Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

Analytical Biochemistry
Pamlea N Brady, Megan A Macnaughtan

Abstract

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmod...Continue Reading

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Citations

Dec 21, 2020·Advances in Colloid and Interface Science·Joëlle Bizeau, Damien Mertz
May 18, 2021·Chemistry : a European Journal·Sanim RahmanSameer Varma
Oct 14, 2019·ACS Biomaterials Science & Engineering·Jeffrey R SimmonsJan K Rainey

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