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)

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.

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 special enzymes.