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In this work, we describe an optimized procedure based on gradual hemolysis  for the isolation of hemoglobin derived from bovine slaughterhouse  erythrocytes in a membrane bioreactor. The membrane bioreactor system  provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its  separation from the empty erythrocyte membranes (ghosts). Ten different  concentrations of hypotonic media were assessed from the aspect of the extent  of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling  and membrane deformations induced by decreased salt concentration. Effective  gradual osmotic hemolysis with an extent of hemolysis of 83% was performed  using 35 mM Na-phosphate/NaCl buffer of pH 7.2-7.4. Under these conditions  most of the cell membranes presented the appearance of the normal ghosts  under phase contrast microscope. The results show that isolation process  yielded predominantly to oxyhemoglobin. Kinetic studies showed that maximal  concentration of hemoglobin was reached after 40 min, but the process cycle  at which recovery of 83% was achieved lasted for 90 min

Optimization of gradual hemolysis for isolation of hemoglobin from bovine erythrocytes