Getting Radical

HeLa lysates were oxidized by the addition of H2O2 (hydrogen peroxide) for 0, 0.5 or 3.5 hours. Afterwards, cell lysates from the control or H2O2-treated samples were treated +/- dinitriphenylhydrazine, then separated by PAGE and transferred to a nitrocellulose membrane. Dinitrophenylated proteins were immunodetected using the anti-DNP antibody provided in the kit. The lanes contain: (1) dinitrophenylated molecular weight standards; (2) DNP-modified HeLa control cells; (3) DNP-modified, 0.5-hour H2O2-treated HeLa cells; (4) DNP- modified, 3.5 hour-H2O2-treated HeLa cells; (5) unmodified HeLa control cells; (6) unmodified HeLa, 0.5-hour H2O2-treatedHeLa cells. Reprinted with permission from Intergen. Free radicals have been implicated in the oxidative modification of a wide variety of macromolecules, including nucleic acids, lipids, carbohydrates, and proteins. "During the last 5 to 6 years, increasing attention has been paid to the oxidation of proteins," comments Robert Hohman, vice president and general manager, Intergen Discovery Products, Purchase, N.Y. Indeed, many researchers are currently investigating the nature and consequences of the oxidative modification of specific proteins involved in different biological processes. Protein oxidation is such a "hot" research topic because it has been implicated in a plethora of biological and pathophysiological processes. For example, oxidative modification is thought to be involved in aging and apoptosis, as well as neoplastic, neurodegenerative, inflammatory, and cardiopulmonary diseases. A number of different processes result in the production of free radicals, which can then go on to modify macromolecules. Examples include metal-catalyzed oxidation, radiation-mediated oxidation, and oxidation via ozone or nitrogen oxides. The oxidative modification of proteins by these and other mechanisms results in the conversion of side chain groups of certain amino acid residues to carbonyl derivatives (aldehydes and ketones). In fact, relative carbonyl content is generally accepted as a hallmark of the oxidation status of proteins. The OxyBlotTM Oxidized Protein Detection Kit exploits this telltale increase in carbonyl groups as a way of detecting proteins that have been modified via oxidation. The kit provides reagents required to convert the carbonyl groups of protein side chains to their dinitrophenylhydrazone (DNP) derivatives. An anti-DNP antibody and secondary antibody conjugate are also included, enabling the detection of dinitrophenylated proteins via standard immunochemical techniques such as Western or dot blot analyses. "We consider the OxyBlotTM technique to be a very powerful tool," says Inge Hanbauer, a researcher studying the roles of oxidative stress in neurological disorders at National Institute of Health's Laboratory of Molecular Immunology, Bethesda, Md. This method is especially advantageous when one has antibodies specific for the oxidation-prone protein of interest. "This enables researchers to estimate the relative levels of carbonyl groups in a specific protein subject to oxidative damage, rather than to simply monitor the total carbonyl content in a sample," explains Intergen's Hohman. Simply immunostaining sister Western blots of DNP-modified proteins with anti-DNP versus a specific antiprotein antibody can provide an approximate comparison of the oxidation status (as estimated via anti-DNP binding) per unit protein (as monitored by the binding of specific antibodies) between different test samples. "A unique feature of the kit is the calibrated molecular weight markers, which controls for all of the variables in the Western blot procedure and permits direct comparison of oxidation levels from experiment to experiment," adds Hohman. Author, Deborah Wilkinson, can be contacted at deborahwilkinson@sprynet.com. For more information, please contact (800) 431-4505, or visit the company web site at www.intergenco.com. E. Shacter, et al., in Free Radicals: A Practical Approach, N.A. Punchard, F.J. Kelly, eds., Oxford, IRL at Oxford University Press, 1996, pages 159-70. E.R. Stadtman, Annual Review of Biochemistry, 62:797, 1993. E.R. Stadtman, Science 257:1220, 1992. B. Halliwell, J.M.C. Gutteridge, Methods in Enzymology, 186:1, 1990.

Getting Radical

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