User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/03

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Qualitative Analysis of Protease Activity Objective Continue the qualitative analysis of protease activity on AuNP fibers. Pepsin, Trypsin, Chymotrypsin, and Proteinase K will be tested. Procedure Stock Solutions Pepsin - 16 mg/4 mL -- 115.6 μL Trypsin - 10.77 mg/4 mL Chymotrypsin - 11.56 mg/4 mL Proteinase K - 13.37 mg/4 mL Solutions Tested Pepsin 57.6 µM (2.5 mL buffer, 2.5 mL enzyme) 11.56 µM (4.5 mL buffer, 0.5 mL enzyme) 1.156 µM (4.95 mL buffer, 0.05 mL enzyme) 0.12 µM (5 mL buffer, 0.005 mL enzyme) Trypsin 57.6 µM (2.5 mL buffer, 2.5 mL enzyme) 11.56 µM (4.5 mL buffer, 0.5 mL enzyme) 1.156 µM (4.95 mL buffer, 0.05 mL enzyme) 0.12 µM (5 mL buffer, 0.005 mL enzyme) Chymotrypsin 57.6 µM (2.5 mL buffer, 2.5 mL enzyme) 11.56 µM (4.5 mL buffer, 0.5 mL enzyme) 1.156 µM (4.95 mL buffer, 0.05 mL enzyme) 0.12 µM (5 mL buffer, 0.005 mL enzyme) Proteinase K (K) 57.6 µM (2.5 mL buffer, 2.5 mL enzyme) 11.56 µM (4.5 mL buffer, 0.5 mL enzyme) 1.156 µM (4.95 mL buffer, 0.05 mL enzyme) 0.12 µM (5 mL buffer, 0.005 mL enzyme) NB - Pepsin has an optimal pH of 1.5-2, so a pH 2.14, 5 mM Citrate, 0.2 M NaCl buffer was made for Pepsin use. That may affect the results. Reaction Conditions The AuNP fibers were spun down at 300 RPM for 4 minutes. The supernatant was removed, and the prepared solutions were added on top of the solid fibers. The tubes were placed in a shaker at 37°C. They will be analyzed tomorrow. Results Proteinase K, Trypsin, and Chymotrypsin all had visible color changes. Pepsin did not have a visible color change. UV-VIS analysis can be found on tomorrow's page.