Protein quantification by bicinchoninic acid (BCA) assay follows complex kinetics and can be performed at short incubation times.
Amongst the available methodologies for protein determination, the bicinchoninic acid (BCA) assay highlights for its simplicity, sensitivity, repeatability and reproducibility. Nevertheless, in spite that the general principle behind this methodology is known, there are still unanswered questions regarding the chemistry behind the assay and the experimental conditions commonly employed. The present work explored the kinetics, and the analytical response of the assay to free amino acids, peptides (containing tryptophan and tyrosine), and proteins. Results revealed kinetic profiles characterized by the absence of plateaus, with behaviors depending on the type of the sample. The latter, along with contribution to the BCA index elicited by oxidation products generated at the side chain of tryptophan and tyrosine, as well as pre-oxidized β-casein, evidenced the presence of complex reaction mechanisms. In spite of such complexity, our results showed that the BCA index is not modulated by the incubation time. This applies for responses producing absorbance intensities (at 562 nm) higher than 0.1. Therefore, we propose that the assay can be applied at shorter incubation times (15 min) than those indicated in manufactures specifications, and usually used by researches and industry (30 min at 37 °C).
['Cortés-Ríos|Javiera|J|', 'Zárate|Ana María|AM|', 'Figueroa|Juan David|JD|', 'Medina|Joaquín|J|', 'Fuentes-Lemus|Eduardo|E|', 'Rodríguez-Fernández|María|M|', 'Aliaga|Margarita|M|', 'López-Alarcón|Camilo|C|']
D000596:Amino Acids D000818:Animals D006801:Humans D007202:Indicators and Reagents D007700:Kinetics D016014:Linear Models D010084:Oxidation-Reduction D010455:Peptides D011506:Proteins D011804:Quinolines D015203:Reproducibility of Results D013053:Spectrophotometry D013997:Time Factors
Protein quantification Bicinchoninic acid BCA assay Myofibrillar protein extracts Tryptophan oxidation Tyrosine oxidation