ABSTRACT
This chapter provides the reader with examples of the utility of joint application of size-exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy to the study of protein stability. It describes the experimental conditions and calibrations involved in the use of SEC in solvent denaturation. The chapter also describes Urea denaturation studies of myoglobin and T4 lysozymes in high and low salt by both CD and SEC. Urea denaturation of myoglobin, as measured by both CD and SEC was reported by R. J. T. Corbett and R. S. Roche. They noted that, as the urea concentration in the mobile phase increased, early and late elution volume peaks were observed. Denaturation by urea of T4 lysozyme, as observed by means of CD in the peptide backbone region of the spectrum, is also sigmoidal in shape. In general, addition of SEC to optical or calorimetric measurements adds an additional dimension of insight into the area of protein stability.
