title: The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay.
abstract: Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu(2+)) to cuprous ions (Cu(1+)), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.
authors.affiliation: Tissue Repair and Regeneration Program, Brisbane, QLD 4059, Australia. Electronic address: dayle.sampson@qut.edu.au.
authors.forename: D L
authors.initials: DL
authors.lastname: Sampson
authors.forename: Y L
authors.initials: YL
authors.lastname: Chng
authors.forename: Z
authors.initials: Z
authors.lastname: Upton
authors.forename: C P
authors.initials: CP
authors.lastname: Hurst
authors.forename: A W
authors.initials: AW
authors.lastname: Parker
authors.forename: T J
authors.initials: TJ
authors.lastname: Parker
mesh_terms: D003300:Copper / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D011804:Quinolines / Q000737:chemistry* / Q000378:metabolism; D012016:Reference Values; D012680:Sensitivity and Specificity; D014557:Urobilin / Q000378:metabolism* / Q000652:urine*
chemical_list: D011506:Proteins; D011804:Quinolines; D014557:Urobilin; D003300:Copper; C047117:bicinchoninic acid
keywords: Bicinchoninic acid; Bile pigment; Bradford; Limits of agreement analysis; Protein determination; Urobilin