A novel procedure for determining protein concentrations from absorption spectra of enzyme digests

A novel procedure for determining molar extinction coefficients ( E M), and hence protein concentrations, has been tested on ribonuclease A, bovine serum albumin, β-lactoglobulins A and B, α-lactalbumin, chymotrypsinogen A, and lysozyme. E M values were obtained from a combination of the absorption spectrum of the native protein and a difference spectrum generated between two aliquots of a thermolysin digest of the protein titrated to pH 1.5 and pH ≥ 13. Enzymatic digestion was shown to minimize conformational contributions to the difference spectrum. The E M of the native protein is then calculated from the tyrosine molarity of the digest and the previously determined tyrosine content of the protein. These E M values displayed a maximum error of −2.2% and an average absolute error of 1.4%. Samples as small as 100 μg give satisfactory results. Accurate sample weight and assumptions with regard to moisture and ash content are not required. In addition, the use of second-derivative (second-order) absorption spectroscopy to access the degree of spectral normalization produced by enzymatic digestion is described.