Factors influencing the electrophoretic patterns of red cell hemolyzates analyzed in cacodylate buffers

Electrophoretic analyses of human red cell hemolyzates in cacodylate, Veronal, and phosphate buffer media at different ionic strengths and pH values yield a variety of patterns. After dialysis against these media and subsequent dialysis against a standard 0.05 M cacodylic acid buffer (Γ/2 = 0.033, pH 6.5), the electrophoretic patterns are of a uniform type. The concentration of a slow-moving component (B) in these patterns is related to the concentration of organic phosphate. The electrophoretic patterns for (a) crystalline hemoglobin, (b) a hemolyzate dephosphorylated by alkaline phosphatase, and (c) a hemolyzate dialyzed against a Veronal buffer at pH 8.6 are almost identical and are characterized by the presence of small concentrations of Component B. Trace amounts of organic phosphates are present in these solutions. The addition of 2,3-diphosphoglycerate, tri-, or dinucleotides to crystalline hemoglobin solutions or to hemolyzates of aged red cells increases the Component B concentration. It is concluded that a hemoglobin-organic phosphate complex is responsible for Component B in the electrophoretic patterns of red cell hemolyzates analyzed in cacodylate buffers.