Tracking Enzymatic Hydrolysis of an Amide Bond Using Highly Simplified 4-nitroanilide Colorimetric Substrates
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Manuscript has been accepted by National High School Journal of Science, 2020
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Alameda County Science and Engineering Fair, 2019 - 2020
- 4th Place in Biochemistry | Microbiology | Molecular Biology Category, March, 2020
ABSTRACT
Proteases, enzymes that hydrolyze peptide bonds, have tremendous implications in the life sciences, ranging from the treatment of various diseases to enabling chemoselective bond cleavage in chemical synthesis in chemical synthesis. The kinetics of proteases can be monitored spectroscopically using nitroanilide substrates, many of which are structurally complex and often require long, multi-step syntheses. This study sought to determine the rate of hydrolysis by various serine proteases—nattokinase, trypsin, and pepsin— on two highly simplified 4-nitroanilide
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colorimetric substrates—4-nitroacetanilide and 4-nitrobenzanilide—via UV-visible (UV-vis) spectroscopy. Each substrate was chemically synthesized in one step via acylation of 4-nitroaniline. Solutions of both substrates were prepared, incubated with buffered enzyme solutions, and enzymatic cleavage of the amide bond was spectroscopically monitored on timecourse. After experimentation, it was determined that nattokinase and trypsin were able to hydrolyze the amide bond of 4-nitroacetanilide at 25e-10 moles per hour and 4e-10 moles per hour respectively, while the results for pepsin were inconclusive as 4-nitroacetanilide is unstable in acidic conditions. None of the proteases were able to hydrolyze the amide bond in 4-nitrobenzanilide. Computational studies suggest that this is a result of differences in amide reactivity between the two substrates rather than differences in binding affinity to each protease. [Full Article Link]