Date of Award
Chemistry and Biochemistry
John Alexander Huffman, Ph.D.
Atmospheric pollution, Ergothioneine, Lactoferrin, Nitrotyrosine, Ocular disease, Protein nitration
Lactoferrin (LF) is an iron-binding glycoprotein of molecular mass ca. 80 kDa that is predominantly found in mammalian body fluids. Lactoferrin is a multifunctional protein that has a wide range of properties such as antibacterial, antiviral, anti-inflammatory, and anti-allergic functions. Tyrosine residues in the protein play a part in many lactoferrin functions. Protein tyrosine nitration modification represents an oxidative and nitrosative stress process which can be caused by the exposure of proteins to oxidants from air pollution or disease. Understanding the way nitrated lactoferrin interacts with the biochemical environment of the body is thus important to the broader goal of understanding and preventing lactoferrin-related diseases, including certain ocular diseases. The main goal of this set of linked studies was to investigate the process by which LF may be nitrated, to observe changes in its protein structure and function, and to investigate a possible route to block the nitration process.
In the first project discussed here, nitrated lactoferrin was synthesized using two different nitration agents: sodium peroxynitrite and tetranitromethane. After nitration, spectroscopic analysis showed formation of nitrotyrosine with an increase in absorbance intensity at 350 nm, as well as attenuation in fluorescence intensity at an excitation wavelength of 280 nm. A sandwich ELISA protocol was developed to quantify the amount of nitrotyrosine in nitrated lactoferrin samples. The results show that the increase in the amount of nitrotyrosine depends on the molar ratio of nitrating agents added to the reaction. This study represents the first time that nitrotyrosine in lactoferrin had been selectively detected. In the second project, a broth microplate antibacterial susceptibility assay was applied to determine changes in the antibacterial function of lactoferrin brought about after nitration. The results reveal a reduction in the naturally present antibacterial activity of LF, which scales linearly with the increase in nitration molar ratios. In the third project, ergothioneine was used to inhibit the nitration reaction using several protein models, as well as to examine it as a potential therapeutic target for nitrosative stress-related diseases. The results illustrated that ergothioneine diminished the nitration production and protected the protein antibacterial activity. Collectively, these findings suggest that the nitration of tyrosine residues within lactoferrin can be important with respect to several physiological processes. Thus, further understanding the interaction between nitrated LF and the biochemical environment may aid in future research toward clinical diagnoses and in the development of therapies for diseases involving nitration of lactoferrin.
Alhalwani, Amani Yahya, "The Investigation of Lactoferrin Nitration: Quantification, Function, and Inhibition" (2018). Electronic Theses and Dissertations. 1452.
Received from ProQuest
Amani Yahya Alhalwani