Enzyme Assay

The general scheme of our fluorescence-based enzyme assay

After we have the CapD_CP mutants, we tested our mutants for their catalytic activity using our fluorescence-based enzyme assay scheme. Fluorescence-based enzyme assay measures the rate at which fluorescence in the testing media is released and the amount fluorescence depends on the rate at which fluorophore-quencher linkage is disrupted. Our substrate PDGA contains a linked fluorophore-quencher component. The faster fluorophore-quencher component is cleaved, the higher the amount of fluorescence is released and thus the greater the enzymatic activity observed.

Run one transpeptidase reaction and one hydrolysis reaction per enzyme (always run control for each set)

Prepare 10x Master Mix with these concentrations. (10uL/rxn)

• HEPES 7.4 pH (250mM)
• 1% Tween
• Substrate (500nM)

Dilute Enzymes

• Measure enzyme concentrations by protein gel band intensity or with spectrophotometer (A280)
• Dilute enzymes to 50nM (~0.0025mg/mL) in strip tubes
  • Make sure you have at least 30uL of your final diluted enzyme

Prepare a 96-well plate

• Pipette diH2O into each well (Two per enzyme)
  • Transpeptidase: 70uL
  • Hydrolysis: 80uL
• Pipette 10uL of 10x Master Mix into each well.
• Pipette 10uL of 50mM L-Glu into each transpeptidation reaction well.

Prepare Spectramax plate reader with desired settings and desired emission and excitation wavelengths

• Pipette 10uL of your enzyme into each well
• Immediately place into Spectramax plate reader and begin reading

Final Concentrations

• Enzyme (CapD) 5nM (0.00025mg/mL)
• Amino Acid (L-Glutamate)0mM or 5mM
• Substrate 50nM
• HEPES (7.4pH) 25mM
• 0.1% Tween
• Final Reaction Volume 100uL

Substrate is 5-FAM-(D-gamma-Glu)5-K(QXL520-NH2 from AnaSpec

Product produced from reaction is 5-FAM-(D-gamma-Glu)5-NH2 from AnaSpec