EXCLI Journal 2009;8:89-96 – ISSN 1611-2156 Received: April 15, 2009, accepted: May 03, 2009, published: May 05, 2009 89 Original article: INSTANTANEOUS MONITORING OF HYDROXYL RADICAL- MEDIATED PROTEIN ALTERATIONS BY GREEN FLUORESCENT PROTEIN Chartchalerm Isarankura-Na-Ayudhya, Nanchaya Seesai, Sittichai Thinjom, Virapong Prachayasittikul * Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand * Corresponding author: E-mail: mtvpr@mahidol.ac.th; Telephone: 662-441-4376, Fax: 662-441-4380 ABSTRACT In the present study, green fluorescent protein (GFP) is successfully applied for instantaneous monitoring of hydroxyl radical-mediated protein alterations. Hydroxyl radical generated from metal-mediated Fenton’s reaction (in the presence of 50 µM copper ions, 10 mM ascorbic acid and 1.05 % hydrogen peroxide) rapidly suppressed the fluorescent emission of 60 % in a few seconds followed by a gradual decrease up to 75 % maximum inactivation was reached. The production of hydroxyl radical was experimentally proven to be specifically derived from copper-catalyzed Fenton’s reaction in which other divalent cations (e. g. Zn 2+ , Cd 2+ , Mn 2+ , Co 2+ and Ni 2+ ) exerted no inhibitory interaction. Supplementation of oxidative scavengers and metal chelators into the assay reaction provided protective effects on the fluorescent intensity. The degree of protection was in the order of EDTA > histidine >>> glutathione ~ sodium azide > thiourea ~ mannitol. The findings herein gain insights not only into the deleterious effect of reactive oxygen species on biological macromolecules but also the potential applica- bility as a versatile antioxidant screening assay. Keywords: Green Fluorescent Protein (GFP), Cadmium-binding peptide (CdBP), Antioxidant screening, Fenton’s reaction, Hydroxyl radical INTRODUCTION Reactive oxygen species (ROS) are highly reactive molecules owing to the presence of unpaired electrons at the va- lence shell. The ROS are implicated in cel- lular activities such as inflammatory re- sponses, cellular dysfunction and apoptosis. Such toxic effects involved in the damage of DNA, lipid peroxidation, oxidations of amino acid, and inactivation of enzymes participated in cellular metabolism (Sorg, 2004; Valko et al., 2007). Direct detection of ROS can be carried out by using electron spin resonance (ESR) or electron paramag- netic resonance (EPR) spectroscopy. How- ever, applications of the technique become limited due to the following reasons: i) re- quirement of sophisticated instrumentation, ii) unstability of the ROS for the measure- ments and iii) interference of electron trap- ping in the liquid phase (Floyd, 2009; Kopani et al., 2006; Swartz et al., 2007). Therefore, several indirect methods e. g. detection of oxidative products, analysis of antioxidative levels and other measurement systems have continuously been developed (Cao et al., 1993; Giammarioli et al., 1999; Mordente et al., 1987; Tangkosakul et al., 2009). Green fluorescent protein (GFP) is an auto-fluorescent protein isolated from Pa-