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Amani Yahya Alhalwani, John E Repine, J Alex Huffman; Environmental nitration of ocular lactoferrin may contribute to dry eye disease. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2863. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Environmental exposure to airborne pollutants in urban areas not only can cause physical damage to non-living surfaces, plants and animals, but also contribute to allergies, inflammation, respiratory distress, and ocular problems in humans. In urban areas, the surface of the cornea is in direct contact with reactive atmospheric species such as nitrogen oxide and ozone. The effects of this interaction are not known but reasonably could include changes in lactoferrin (LF), an ocular protein whose reduced concentration correlates with dry eye syndrome (DES). Protein nitration forms 3-nitrotyrosine and is a marker of nitrosative stress exposure. Our aim is to detect chemical changes in LF and nitrated lactoferrin (NLF) that can be used to create and optimize an assay to quantify nitrated tyrosine and other proteins that are relevant to the pathophysiology of DES.
Human LF was nitrated by exposure to tetranitromethane (TNM) in Tris-HCl buffer for 23 hours in vitro. Separately, samples of LF were then exposed to peroxynitrite as a surrogate endogenous nitrating agent. Each reaction mixture was subsequently purified using a PD-10 size exclusion chromatography column and eluted with reaction buffer. Fluorescence and UV-vis spectroscopy, SDS-PAGE, Western Blot and ELISA analyses were used to detect and quantify LF, NLF, and lysate protein from animal corneal tissue.
Our analyses highlighted the chemical differences of LF and NLF. Absorbance peaks were observed around 280 and 350 nm for tyrosine and nitrated tyrosine, respectively. Fluorescence spectroscopy also showed reduced emission intensity of NLF compared to LF. In addition, NLF has a higher molecular weight and greater charge compared to LF resulting in a different band position in SDS-PAGE. We quantified the total amount of proteins in corneal tissue to be 2 mg/mL. Based on the total protein concentration, the concentration of LF using ELISA was 0.01 mg/mL.
Lactoferrin was nitrated via reaction with TNM and peroxynitrite. Our initial results support the possibility that environmental nitration of lactoferrin and perhaps other key proteins that may contribute to DES. We also developed a new system to test therapeutic strategies for reducing the effect of nitrosative stress on the development of DES.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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