Protein Unfolding of metMyoglobin Monitored by Spectroscopic Techniques

Abstract

In this experiment the unfolding of the protein, myoglobin, will be monitored using both fluorescence and UV-vis absorption spectroscopy. Changes in the absorbance at 409.5 nm, the absorption maximum of the native state, will be monitored in order to probe changes in the protein conformation after initiation of unfolding by addition of a chemical denaturant. Protein unfolding will also be monitored after exciting the sample at 280 nm and following protein fluorescence emission at 345 nm, the fluorescence maximum for the unfolded state. The absorption run follows the time-dependent decrease in the concentration of the native-state species, whereas the fluorescence experiment monitors the increase in the concentration of the unfolded state. Kinetic rate constants obtained using the two techniques will be compared.