A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases

  • Gregory Arnal
  • Mohamed A. Attia
  • Jathavan Asohan
  • Harry BrumerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1588)


The quantitation of liberated reducing sugars by the copper-bicinchoninic acid (BCA) assay provides a highly sensitive method for the measurement of glycoside hydrolase (GH) activity, particularly on soluble polysaccharide substrates. Here, we describe a straightforward method adapted to low-volume polymerase chain reaction (PCR) tubes which enables the rapid, parallel determination of GH kinetics in applications ranging from initial activity screening and assay optimization, to precise Michaelis–Menten analysis.

Key words

Glycoside hydrolase (GH) Glycosidase Carbohydrate-active enzymes (CAZymes) Copper-bicinchoninic acid (BCA) Polysaccharide Enzymology Reducing sugar 



Funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) via the Strategic Partnership Grants for Networks (for the Industrial Biocatalysis Network) and Discovery Grant programs is gratefully acknowledged. Equipment infrastructure was funded by the Canada Foundation for Innovation and the British Columbia Knowledge Development Fund. We thank Sean McDonald (Brumer group, UBC) for comments on an early version of this chapter.


  1. 1.
    Sinnott M (2013) Carbohydrate chemistry and biochemistry: structure and mechanism, 2nd edn. RSC Publishing, LondonGoogle Scholar
  2. 2.
    Mopper K, Gindler EM (1973) New noncorrosive dye reagent for automatic sugar chromatography. Anal Biochem 56:440–442CrossRefPubMedGoogle Scholar
  3. 3.
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefPubMedGoogle Scholar
  4. 4.
    McFeeters RF (1980) A manual method for reducing sugar determinations with 2,2'-bicinchoninate reagent. Anal Biochem 103:302–306CrossRefPubMedGoogle Scholar
  5. 5.
    Waffenschmidt S, Jaenicke L (1987) Assay of reducing sugars in the nanomole range with 2,2'-bicinchoninate. Anal Biochem 165:337–340CrossRefPubMedGoogle Scholar
  6. 6.
    Doner LW, Irwin PL (1992) Assay of reducing end-groups in oligosaccharide homologs with 2,2'-bicinchoninate. Anal Biochem 202:50–53CrossRefPubMedGoogle Scholar
  7. 7.
    McIntyre AP, Mukerjea R, Robyt JF (2013) Reducing values: dinitrosalicylate gives over-oxidation and invalid results whereas copper bicinchoninate gives no over-oxidation and valid results. Carbohydr Res 380:118–123CrossRefPubMedGoogle Scholar
  8. 8.
    Garcia E, Johnston D, Whitaker JR, Shoemaker SP (1993) Assessment of endo-1,4-beta-d-glucanase activity by a rapid colorimetric assay using disodium 2,2'-bicinchoninate. J Food Biochem 17:135–145CrossRefGoogle Scholar
  9. 9.
    Fox JD, Robyt JF (1991) Miniaturization of 3 carbohydrate analyses using a microsample plate reader. Anal Biochem 195:93–96CrossRefPubMedGoogle Scholar
  10. 10.
    Kenealy WR, Jeffries TW (2003) Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening. Biotechnol Lett 25:1619–1623CrossRefPubMedGoogle Scholar
  11. 11.
    Meeuwsen PJA, Vincken JP, Beldman G, Voragen AGJ (2000) A universal assay for screening expression libraries for carbohydrases. J Biosci Bioeng 89:107–109CrossRefPubMedGoogle Scholar
  12. 12.
    Stoll VS, Blanchard JS (1990) Buffers: principles and practice. In: Deutscher MP (ed) Methods in enzymology: guide to protein purification. Academic Press, Cambridge, MA, pp 24–38.Google Scholar
  13. 13.
    Deutscher MP (1990) Maintaining protein stability. In: Deutscher MP (ed) Methods in enzymology: guide to protein purification. Academic Press, Cambridge, MA, pp 83–89CrossRefGoogle Scholar
  14. 14.
    Cornish-Bowden A (2012) Practical aspects of kinetics. In: Fundamentals of enzyme kinetics, 4th edn. Wiley-Blackwell, Hoboken, NJ, pp 85–106Google Scholar
  15. 15.
    Attia M, Stepper J, Davies GJ, Brumer H (2016) Functional and structural characterization of a potent GH74 endo-xyloglucanase from the soil saprophyte Cellvibrio japonicus unravels the first step of xyloglucan degradation. FEBS J 283:1701–1719CrossRefPubMedGoogle Scholar
  16. 16.
    Kongruang S, Han MJ, Breton CIG, Penner MH (2004) Quantitative analysis of cellulose-reducing ends. Appl Biochem Biotechnol 113:213–231CrossRefPubMedGoogle Scholar
  17. 17.
    Abdelakher M, Hamilton JK, Smith F (1951) The reduction of sugars with sodium borohydride. J Am Chem Soc 73:4691–4692CrossRefGoogle Scholar
  18. 18.
    Skoog DA, Holler FJ Crouch SR (2006) Appendix I. In: Douglas A et al (eds) Principles of instrumental analysis, 6th edn. Brooks Cole, Pacific Grove, CA.Google Scholar
  19. 19.
    Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, New York City, NY, pp 571–607CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Gregory Arnal
    • 1
  • Mohamed A. Attia
    • 1
    • 2
  • Jathavan Asohan
    • 1
  • Harry Brumer
    • 1
    • 2
    • 3
    Email author
  1. 1.Michael Smith LaboratoriesUniversity of British ColumbiaVancouverCanada
  2. 2.Department of ChemistryUniversity of British ColumbiaVancouverCanada
  3. 3.Department of Biochemistry and Molecular BiologyUniversity of British ColumbiaVancouverCanada

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