Photometric analysis of proteins and peptides at 191–194 mμ

Abstract

The light absorption band of the peptide bond at 191–194 mμ has been used for quantitative measurement of purified peptides and proteins with the Beckman DBG spectrophotometer. In comparison to measurement of proteins at 280 mμ those conducted at 191–194 mμ are approximately 40–80 times more sensitive, depending on the protein. This is a significant advantage, especially in monitoring of effluents from chromatographic columns. Specific absorbance values of different proteins at 191–194 mμ vary less widely than those at 280 mμ and, therefore, in cases in which proteins cannot be analyzed in terms of nitrogen or protein weight, analysis by photometry at 191–194 mμ is subject to less uncertainty than that at 280 mμ. The average specific absorbance ( A 1cm 1%) for 5 proteins was 686 with the Beckman DBG spectrophotometer. With this instrument we have observed direct proportionality between absorbance and concentration up to an absorbance of about 0.7. All anions except fluoride interfere since they absorb near 190 mμ. Therefore, the only electrolytes which can be used in appreciable concentration are those containing fluoride as anion.