Studies on genetic variants of human plasma transferrin.
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The work presented in this thesis is concerned with the characterisation of human plasma transferrins showing abnormal electrophoretic mobilities on polyacrylamide gels. The work is divided into three studies: (i) a study of transferrin variants detected by nondenaturing polyacrylamide gel electrophoresis; (ii) a study of the plasma concentrations of individuals showing transferrin phenotypes associated with the three most common Tf C alleles, Tf Cl, Tf C2 and Tf C3; and (iii) a study of a reported nondegenerate nucleotide difference in the sequence of the cloned human transferrin gene. In the first study, six transferrin variants (3 Tf Br,,.,,, s and 3 Tf D. 5) showing abnormal electrophoretic mobilities on nondenaturing polyacrylamide gels, and the two Tf C variants, Tf Cl and Tf C2 which occur at polymorphic levels (> 1%) in human populations, were isolated and purified from human plasma. Transferrins were purified by a combination of DEAE Sephacel anion-exchange chromatography, SP. Sephadex cation-exchange chromatography and G-200 gel-filtration chromatography. A series of comparative studies were then carried out on the isolated transferrins to determine whether the six transferrin variants detected in this thesis and the Tf C2 variant, showed similar characteristics to the wild-type Tf Cl. Transferrins were studied for sialic acid content of the two glycan chains, and for molecular weights and isoelectric points of the iron-free (apo) and iron-saturated (holo) transferrin forms. Metal-binding properties were examined by studying the binding of Fe", Cu", Al" and Ga"'. Iron-binding was studied at physiological and endosomal pH (7.5 and 5.5 respectively) using FENTA as the iron donor. Binding of Cue*, Al"' and Ga'* were examined at physiological pH using CUNTA, ALNTA and GANTA respectively. The ability of transferrins to retain bound iron was examined by studying pH-induced iron release over a pH range of 6.0-4.0. Conformational stabilities were determined by studying the iron-binding abilities of apotransferrins following exposure to urea or thermal denaturation, and by studying iron loss from holo transferrins following exposure to urea or thermal denaturation. Other than expected differences in isoelectric points, and slightly faster rates of iron loss from variant holo transferrins titrated at physiological pH, all variant transferrins were found to show similar characteristics to Tf Cl, with identical molecular weights and sialic acid content, similar metal-binding properties, and similar stabilities to urea or thermal denaturation. The results indicate that the structure and function of the variant transferrins are not adversely affected by their differences in primary structure. The second study examined the relationship between plasma transferrin concentration and transferrin phenotypes representing five of the six most common Tf C phenotypes (i. e. Tf Cl, Tf C2, Tf C2-1, Tf C3-1 and Tf C3-2), to determine whether plasma concentrations were dependent on transferrin phenotype as suggested in literature. Transferrin phenotypes of 931 unrelated individuals were determined by electrophoresis of plasma on polyacrylamide isoelectric focusing gels. Plasma transferrin concentrations were determined by single radial immunodiffusion using rabbit anti-human transferrin IgG. A significant difference was found between the plasma concentrations of the five transferrin phenotypes (p < 0.01) indicating that transferrin concentration was dependent on phenotype. The results suggest that the two Tf C alleles, Tf C2 and Tf C3 are associated with low plasma concentrations. The third study was initiated to investigate a report in literature that a nondegenerate nucleotide difference of adenine for guanine at base 1086 in exon 8 of the human transferrin gene, may indicate the presence of a hitherto unrecognised transferrin variant with Asn rather than a wild-type Asp at position 310 of the amino acid sequence. The nucleotide sequenceo f exon 8 from 220 unrelatedi ndividuals showing transferrin phenotypes associated with the three most common Tf C alleles, Tf Cl, Tf C2 and Tf C3, and from 5 individuals showing abnormal transferrin phenotypes in the first study, were amplified by polymerase chain reaction. Amplified products were digested with the restriction endonuclease, Fok I which has a single recognition site in exon 8 containing the proposed wild-type guanine at base 1086, or with Bsm I which also shows a single recognition site containing the proposed adenine nucleotide. The study failed to detect the presence of the proposed transferrin variant, confirming that the wild-type guanine was present in all 225 individuals.
AuthorsOkyere-Boakye, Ivan W
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