The Science Seminar Series: October 4, 2012
Nitric Oxide: Biochemistry and Detection
Dr. Yakov Y. Woldman
Department of Chemistry
Valdosta State University
Time: 4:00 -5:00pm
The presentation is an introduction to biochemistry of nitric oxide (NO) and methods of its detection in biological systems. Emphasized is chemiluminescent method for quantitation of NO generation in cell cultures proposed by author. The method is based on the reaction of soluble guanylyl cyclase, which is activated by NO; the product of the reaction is enzymatically converted to ATP, detected by chemiluminescence in luciferin-luciferase system. The method has been applied to the measurement of NO generation by activated murine macrophages and bovine aortic endothelial cells. For activated macrophages the rate of NO production is about 100 amol/(cell·min); the same rate was found from the measurements of nitrite, the final product of NO oxidation, for the same cells. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); it is approximately doubles upon activation by bradykinin, Ca2+ ionophore A23187 or mechanical stress (shaking). For both types of cells the measured NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing to measure NO generated by 102 – 104 cells, depending on the rate of NO production. The direct comparison shows the chemiluminescent method to be two orders of magnitude more sensitive than fluorescent detection using NO-reactive dye DAF-FM.