Second-Derivative Spectroscopy of Proteins. A Method for the Quantitiative Determination of Aromatic Amino Acids in Proteins

Abstract

Second derivative spectroscopy was used to resolve the complex protein spectrum in the near-UV region and the contributions of the 3 aromatic chromophores were evaluated. A method for the direct quantitative determination of phenylalanine and tryptophan in proteins was carried out. Phenylalanine determination was carried out in the spectral region between 250 and 265 nm, where there are no significant contributions from other aromatic chromophores. Tryptophan determination was performed in the 290-295 nm region and the experimental values have been corrected for the presence of tyrosine. The results obtained on 10 highly purified proteins [bovine serum albumin, .alpha.-chymotrysinoqen, human Hb, sperm whale myoglobin, egg lysozyme, bovine liver catalase, horse heart cytochrome c, papain, beef ferrimyoglobin and apomyoglobin] were in good agreement with those obtained from sequence analysis.