Abstract

Human serum transferrin is a 79 kDa glycoprotein responsible for the safe transport of iron throughout the blood stream. Transferrin binds up to two ferric ions in two domains known as the N-lobe and C-lobe respectively. Thus, there are four transferrin metalloforms: apo-Tf (with no iron bound), FeN-Tf (with one iron bound in the N-lobe), FeC-Tf (with one iron bound in the C-lobe) and holo-Tf(with two iron ions bound, one in each of the lobes). The two domains are homologous but feature differences in the kinetics and thermodynamics of iron binding and release. The two lobes are also different in terms of the external factors that affect their iron binding. This dissertation shows that pyrophosphate affects the proportions of transferrin metalloforms, even at physiological concentrations which are small relative to iron, transferrin, and carbonate. Specifically, it decreases the amount of FeN-Tf while increasing the amount of FeC-Tf. It was also found that this is mediated at least partially through iron release, as the same effect was observed in already loaded transferrin which featured a mixture of all four metalloforms. This effect was also still observed in the presence of various blood serum components at physiological concentrations. In testing serum components, it was found that pyrophosphate decreases the extent of loading in the presence of 1 mM phosphate. Curiously, calcium ions were also found to consistently decrease the extent of iron loading in the same circumstances. It was found that zoledronic acid also decreases FeN-Tf while increasing the amount of FeC-Tf when included in iron loading experiments, and at a physiological zoledronic acid:Fe3+ molar ratio. Over a dozen blood serum ions were also evaluated for lobe-specific effects on transferrin loading. Surprisingly, lactate increased the proportion of holo-Tf by approximately 15 percentage points and decreased apo-Tf by 10 percentage points. Glyoxylate diminished iron loading, raising the fraction of apo-Tf by over 10 percentage points and decreasing the fractions of all three ferric metalloforms. Homocysteine preincubation induced loading under ambient carbonate levels. In order to make these findings, an existing micellar electrokinetic chromatography technique was validated against the traditional method of transferrin metalloform separation: urea gel electrophoresis. The micellar electrokinetic chromatography was found not to interfere with transferrin metalloform proportions and to be more internally consistent compared to gel electrophoresis. A method was also developed for top-down transferrin analysis on an IM-capable mass spectrometer.

Degree

PhD

College and Department

Chemistry and Biochemistry; Computational, Mathematical, and Physical Sciences

Rights

https://lib.byu.edu/about/copyright/

Date Submitted

2024-12-19

Document Type

Dissertation

Keywords

transferrin, micellar electrokinetic chromatography, pyrophosphate, zoledronic acid

Language

english

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