Submitted to: Journal of Chromatography A
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 7, 2005
Publication Date: January 27, 2006
Citation: Tilley, M., Bean, S.R., and Tilley, K.A. 2006. Capillary electrophoresis for monitoring dityrosine and 3-bromotyrosine synthesis. J. Chromatography A. 1103:368-371. Interpretive Summary: Amino acid side chains of proteins are very important. They can interact by crosslinking within a protein and between different proteins. In some proteins they are an important for developing larger structures that are functionally necessary. However they can also form in proteins damaged by free radicals and other oxidants. Often times the formation of certain crosslinked amino acids is indicative of diseases. Crosslinking of the side chains of two tyrosines form a compound called dityrosine (DY). DY has various biological functions in extensible proteins found in insect eggs, fibers such as silk and wool and may have a functional role in food processing. Excessive crosslinking of tyrosine is a sign of protein damage in diseases such as cataracts, Parkinson's and Alzheimer's diseases. Adding a bromine atom to the tyrosine side chain will form 3-bromotyrosine, a molecule that is formed in conditions such as asthma and allergic inflammation. Similarly, 3-bromotyrosine is a specific indicator of protein oxidation eosinophil-mediated tissue injury. In order to adequately study dityrosine and 3-bromotyrosine, an efficient and high yielding method for production of sufficient quantities of standard compounds was previously developed. Here, we report the development of capillary electrophoresis (FZCE) separation as a rapid and simple method for the analysis of tyrosine, dityrosine and 3-bromotyrosine which can be used in a number of applications in diverse fields.
Technical Abstract: Protein oxidation affects the structure of many amino acids. Variants of tyrosine are increasingly important in medical and food sciences. The synthesis of standards is essential for monitoring the disease state of patients with various illnesses and the quality of a number of food products. A method for monitoring standard synthesis of dityrosine and 3-bromotyrsoine from tyrosine using free zone capillary electrophoresis is presented. Optimum separation was achieved using an isoelectric buffer consisting of iminodiacetic acid (100mM) + 75 mM lauryl sulfobetaine (SB3-12) + 0.02% hydroxypropylmethyl cellulose (HPMC) in a 27cm x 75um capillary at 22kv and 45 °C. Using these conditions the two adducts could be easily separated in less than 4 minutes. The electropherograms are similar to lengthy chromatograms, thus making analysis time distinctly shorter.