Characterization of a new horse transferrin variant : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
Transferrin is a glycoprotein with a molecular weight of approximately 80 kd. Its single polypeptide chain is formed into two lobes and it is able to bind two ferric (Fe III) ions per protein molecule. Horse serum transferrin, like the transferrins of most vertebrate species, exhibits extensive genetic polymorphism. Transferrin is one of several protein systems used for blood-typing horses. During routine blood typing a new band (designated*) was found. This variant originated from a thoroughbred stallion which was of considerable value as a sire and so it was of interest to characterize this new transferrin variant. Thoroughbred horses carry genes for only six of the fourteen known transferrin isoforms; D, Fl, F2, H2, 0 and R. The aim of this project was to characterize, by classical amino acid sequence analysis, the transferrin variant and the parental variants D and Fl, from one of which must have arisen. All three variant forms (D, Fl and*) were purified. Tryptic digests of the variants were analysed by HPLC and those peaks appearing to differ between the HPLC profiles were sequenced by automated protein sequencing. The sequences obtained confirmed that the protein isolated was a transferrin variant. Further sequencing allowed deduction of the parent transferrin variant. Two clear sequence differences between the D and Fl variant have been identified. The Fl variant was found to contain an arginine residue at amino acid position 553, whereas the D variant contains a cysteine residue at this position. At position 418 of the Fl variant a serine residue was found and at the same position in the D variant a proline residue was found. Sequence determination of peptides from the * tryptic digest revealed that a proline residue and a cysteine residue were found at positions 418 and 553 respectively, clearly indicating that the new * phenotype has arisen from the D allele and not the Fl allele.
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