Methods: Protein Determination

1. Actions of Pesticides on Detoxification Mechanisms of Corn Earworms, Helicoverpa ZeaStefania Blevins and Shirley L. ChaoFayetteville State University Apprentice, Stefania Blevins, a Junior at Douglas Byrd High School, conducted experiments to understand the mechanisms of toxicity of two pesticides on the developing corn earworm, a major pest of various crops. Diazinon, an insecticide and Nicosulfuron, a herbicide were tested. Her experience this summer involved: planning experiments, conducting biochemical experiments, analyzing/graphing data, and writing a scientific paper to be submitted for publication Methods: Protein Determination Corn earworm samples were first weighed. After thawing, each treated Helicoverpa Zea (whole body corn earworm) larva was homogenized in a Power Gen 700 polytron in 1mL of buffer containing the following: NaCl at 0.1M, Tris-HCl at 0.05M (pH8.0), MgCl2 at 0.02M. Each sample was kept on ice at all times. The homogenates were then centrifuged at 14000 X g or 3 minutes. The supernatant was then removed for protein determination and the pellet was discarded. Approximately 3mL of Bradford reagent was added to each 30uL of sample to measure the amount of protein contained in the supernatant. Following at least a five minute incubation, the absorbance of each sample was read at 595nm using a spectrophotometer (Varian, Cary, NC). Glutathione s-Transferase Determination The supernatants used for the protein assay were also used for determining Glutathione S-Transferase activity. Glutathione and CDNB (1-chloro-2,4-dinitrobenzene) were added at 4.33mM and 1.12mM respectively. The negative control, a control sample without the substrate added, was also tested with treated samples. The total volume for this assay was 3mL containing: 30ul of supernatant from homogenized samples (approximately 40-100ug of protein), 15uL of CDNB, 130ul of glutathione, and 0.1M TRIS-HCl at pH8.0. Absorbance was read at 340 nm wavelength at the following timepoints: 2,4,6, and 8 minutes.

2. Actions of Pesticides on Detoxification Mechanisms of Corn Earworms, Helicoverpa ZeaStefania Blevins and Shirley L. ChaoFayetteville State University Results: . Conclusion: Binary mixtures of Diazinon and Nicosulfuron did not significantly alter Glutathione S-Transferase activity. While Glutathione S-Transferase is involved in the detoxification of many toxicants, the enzyme does not appear to play a role in the toxicity of these pesticides.