Lidstrom:Enzyme Assay Basics

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Cell Pellet Prep

General guidelines:

• 50 mL of E. coli in LB/TB is usually plenty. (stationary phase)
• If using a methylotroph, you may need more culture volume. Some strains are "sick" and don't grow very turbid. Use:
  • 100 mL of at OD 0.6-0.8 ~or~
  • 200 mL at OD = 0.4 ~or~
  • 300 mL at OD = 0.2

Of course the amount of biomass depends on how well your enzyme is expressed and what its specific activity is.

Lysis

• Use a similar mass of cells for different strains you are breaking.
  • This allows for more accurate BCA results. JM (10/2013) finds strong dependency on protein concentration calculations depending on the dilution used when dilutions span an order of magnitude.
• Resuspend in 2 mL of an appropriate lysis buffer
• French press 2-3 times.
• Centrifuge out debris. Perhaps ultracentrifuge.

Analyze protein concentration

• 1000 ug/mL is good, says Ceci

Assay cells

• Ceci/Amanda always do 200 uL in assays. There is no reason not to do 100 or 150 uL though. JM (10/2013)
• Ceci adds 50 uL per rxn. She said you don't want to dilute the enzymes more than you need to, as they are most happy when concentrated.
  • This may require that you use more substrate. Hopefully your substrate is cheap.
  • If you are doing an NADH-linked assay, there is a limit to the amount of NADH you can add, as you will saturate the spectrophotometer.

Calculating Enzyme Rates

• The slope provided by the plate reader needs to be converted to moles/time/mg protein or similar units.
• Convert absorbance to M
  • If using NADH, the extinction coefficient at 340nm is 6220 M^-1cm^-1
    • If there is less volume in the well, the conversion factor is greater because the path length is larger.
• Factor in the path length.
  • If using the crystal plate in the plater reader, 200 uL is 0.51 cm. (Elizabeth "Betsy" Skovran figured this out)
• Normalize for protein concentration.
• How to convert units from plate reader to mM/min or uM/min:

Sample calculation for converting the milli-units/min provided by the plate reader to concentration change over time. Calculations assume NADH is being monitored at 340 nm and allows for a flexible number of microliters of total reaction in the well.

Controls

• The basic set of tests you should do:
  • + enzymes + substrate
  • + enzymes - substrate
  • - enzymes + substrate (strain = empty vector control or equivalent)
  • - enzymes - substrate (strain = empty vector control or equivalent)
• Mary likes to see a plot like

  

  cartoon of a good way to depict enzyme assay data. Each bar is Vmax for reaction with substrate - Vmax for rxn without substrate.

• You could also subtract the empty vector height from the control height, but Mary said she prefers to see them separately. JM 10/31/2013

Example of Preparing for an assay that monitors NADH consumption

• First, determine how fast NADH oxidizes in your assay environment. It is pH and buffer dependent. It may not be zero.
• Determine how fast the reaction proceeds in the absence of your enzymes at various substrate concentrations.
  • Example: look at Vmax as you increase [formate] for an assay that has formate as a substrate and NADH as the cofactor and substance you are monitoring. You should have an increase in Vmax as you increase [formate] because there are formate dehydrogenases present. As you increase [formate] you will saturate these enzymes (curve 1 in the picture below). You may see that increases in [formate] lead to decreases in Vmax (curve 2 in the cartoon below); this is caused by inhibition. You want to chose a value of formate that is high enough to saturate the background FDHs if you want to observe the rate caused by enzymes you are adding.

cartoon of adding increasing [formate] to a strain not expressing the enzymes you are testing for.