Purification and partial characterization of rat serum transferrin (author's transl)

Abstract

Rat transferrin was isolated from serum. At the second step of the purification procedure, rat transferrin was separated clearly into two fractions by the chromatography on DEAE-Sephadex A-50, with a linear gradient of a narrow range from 0 to 100 mM NaCl in 20mM P-K buffer pH 8.2. The purification after this step was followed individually in each fraction by CM-cellulose 52 ion-exchange chromatography, Sephadex G-150 gel filtration and Wheat germ Lectin-Sepharose 6 MB affinity chromatography. As the final step of the purification procedure of the front fraction, the transferrin fraction was deferrinized and applied on DEAE-Sephadex A-50 column. The elution of deferrinized transferrin shifted to the position of the back fraction. Anti-rat transferrin was obtained from albino rabbit. The purity of rat transferrin was certified by immunoelectrophoresis against anti-rat whole serum-rabbit serum and anti-rat transferrin just prepared.The amino acid composition of rat transferrin was determined and each amino acid composition from 2 series of procedure was almost the same, and the molecular weight of rat transferrin was 74, 000 by gel filtration and 72, 000 by SDS polyacrylamide disc gel electrophoresis. Reduction and alkylation of rat transferrin indicated that rat transferrin was composed of a single polypeptide chain. Isoelectric points of rat transferrin showed within a range of pI 5.40--6.25 in holo-transferrin and pI 5.75-6.45 in apo-transferrin. Five to eight protein bands were identified with isoelectric focusing individually in each form of rat transferrin. Rat transferrin and hemopexin were separated by Wheat germ Lectin-Sepharose 6 MB affinity chromatography. The absorption spectrum of rat hemopexin was at 410 nm and 530 nm, while rat transferrin had faint absorption at 470nm. Con-A Sepharose could not bind rat transferrin, but bind hemopexin. The rat transferrin, whose histidine residues were modified with diethylpyrocarbonate, could not bind iron. While the transferrin used as the source of this chemical modification was able to bind 2 iron atoms.