Quantitation of Protein Tyrosine, 3-Nitrotyrosine, and 3-Aminotyrosine Utilizing HPLC and Intrinsic Ultrviolet Absorbance

Abstract

Nitrotyrosine has been found in the urine of humans with no known exposure to exogenous nitrating agents. We have shown that peroxynitrite, a nitrating agent formed by the near diffusion-limited reaction of nitric oxide with superoxide, is formed by activated macrophages. Using an antibody which recognizes nitrated tyrosine residues in proteins, we have obtained immunohistological evidence for nitration in a number of pathological conditions amenable to peroxynitrite formation. We have developed an HPLC method as a means of confirming the presence of nitrotyrosine. Quantitation of small amounts of nitrotyrosine in complex protein mixtures presents special problems of sensitivity and specificity which are often exacerbated by traditional amine-derivatization of all amino acids. An HPLC method for proteins hydrolysates is described which relies on intinsic UV absorbance of nitrotyrosine at 280 nm (which is fivefold higher than tyrosine at pH 3.5) for quantitation and on its unique absorbance at 355-365 nm for partial identification. Since only aromatic acids are detected, sample sizes can be increased to permit detection of small amounts of nitrotyrosine without introducing interference. Treatment of BSA with 4mM peroxynitrite resulted in nitration of 12.8% of total tyrosine residues; a small fraction (0.9%) of the total was detected as aminotyrosine. A total of 9.6% of the tyrosine residues in fresh plasma were nitrated as a result of treatment with 4mM peroxytyrosinee during hydrolysis. The advantages of this method over traditional amine-dervatized amino acid analysis are discussed and modifications which would further increase sensitivity to aminotyrosine are presented.