Development of a protein assay with copper chelator chromeazurol B, based on the biuret reaction

Eisaku Hokazono, Eri Ota, Taiki Goto, Saori Fukumoto, Yuzo Kayamori, Takeshi Uchiumi, Susumu Osawa

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1 Citation (Scopus)


This study aimed to provide a novel and highly sensitive protein assay based on the biuret reaction and using chromeazurol B, a metal chelate compound. The method consists of two reagents and an automated analyzer. First, a complex of copper and protein (biuret reaction) is formed. Second, a chelating reagent containing chromeazurol B forms a three-dimensional complex of protein, copper, and chromeazurol B at neutral pH, resulting in highly sensitive coloration. The intra-assay (n = 20) variation for the three levels was 3.54 % or lower at each concentration. Each response with α, β-, and γ-globulin was 103.8 % and 104.3 %, respectively, against albumin. The molar absorption coefficient (ε) of the present method was 2.5 × 105 m2/mol against human albumin, higher than that of the commercially available Lowry method (ε = 8.7 × 104 m2/mol), which is based on the same principle. The correlation test for the pyrogallol method with 30 urine samples showed good performance (r = 0.961). The method described here (the Biuret-based CAB method) is a more sensitive and rapid assay than the Lowry method, and it may also be applied to biological samples because of its similar reactivity towards various proteins.

Original languageEnglish
Article number114320
JournalAnalytical Biochemistry
Publication statusPublished - Oct 1 2021

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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