The enzyme yeast alcohol dehydrogenase (YADH) is used in the undergraduate biochemistry lab class at Miami University to teach students essential biochemical methods and concepts. Specifically, the students compare the wild type enzyme to overexpressed His-tagged YADH. An experiment was designed for students to monitor protein stability over time using recently “upcycled” HP 8453 UV/Vis spectrophotometers. The instruments were equipped with Peltier cell holders and new software by Olis (Athens, GA). A 927 nM YADH solution in a 50 mM phosphate buffer at pH 7.0 was incubated at temperatures of 10, 25, 37, and 55 °C. The denaturation of the protein was accompanied by aggregation and turbidity, which was monitored by collecting absorbance readings at 310 nm every 20 minutes for 3 hours and 20 minutes. The experiment worked well in graduate student hands, but several issues without a clear source became apparent once the experiment was performed in the lab. Discrepancies in the students’ showed problems with the reproducibility of the data. Additionally, the starting absorbance of the protein solution was very high in certain experimental runs when it should have always been close to zero. I am optimizing the previous method for this experiment so as to solve these issues and make any other improvements in the protocol for using the thermally controlled UV/Vis. I have been focused on improving the conditions of the initial YADH solution to keep the starting absorbance measurement low as well as preventing unwanted precipitate from forming throughout the experiment. In doing this research, I hope to provide valuable information about the protocol using the upcycled instruments that can be standardized across instruments and improve students’ data collection.
Author(s): Kathryn Williams, Chemistry Major
Advisor(s): Ann Hagerman, Department of Chemistry and Biochemistry
Alison Bates, Department of Chemistry and Biochemistry
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