Monitoring of protein unfolding and folding with UV/Vis spectroscopy

In this application note, PerkinElmer demonstrates the ability of the LAMBDA^™ 365+ with Peltier Accessory and UV WinLab to monitor the unfolding and folding of a protein as the temperature is increased and decreased. α-chymotrypsinogen is a precursor, or zymogen, of chymotrypsin. Chymotrypsin fulfills the role of breaking down proteins into smaller peptides in the digestive tract. It also displays an interesting property in that, when it unfolds due to thermal denaturation, it is able to refold when cooled back to room temperature. A 0.5-mg/mL solution of α-chymotrypsinogen in 10-mM NaCl buffer (made to pH 3.0 with 1M HCl). This was measured using the PerkinElmer LAMBDA 365+ with Peltier accessory. In order to determine the correct wavelength for monitoring the stage of denaturation, two samples of the solution were measured between 200-500 nm at 25ºC and 85ºC. The two resulting spectra were subtracted to determine the wavelength showing the most variation. This wavelength was used to monitor denaturation over a range of temperatures.