Evaluation of amino acid analysis as reference method to quantitate highly purified proteins.

The applicability of amino acid analysis for accurate quantitation of reference standard preparations of proteins has been evaluated. This approach is very useful since, in addition to absolute quantitative information, it also provides a measure of composition, partial identity, and purity in a single experiment. Comparisons with Kjeldahl nitrogen assay and/or UV measurements shows that amino acid analysis is reliable for the quantitation of small-to-medium size proteins in the molecular weight range of 6-22 kDa. For larger proteins such as immunoglobulins (150 kDa), amino acid analysis may "underestimate" the total protein concentration. These results also show the effect of recovery of individual residues on protein quantitation. As expected, the recovery of more than one stable residue could be used to calculate total protein content of samples, which is in good agreement with the results obtained by Kjeldahl nitrogen assay. However, the protein concentrations calculated from the total mass of the recovered residues appear to give relatively low estimates in almost all cases. Thus, it is concluded that amino acid analysis is an appropriate reference method only when stable residues are employed for quantitation, particularly for highly purified proteins of rDNA origin.