Defense Date


Graduation Date



Immediate Access

Submission Type


Degree Name



Chemistry and Biochemistry


Bayer School of Natural and Environmental Sciences

Committee Chair

David W. Seybert

Committee Member

Charles T. Dameron

Committee Member

Mitchell E. Johnson

Committee Member

William Brown


biochemistry, human serum albumin, human serum transferrin, lipid peroxidation by-products, MALDI-TOF MS, protein modification


Lipid peroxidation (LP) proceeds by a free radical chain reaction mechanism in which molecular oxygen is incorporated into polyunsaturated fatty acids (PUFA) to yield lipid hydroperoxides (LOOH). The ultimate end-products of LP are the production of alpha, beta unsaturated aldehydes, such as acrolein, 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA). These aldehydes are very reactive and exhibit facile reactivity with various biomolecules, particularly proteins, resulting in the formation of fluorescent adducts that could be useful biological marker in diseases. These fluorescent adducts typically emit light between 400 and 600 nm when excited at wavelengths ranging from 300-400 nm. Lysine residues are the major amino-acids that are predicated to react with aldehydes to yield fluorescent adducts. LP has been implicated in several diseases such as atherosclerosis, Parkinson, Alzheimer, and diabetes.

Water-soluble fluorescent species (WSF) are fluorescent species that exhibited similar fluorescence characteristics for excitation and emission to those from aldehyde-modified proteins and amino-acids analogues. WSF showed also direct correlation with age, so it suggested that its components could serve as a useful biomarker for in vivo LP.

We determined that WSF was composed of two main components, a high molecular weight protein component and a low molecular weight non-protein component. The non-protein component was unreactive towards proteins. We were able to identify four major protein species in the protein component that contributed to the fluorescence. These protein species were HSA, HST, Ig-G and carbonic anhydrase, with HSA and HST being the major species contributing to the overall fluorescence in the protein component. The fluorophore was determined to be covalently bound to the protein component.

Using CNBr digestion on isolated HSA and HST, we were able to determine multiple fluorophore binding sites within both proteins. We determined by MALDI TOF/MS that there were indeed MW increases in HSA and HST after aldehyde modification due to the fluorophores formation. In vitro studies on HSA determined a change in the pI due to aldehyde modifications. In vitro functional studies on HST suggested that there might be a loss of one the HST Fe binding sites due to aldehyde modification.