Lidstrom:Enzyme Assay Basics

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Background

Resources:

• Basics of Enzymatic Assays for HTS. Though written for high-throughput screening, the principles apply to any enzyme assay.

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

Factors to control for

From Wikipedia 2013/11/1

• Salt Concentration:
  • Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1-500 mM. As usual there are exceptions such as the halophilic algae and bacteria.
• Effects of Temperature:
  • All enzymes work within a range of temperature specific to the organism. Increases in temperature generally lead to increases in reaction rates. There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates. This is due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme active site.[11] The "optimum" temperature for human enzymes is usually between 35 and 40 °C. The average temperature for humans is 37 °C. Human enzymes start to denature quickly at temperatures above 40 °C. Enzymes from thermophilic archaea found in the hot springs are stable up to 100 °C.[12] However, the idea of an "optimum" rate of an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates, the reaction rate and the denaturation rate. If you were to use an assay measuring activity for one second, it would give high activity at high temperatures, however if you were to use an assay measuring product formation over an hour, it would give you low activity at these temperatures.
• Effects of pH:
  • Most enzymes are sensitive to pH and have specific ranges of activity. All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds. Most enzymes function between a pH of 6 and 8; however pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8.
• Substrate Saturation:
  • Increasing the substrate concentration increases the rate of reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate will plateau.
• Level of crowding
  • Large amounts of macromolecules in a solution will alter the rates and equilibrium constants of enzyme reactions, through an effect called macromolecular crowding.[13]

Preparing Samples

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

Assaying

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.

Validity Check

• If you reduce the cell extract 2x, do you see Vmax reduce by 2x? (a desired result)
  • Note: crowding does change, so your numbers may not be ideal.
• If you double the substrate or cofactor concentration(s), does the rate remain unchanged?
  • If the rate changes, it is possible inhibition is occurring. For example, assays that use ATP have ATP degrade to ADP at some rate. If ADP inhibits your assay, then increased in ATP may lead to decreases in Vmax.

Data Processing

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. Normalizing by protein concentration should be done subsequently.

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.

Open Questions

How high can the NADH concentration be before the detector is saturated?

• Will be dependent on the path length.
• Can be tested by making a calibration curve with increasing [NADH]