Lab 10: PROCEDURE

Setting up the spectrophotometer

• Turn on the Shimazdu Spectrophotometers and allow it to warm up (this takes about 15 min.)
• Set the following parameters on the Shimazdu Spectrophotometers:
  • From the main menu, select Kinetics (# 4)
  • From the next menu, select 1 cell kinetics/λ (#1)
  • You will need to alter the program parameters. Use the arrows to move through the different fields.
    • Measure time = 40 seconds Δ = 0.1
    • Factor – leave this alone
    • Recording range = -0.5 – 1.0 A
    • Temp con. None
    • Time scale: sec
  • Next hit the “Goto WL” button. Then type in 405. The spectrophotometer should change the wavelength to 405 nm.
• Note there are also spectrophotometer instructions in the Appendix (link opens in a new tab): Appendix 5: Operation of the Shimadzu spectrophotometers

Setting up the assay: Pilot reactions

Since we do not know what effect your mutation will have on HCAII’s activity, we recommend that you assay the wild type HCAII first. That way, you will know that any change in activity you see in the mutant is due to the mutation and not to the assay itself.

The following video demonstrates the process of running a kinetics experiment:

• First you will run a test experiment, or pilot, to make sure you are using the correct amount of enzyme and that your assay is working. The pilot reaction should contain:
  • A final protein concentration of ~0.1 µM
  • 20 µL of 50 mM PNPA (for a final concentration of 1 mM)
  • Buffer to a total of 1 mL
• We recommend assembling the reaction directly in the cuvette (this also saves plastic). Add buffer first, then protein. Always add PNPA last, when you are ready to measure.
• Once the PNPA has been added to the reaction you will need to act quickly. Make sure your reaction is well mixed before measuring  – mix by gently pipetting up and down with a p1000.
• Your reaction should be in the front slot of the spec and your blank (what are you using as a blank?) should be in the back.
• Once both samples are in the spectrophotometer hit “START” immediately.
• Once the reaction has finished, look at the graph. If the line runs off the top of the graph (i.e. the absorbance went above 2) within the first 10 seconds, the reaction rate is too fast and you need to reduce the amount of enzyme you are using. Next, press the “Datalist” button to see the slope of your reaction. Your slope should be around 0.1. If it is not, increase or decrease the amount of enzyme you are using. (If you are in the correct range, you can repeat the assay twice more and use this as one of the substrate concentrations in your data set.) If you do not have enough enzyme to do the remaining assays, reduce the amount of enzyme you are using as much as possible. Alternatively, you may have to collect fewer data points. Discuss with a TA if you are unsure what to do.

• Once you have determined the amount of enzyme you will use for each reaction, run the rest of the assays.
• Since you are going to be calculating the K_M and V_max for your mutant and wild-type enzymes, you will want to test a range of PNPA concentrations above and below the K_M. Select 8 substrate concentrations to test initially. Plan to add at least two more after looking at the plot of your initial 8.
  • Note that for technical reasons you cannot test [PNPA] >5 mM.
  • You will need a separate blank for each [PNPA].
  • You will run each [PNPA] in triplicate. It is possible to use the same blank for all three replicates as long as the PNPA in the blank has not hydrolyzed too much (i.e. turned yellow); however, it is challenging to move quickly enough (especially at high [PNPA]), and you may get cleaner data by making a fresh blank for each measurement.

• SAVE YOUR PROTEIN SAMPLES AT -20 ºC.