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Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights
- Source :
- Analytical Biochemistry. 491:43-51
- Publication Year :
- 2015
- Publisher :
- Elsevier BV, 2015.
-
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 unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.
- Subjects :
- Reductive methylation
Lysine
Biophysics
Methylation
Biochemistry
Article
Acid dissociation constant
Absorbance
Rosaniline Dyes
Animals
Bicinchoninic acid assay
skin and connective tissue diseases
Colorimetry
Molecular Biology
Bradford protein assay
Chromatography
Chemistry
Proteins
Serum Albumin, Bovine
Cell Biology
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Quinolines
Cattle
Subjects
Details
- ISSN :
- 00032697
- Volume :
- 491
- Database :
- OpenAIRE
- Journal :
- Analytical Biochemistry
- Accession number :
- edsair.doi.dedup.....ccc9064ef2f930a3baedc5079136d2d5