Two major modifications of the biuret test are commonly applied in modern colorimetric analysis of peptides: the bicinchoninic acid (BCA) assay and the Lowry assay. In these tests, the Cu+ formed during the biuret reaction reacts further with other reagents, leading to a deeper color.

In the BCA test, Cu+ forms a deep purple complex with bicinchoninic acid (BCA),[1] which allows proteins in the range of 0.0005 to 2 mg/mL to be determined. This assay is often referred to as "Pierce assay" after the manufacturer of a reagent kit. The complex of Cu+ with BCA absorbs around 562 nm, producing the signature violet color. The BCA protein assay increases the sensitivity of the biuret test by a factor of around 100, and gives the important benefit of compatibility with samples that contain up to 5% surfactants. The water soluble BCA/copper complex absorbs much more strongly than the peptide/copper complex.

In the Lowry protein assay Cu+ is oxidized back to Cu2+ by MoVI in Folin-Ciocalteu's reagent, which forms molybdenum blue (MoIV). Tyrosine residues in the protein also form molybdenum blue under these circumstances. In this way, proteins can be detected in concentrations between 0.005 and 2 mg/mL.[2] Molybdenum blue in turn can bind certain organic dyes such as malachite green and Auramin O, resulting in further amplification of the signal.[3]

1. Smith, P.K. et al.: Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1985) 76-85.
2. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall: Protein Measurement With the Folin Phenol Reagent, J. Biol. Chem. 193 (1951) 265 - 275.
3. Sargent, M.G.: Fiftyfold amplification of the Lowry protein assay. Anal. Biochem. 163 (1987) 476-481.

【wkh, 2014-12-03 12:44:09】 【责任人 wkh】 [已阅读 1172 次]