) have been measured in both control and pathological erythrocytes. Dithiothreitol (DTT) has been used as an antioxidant compound, while N-ethyl-maleimide (NEM) has been employed as a pro-oxidant compound for positive control. Our results show that sulphydryl groups oxidation in pathological erythrocytes is significantly higher than in control erythrocytes, with a consequent significant reduction in anion transport kinetics, as revealed by the rate constant evaluation. The oxidative state of band 3 protein and, hence, its function were restored by an antioxidant treatment, leading to conclude that: i) band 3 protein-mediated anion transport is reduced in leishmanial infection; ii) an impaired band 3 protein function is associated to sulphydryl groups oxidation; iii) sulphydryl groups oxidation is a reversible event.