Determination of absorption coefficients of purified proteins by conventional ultraviolet spectrophotometry and chromatography combined with multiwavelength detection.

The direct, ultraviolet spectrophotometric determination of protein absorption coefficients was found to be more reproducible and accurate when diluting was replaced by chromatography and multiwavelength detection. Four different ultraviolet spectrophotometric methods, described in the literature, were compared by calculating A0.1%280 values from the spectra of 25 proteins, obtained by chromatography. Only two methods, i.e., one based on the absorbance at 210 nm and the other on the absorbance at 205 nm, corrected for the absorbance of aromatic amino acids at that wavelength, were sufficiently accurate to be of potential use for the determination of unknown proteins. It was found, however that with uncorrected A203 values even better results could be achieved. Using 7 well-defined proteins the equation A0.1%280 = 38.69 X A280/A203 - 0.01 was established by linear regression. A0.1%280 values for 14 pure proteins calculated with this equation showed a mean deviation of only 4% from literature values. Since similar deviations were seen with 5 chromophoric and 7 glycoproteins, 3 and 7% respectively, the method may have universal applicability. In the configuration used, only 40 micrograms of a protein is required for the chromatographic determination of its absorption coefficient.