Application of the Ninhydrin Reaction for Quantification of Total Protein Contents: Establishment of Conversion Formulas

The ninhydrin assay is an interesting alternative for protein quantification to the widely used Kjeldahl and elemental analysis methods. The two latter methods only measure the nitrogen content that must be converted into the protein content using empirical factors. In contrast, the ninhydrin reaction is more selective and reacts with primary amines including α-amino acids but not with nitrate and nucleic acids, the main source of nonprotein nitrogen in biological samples. Here, we determined the protein contents of a number of samples with the Kjeldahl method and elemental analysis and measured the amount of liberated amino acids using the ninhydrin assay. These results allowed establishing a universal formula ω­(P) = (AA – 0.21) × 11.2 for conversion of the amino acid content (AA, in mmol g–1) into the protein content (ω­(P) in % (w/w)) for solid samples. For liquid samples, the formula c­(P) = (AAH – AA0) × 120 was established, where c­(P) is the protein concentration in mg mL–1 and AAH and AA0 are the measured amino acid equivalents in mmol mL–1 with and without hydrolysis, respectively. The method showed good intraday and interday reproducibility and gave more accurate results than the Kjeldahl assay when applying the default conversion factor of 6.25.