Sulfate influx on band 3 protein of equine erythrocyte membrane (Equus caballus) using different experimental temperatures and buffer solutions

The aim of this study was to assess the anion transport in equine erythrocytes through the measurement of the sulfate uptake operating from band 3 using different experimental temperatures and buffer solutions. Blood samples of six clinically healthy horses were collected via jugular vein puncture, and an emochrome-citometric examination was performed. The blood was divided into four aliquots and by centrifugation and aspiration the plasma and buffy coat were carefully discarded. The red blood cells were washed with an isosmotic medium and centrifuged. The obtained cell suspensions were incubated with two different experimental buffer solutions (buffer A: 115mM Na2SO4, 10mM NaCl, 20mM ethylenediaminetetraacetic acid, 30mMglucose; and buffer B: 115mMNa2SO4, 10mM NaCl, 20mM ethylenediaminetetraacetic acid, 30mMMgCl2) in a water bath for 1 h at 25 C and 37C. Normal erythrocytes, suspended at 3% hematocrit, were used tomeasure the SO4= influx by absorption spectrophotometry at 425 nm wavelength. Unpaired Student’s t-test showed a statistically significant decrease (P<0.01) of rate constants in equine erythrocytes at 25 C versus 37C using both experimental buffer solutions. Comparing the buffer A with buffer B unpaired Student’s t-test showed statistically lower values (P<0.0001) for A solution versus B solution both at 25 C and at 37C. The greater inhibition of SO4= influx measured in equine erythrocytes indicates the increased formation of the sulfydryl bonds in band 3 and the modulation of the sulfydryl groups, culminating in the conformational changes in band 3.