9727613 Branchaud Recently, irreversible oxidative amino acid sidechain crosslinks have been found in several proteins. Such crosslinks can occur spontaneously under mildly oxidative physiological conditions. Oxidative amino acid sidechain crosslinks can serve useful purposes, to provide structural stability to proteins (tyrosinase) or to provide novel built- in redox cofactors at enzyme active sites (galactose oxidase, glyoxal oxidase, and methylamine dehydrogenase). Oxidative amino acid sidechain crosslinks are also an undesired but inevitable consequence of oxidative metabolism. For example, ortho-tyrosine and dityrosine are believed to be markers for oxidative stress and for aging processes. Irreversible oxidative crosslinking of amino acid sidechains may be more widespread than currently believed. Such crosslinks could be easily overlooked using existing methods to analyze for amino acid composition of proteins. This research will develop a novel, general and highly sensitive method for fluorescence detection of sidechain crosslinked amino acids in proteins utilizing excimer formation by pyrene to make the sidechain crosslinked amino acids "light up" and uniquely stand out against a background of normal amino acids in fluorescent HPLC analysis of complete protein digests. Mass spec troscopic analysis of isolated purified HPLC fractions will identify the amino acid compositions and chemical structures of crosslinks. Combustion of food provides the energy necessary to support life. Approximately 1-6% of the oxygen used to burn food is not completely consumed but instead produces reactive oxygen species (ROS). ROS damage molecules, cells and organs in the body. This research project will develop a new method to detect oxidative damage to proteins. Proteins are major building blocks of living things. There are many possible applications of the method both in fundamental biochemical and applied biomedical research.
