Abstract
Abstract Human transferrin C and a genetic variant found by a starch gel electrophoresis screening program were purified and compared structurally. Isolation combined two bulk fractionations, with the use of rivanol and ammonium sulfate, with the more selective procedures of Sephadex G-100 gel filtration and diethylaminoethyl Sephadex A-50 ion exchange chromatography. Transferrin C and the variant were separated during the latter. The product obtained was homogeneous in the ultracentrifuge (s20,w = 5.1 S), in free boundary electrophoresis at several pH values (mobility = -3.03 and -3.63 units for apo- and ferritransferrins C, respectively, in pH 8.6 Veronal; isoelectric point, pI, of apotransferrin C = 5.5), in immunoelectrophoresis, and in starch gel electrophoresis. Amino acid analyses agreed with published data but failed to reveal differences between the two varieties. The peptide map of the Caucasian variant D-G.F. contains all the peptide spots seen in the map from transferrin C and a single additional spot not seen in the latter. The peptide is neutral at pH 6.5 and fails to stain for arginine, histidine, or tyrosine. This variant peptide is assumed to arise from one of two overlapping peptides, from the unresolved core, or from a peptide produced from two distinct parts of the molecule.
Highlights
Acid-citrate-dextrose or EDTA plasma was obtained from healthy donors;[4] the donors of variant-containing plasma had been identified by a screening program based on starch gel electrophoresis and radioautography
Isolation of Transferrin-The procedure developed for the purification of human transferrin and the separation of the genetic varieties from the plasma of heterozygotes is based on the following principles: (a) stabilization of the transferrin by saturation with iron; (6) batchwise removal of most of the unwanted proteins by two methods which would not differentiate between transferrin varieties, namely, rivanol precipitation of albumin and most of the other plasma proteins, and ammonium sulfate fractionation of the supernatant solution from which the rivanol had been removed, and (c) final purification by two methods differing in principle, namely, gel filtration based on molecular size and ion exchange chromatography based on molecular charge
The individual variants were identified by comparison of their starch gel electrophoretic mobility with that of Starch gel electrophoresis was carried out in the discontinuous Tris-citrate-borate buffer system
Summary
Isolation of Transferrin-The procedure developed for the purification of human transferrin and the separation of the genetic varieties from the plasma of heterozygotes is based on the following principles: (a) stabilization of the transferrin by saturation with iron; (6) batchwise removal of most of the unwanted proteins by two methods which would not differentiate between transferrin varieties, namely, rivanol precipitation of albumin and most of the other plasma proteins, and ammonium sulfate fractionation of the supernatant solution from which the rivanol had been removed, and (c) final purification by two methods differing in principle, namely, gel filtration based on molecular size and ion exchange chromatography based on molecular charge. In order to locate individuals with transferrin variants, samples of blood from blood bank donors were obtained and examined by starch gel electrophoresis.
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