Interference study should be performed for every protein measurement method used

Abstract

It is particularly important, when analyzing biological material, for the measurement procedure to be specific to the analyte and not to suffer interference by the matrix effect. Tissue fraction studies also require rapid and accurate methods to estimate the concentration of protein in solutions as well as many measurement methods used in medical laboratories. The design of this study is based on a comparison of the Lowry and the bicinchoninic acid (BCA) methods for the measurement of the total protein concentrations of rat liver subcellular fractions. In our experiment, subcellular fractions enriched in peroxisomes (POs) obtained by differential centrifugation were then further separated by means of density gradient centrifugation. We performed the protein measurement assays on all fractions obtained during the purification steps. The protein contents of the fractions obtained were determined by the two methods. The method comparison statistics were performed by linear Deming regression analysis and Altman and Bland bias plot. The regression equation was unacceptable, indicating that the last three fractions separated by means of Nycodenz discontinuous density gradient centrifugation gave remarkably divergent results. For the Lowry method, the Nycodenz effect could not be eliminated with the use of interference blank. In addition to Nycodenz, the potentially interfering compound used in the isolation procedure as isolation medium was 3-(morpholino)propane sulfonic acid (MOPS). In decreased concentrations of MOPS (10 mM), interference blank should be used for correct measurement with Lowry, but in practical use 10 mM does not provide buffering potency. In the BCA method, interference blank correction seemed to eliminate the measurement error in all concentrations of Nycodenz. There was no MOPS effect on the BCA measurement assay. Referring to deviations as sample-inherent matrix effects, we concluded that not only one, but more measurement methods should be used in order to make a correct protein measurement.