Electrophoretic and Spectroscopic Observations of Bilirubin in the Presence of Ethylenediamine Tetraacetic Acid

Abstract

IN the absence of proteins, unconjugated (indirect) bilirubin is known to be immobile in an electric field. However, in a buffer system containing ethylenediamine tetraacetic acid (EDTA), for example, the EDTA-tris-boric acid buffer described by Aronsson and Grönwall¹ or in a buffer solution containing only 0.021 mol. EDTA (ref. 1) adjusted above pH 7.0 by adding sodium hydroxide, I found bilirubin (alkaline solution) to move towards the anode when submitted to paper electrophoresis. Moreover, applying an EDTA containing alkaline solution of bilirubin in any alkaline buffer system, for example, Michaelis barbital buffer to electrophoresis, the same phenomenon occurs (Fig. 1). After terminating electrophoresis in EDTA the displaced spots of bilirubin were found yellow and yielding the diazo reaction by Verschure's method² even more readily than bilirubin without EDTA; however, in contrast to the latter the spots of bilirubin in EDTA turned to green in the lapse of some days. The motility of bilirubin in EDTA was found undisturbed by the presence of excess amounts of Ca⁺⁺, or by pretreating bilirubin with ditizon dissolved in carbon tetrachloride, to remove metal traces if present. (This pretreated bilirubin failed to move in an alkaline buffer not containing EDTA)