2.3. Erythrocyte Membrane and Hemolysate Preparation and Protein Determination

The erythrocyte membranes were prepared using the method described by Dodge et al. [14]. The concentration of the plasma and membrane proteins was determined spectrophotometrically with the Folin and Ciocalteu's phenol reagent according to the method outlined by Lowry et al. [15]. The method was based on the reaction of Cu⁺, produced by the oxidation of peptide bonds, with Folin–Ciocalteu reagent. The products of this reaction are optically active with a maximum absorption at 750 nm. The amount of protein in each sample was estimated using a calibration curve plotted by taking different concentrations of bovine albumin as a standard.

The hemolysate was prepared from the erythrocytes after they were lysed with cold water at a ratio 1 : 1.5 and vortexed for 10 min. Hemolysate was centrifuged at 16000 × g for 10 minutes, for the separation of erythrocyte membranes. In the obtained hemolysate, the concentration of hemoglobin (Hb) was estimated using Drabkin's method based on the oxidation of hemoglobin to methemoglobin in the presence of alkaline potassium ferricyanide [16]. Methemoglobin reacts with potassium cyanide to optically active (540 mN) stable cyanmethemoglobin. The molar absorption coefficient of hemoglobin was used to calculate the protein concentration in the samples (ε = 44 mmol⁻¹·L·cm⁻¹).