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. Author manuscript; available in PMC: 2022 Oct 28.
Published in final edited form as: Nat Microbiol. 2022 Apr 28;7(5):680–694. doi: 10.1038/s41564-022-01103-1

Table 2.

Enzymatic kinetics of trypsin inhibition by metabolites in bilirubin deconjugation pathway

Inhibitor Name Structure Kobs (s−1) k2/Ki (M−1s−1) IC50 (μM)
Biliverdin graphic file with name nihms-1787854-t0014.jpg 0.001822 181.8182 12.68
Conjugated Bilirubin graphic file with name nihms-1787854-t0015.jpg 0.004937 500 4.679
Unconjugated Bilirubin graphic file with name nihms-1787854-t0016.jpg 0.006642 666.6667 3.478
Mesobilirubin graphic file with name nihms-1787854-t0017.jpg 0.005981 588.2353 3.862
Urobilinogen graphic file with name nihms-1787854-t0018.jpg 0.004785 476.1905 4.828

Inhibition constants were measured in a 0.046M Tris-HCl, 0.0115M CaCl2, held at a pH 8.1 and 37°C. 10ug/mL of bovine pancreatic trypsin was used with the substrate N-p-Tosyl-Gly-Pro-Arg 7-amido-methylcoumarin HCl stock at 100μM. Each well has a final volume of 150μL: 3μL trypsin, 3μL of inhibitor, 69μL of buffer, 75μL of buffered substrate. The second-order inhibition rate (k2/Ki (M−1s−1)) was generated by carrying out a series of experiments at different concentrations of the potential inhibitory metabolite(s). Non-linear regression modelling was done to determine the IC50 values for the metabolites within the bilirubin deconjugation pathway (n=3 replicates/metabolite).