| dc.description.abstract | 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. | en_US |
