Abstract
Publisher Summary Knowing the extinction coefficient, ɛ , of a protein is important for many biochemical and biophysical studies. In size-exclusion chromatography with online light-scattering, uv absorbance, and refractive index detectors (SEC-LS/UV/RI) used in the study of molecular weights of glycosylated proteins and protein–carbohydrate complexes, it is apparent that when ɛ of the polypeptide is known, it is possible to combine the information from all three detectors to obtain the polypeptide molecular weight of the complex. Also for many other reasons, it would be useful to have a convenient method to measure ɛ . For non-glycosylated proteins, it has been found that the signals from refractive index (RI) and absorbance (UV) detectors provide a simple chromatographic method for ɛ determination with a reasonable accuracy, and thus when SEC-LS/UV/RI experiments are done, the data for determining ɛ are available without extra effort. This chapter describes a method of using RI and UV detectors to determine the extinction coefficients of non-glycosylated proteins and present results for several commercial proteins. Then it discusses applications of this method for studying protein interactions when using SEC-LS/UV/RI. Focus lies on the on-line chromatographic determination of extinction coefficients of non-glycosylated proteins and its applications in SEC-LS/UV/RI.
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