title: Direct measurement of catalase activity in living cells and tissue biopsies.
abstract: Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies - can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings.
authors.affiliation: Metabolic Photonics Laboratory, Departments of Surgery and Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
authors.forename: Christine N
authors.initials: CN
authors.lastname: Scaglione
authors.affiliation: Metabolic Photonics Laboratory, Departments of Surgery and Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
authors.forename: Qijin
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Metabolic Photonics Laboratory, Departments of Surgery and Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA. Electronic address: Ramanujanv@csmc.edu.
authors.forename: V Krishnan
authors.initials: VK
authors.lastname: Ramanujan
mesh_terms: D000818:Animals; D002374:Catalase / Q000378:metabolism*; D004796:Clinical Enzyme Tests / Q000379:methods*; D003199:Computer Systems; D004789:Enzyme Activation; D006801:Humans; D008099:Liver / Q000201:enzymology*; D061986:MCF-7 Cells; D008297:Male; D051379:Mice; D009374:Neoplasms, Experimental / Q000201:enzymology*; D010092:Oximetry / Q000379:methods*; D010100:Oxygen / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D002374:Catalase; D010100:Oxygen
keywords: Aging; Antioxidants; Breast cancer; Catalase activity; Oxidative stress; Oxygen sensing