A simple one-column procedure for the separation of swine and human serum transferrins.
A rapid method for the separation of transferrin from swine or human serum is described. Serum (human or swine) is brought to 50% of saturation with ammonium sulfate for removal of immunoglobulins, the resulting precipitate discarded and the supernatant brought to 70% of saturation. The resulting precipitate was dissolved in and dialyzed against 1.54 mM sodium azide (I = 0.00154). Chromatography of the low ionic strength ammonium sulfate fractions (= 20 ml of swine or human serum, 70% of saturation) on columns of Bio-Gel A-1.5 m-Reactive Blue 2, equilibrated with 1.54 mM sodium azide, resulted in two peaks, a breakthrough peak and pure transferrin which was eluted with a linear gradient with 0.5 M potassium phosphate buffer, pH 7.1, as limit buffer. Yields varied between 53 and 55% from whole serum and 70-76% from the ammonium sulfate fractions. Transferrins from both species were found to be homogeneous when subjected to immunoelectrophoresis (anti whole serum antibody) and anionic and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. Hemopexin, a frequently found contaminant in transferrin preparations, is tightly bound by the gel-dye complex under the experimental conditions. Swine serum transferrin possesses many physicochemical properties practically identical to the human protein. Although small differences in physicochemical properties were apparent the extinction coefficients, molecular weights, electrophoretic mobilities, absorbance maxima of the diferric proteins (470 nm), isoelectric points and the absorbance ratios (465 nm/410 nm) of the diferric proteins were practically identical. Both swine and human transferrin produced a reaction of identity (complete coalescence) when reacted with antibody to either transferrin.
Baumstark
J S
JS
Department of Obstetrics and Gynecology, Creighton University, School of Medicine, Omaha, NE 68131.
D000818:Animals; D000906:Antibodies; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D007525:Isoelectric Focusing / Q000379:methods; D008970:Molecular Weight; D013045:Species Specificity; D013552:Swine; D014168:Transferrin / Q000302:isolation & purification*
D000906:Antibodies; D007202:Indicators and Reagents; D014168:Transferrin