Tracking Enzymatic Hydrolysis of an Amide Bond Using Highly Simplified 4-nitroanilide Colorimetric Substrates



  1. López-Otín, C., & Bond, J. S. (2008). Proteases: Multifunctional Enzymes in Life and Disease. Journal of Biological Chemistry, 283(45), 30433–30437. doi: 10.1074/jbc.r800035200
  2. Hedstrom, L. (2002). Serine protease mechanism and specificity. Chemical reviews, 102(12), 4501-4524. doi: 10.1021/cr000033x
  3. Chen, H., McGowan, E. M., Ren, N., Lal, S., Nassif, N., Shad-Kaneez, F., … & Lin, Y. (2018). Nattokinase: a promising alternative in prevention and treatment of cardiovascular diseases. Biomarker insights, 13, 1177271918785130. 10.1177/1177271918785130
  4. Guo, H., Ban, Y.-H., Cha, Y., An, E. S., Choi, J., Seo, D. W., … Kim, Y.-B. (2019). Comparative anti-thrombotic activity and haemorrhagic adverse effect of nattokinase and tissue-type plasminogen activator. Food Science and Biotechnology, 28(5), 1535–1542. doi: 10.1007/s10068-019-00580-1
  5. Johnston, N., Dettmar, P., Bishwokarma, B., Lively, M. O., Koufman, J. A. (January 2009). Activity/Stability of Human Pepsin: Implications for Reflux Attributed Laryngeal Disease. The Laryngoscope, 117(6), 1036-1039. doi: 10.1097/MLG.0b013e31804154c3
  6. Heinrich, H. C., Gabbe, E. E., & Icagic, F. (1979). Immunoreactive serum trypsin in diseases of the pancreas. Klinische Wochenschrift, 57(22), 1237–1238. doi: 10.1007/bf01489252
  7. Fahrenkrug, J., and E. Magid. “Concentration of immunoreactive trypsin and activity of pancreatic isoamylase in serum compared in pancreatic diseases.” Clinical chemistry 26.11 (1980): 1573-1576. doi: 10.1093/clinchem/26.11.1573
  8. Acker, M. G., & Auld, D. S. (May 2014). Considerations for the design and reporting of enzyme assays in high-throughput screening applications. Perspectives in Science, 1(1-6), 56–73. doi: 10.1016/j.pisc.2013.12.001
  9. Charney, J., & Tomarelli, R. M. (1947). A colorimetric method for the determination of the proteolytic activity of duodenal juice. J Biol Chem, 171(2), 501-505.
  10. Yang, D., Park, H., Yoo, T. (September 2016). A colorimetric protease activity assay method using engineered procaspase-3 enzymes. Analytical Methods, 33, 6247-6374. doi: 10.1039/C6AY01256F.
  11. Aldersley, F. M., Kirby, J. A., Lancaster, W. P., McDonald, S. R., Smith, R. C. (1974). Intramolecular catalysis of amide hydrolysis by the carboxy-group. Rate determining proton transfer from external general acids in the hydrolysis of substituted maleamic acids. Journal of the Chemical Society, Perkin Transactions 2, 12, 1487-1495. doi: 10.1002/chin.197451193
  12. Neese, F. (2012). The ORCA program system, Wiley Interdiscip. Rev.: Comput. Mol. Sci., 2, 73-78.