Analytic Core

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CORE E: ANALYTICAL REDOX BIOCHEMISTRY Director: Danyelle Townsend, PhD ABSTRACT Understanding the complexities of redox mediated signaling events requires a multidisciplinary approach. The SC COBRE in Oxidants, Redox Balance and Stress Signalling has assembled a cohort of promising junior faculty with expertise in relevant biomedical model systems. Analytical biochemistry specific to the detection and quantification of redox sensitive molecules and coordinate protein changes that drive homeostasis is a unique niche. As such, development of the Analytical Redox Biology Core (ARBC) is important. The primary objective of the Core is to provide comprehensive analytical redox biochemistry methods and mentoring support for the COBRE junior faculty with the goal to advance their research endeavors, publications and fundability. The specific aims of the ARBC are: 1) Provide ROS/RNS identification and quantification using state-of-the-art techniques; 2) Perform quantitative analysis of ROS/RNS (redox molecules and metabolites), including those associated with calcium mobilization and changes in energy metabolism; 3) Provide expertise and technology for in depth biochemical analysis of thiol-centered enzyme activities and define protein:protein interactions. Since oxidative (nitrosative) stress often is associated with a conditional increase in antioxidant protection, the Core has established methods to detect and measure various antioxidant enzyme activities as a function of oxidant stress/antioxidant protection equilibrium. Comprehensive analysis of redox status also includes measurement of intracellular GSH, GSSG, protein surface and “buried” thiols utilizing both endpoint and/ or real-time kinetic measurements with millisecond resolution. In complex studies of redox signaling, certain protein:protein interactions appear to be redox dependent and attributed to post-translational modifications, including S-nitrosylation and S-glutathionylation. The ARBC has developed fluorescent labeling and FRET analysis to evaluate redox dependent protein:protein interactions with subsequent in silico molecular modeling using ZDOCK, GOLD Suite (v 5.2) software. Collectively, these technologies will provide a multidisciplinary approach to advance the understanding of redox mediated signaling events specific to the model systems presented by the junior faculty in their research.