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Nitrilase activity assay

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Table of Contents

1. Picric acid colorimetric assay for the quantitative determination of cyanide

For activity assays of cyanide-degrading Nitrilases.

Solutions required

  • 500 mM Tris-HCl, pH 8.0.
  • Substrate stock solution (100 mM KCN in 500 mM tris-HCl, pH 8.0). Dissolve 0.065 g KCN in 10 ml 500 mM Tris-HCl in a fumehood. The pH will have increased dramatically upon addition of the KCN and so the solution requires acidification. Add approx. 150 μl 32% HCl per 10 ml buffered KCN. Check pH with strips and then store the solution in the fridge, within a second bottle. The pH will drift quite rapidly [within 1-2 weeks], so check the pH periodically.
  • 500 mM Na2CO3, in distilled water [pH not adjusted].
  • 1.5% picric acid in distilled water. Keep away from direct light.

Equipment required

  • boiling waterbath
  • ELISA plate [wash out and reuse after spectrophotometry]
  • ELISA platereader. This is located on the 4th floor in MCB in the tissue culture room. If you use the reader after hours, you need to get the tissue culture room key from Nicci Illing’s lab on the 4th floor. The person to speak to is Faezah. She only works half-day, so ensure that you get the key in the morning.

Procedure

  • Dispense 80 μl protein fractions into 1.5 ml Eppendorf tubes. Add 20 μl of 100 mM buffered KCN at pH 8.0.
  • For the positive control, add 20 μl KCN to 80 μl distilled water.
  • For the negative control, add 100 μl distilled water in a 1.5 ml Eppendorf tube.
  • Incubate for 30 min – 1 hour at optimum temperature [the duration will depend on the activity, however, once you have an idea of the activity, you should standardise the assays according to time. If your activity is very high, rather dilute and assay for the standard period rather than reducing the incubation time].
  • Switch on the boiling water bath to ensure that it is ready when the incubation ends.
  • Atthe end of the incubation period, remove the tubes from the incubator and keep on the lab bench while making up the picric/carbonate mix.
  • To make the picric/carbonate mix, add equal volumes of picric acid and Na2CO3 just before use.
  • Add 80 μl picric/carbonate to the 100 μl samples. Mix thoroughly, place plastic cap protectors on the Eppendorf lids to prevent them popping open while in the boiling water.
  • Place the tubes in a float and lower into the boiling water. Wait 5 minutes.
  • Remove the tubes and allow to cool to room temperature. You will notice that the positive control [20 mM KCN only] is a deep blood/ruby red colour while the negative control [distilled water only] is canary yellow. Partial HCN degradation will result in a range of colours from light to very dark orange.
  • Dispense 100 μl of each fraction into ELISA plate wells, taking care not to add any solution to the first column [i.e., 1] of the ELISA plate. The platereader will not tolerate any coloured solution in the first column.
  • Switch on the platereader and insert the 492 nm filter [this is kept in a rectangular box in one of the small drawers directly below the platereader]. Read absorbances and collect the printout. Switch off the machine, replace the cover and write down your name and date and filter in the notebook provided. Subtract absorbances from the negative control [blank].
  • Dispose of all cyanide containing waste into clearly marked Winchester bottles and keep in the fumehood. When full, speak to Andile, a Departmental Assistant in MCB and he will show you where the specially marked cyanide waste bin is located in the chemical waste storeroom.

This method assumes that the nitrilase you are working with will tolerate KCN concentrations of 20 mM – this may not always be the case and you may identify substrate poisoning. If you experience this, rather assay with lower KCN concentrations [around 12.5 mM]. The colour reaction will still remain intense. For each enzyme though, you would have to do a progress curve to ensure that your substrate is not limiting. This is an important consideration if you are performing any kind of kinetic analyses, including construction of the purification table. Perform doubling dilutions of your enzyme solution until you reach a concentration where you are assured that substrate remains in excess at the end of the assay period.

2. Indophenol blue method for the quantitative detection of ammonia/ammonium

For activity assays of nitrilases that hydrolyze higher nitriles (benzonitrile among others) resulting in the production of ammonia among other products.

Solutions required

  • Phenolic alcohol. Dissolve 1 g phenol in 95% ethanol and make to a final volume of 100 ml. This solution is highly corrosive; be careful.
  • Na-nitroprusside. Dissolve 0.1 g Na-nitroprusside in distilled water and make to a final volume of 20 ml. Wrap bottle in tin foil. This has a maximum shelf-life of 1 month.
  • Alkaline complexing reagent. Dissolve 10 g trisodium citrate and 0.5 g NaOH in distilled water and make up to a final volume of 50 ml.
  • Sodium hypochlorite (NaOCl). Use Jik or any other commercial bleach.
  • Oxidising solution. Add 1 part NaOCl to 4 parts alkaline complexing reagent. Prepare this just before use.

Equipment required

  • ELISA plate [wash out and reuse after spectrophotometry]
  • ELISA platereader. This is located on the 4th floor in MCB in the tissue culture room. If you use the reader after hours, you need to get the tissue culture room key from Nicci Illing’s lab on the 4th floor. The person to speak to is Faezah. She only works half-day, so ensure that you get the key in the morning.

Procedures

1. Ammonia standard curve

  • One can use either (NH4)2SO4 or NH4Cl to prepare standard solutions. However, remember that 1 mole of NH4Cl contains 1 mole NH4 while 1 mole (NH4)2SO4 contains 2 moles of NH4.
  • Make up a 1 mM stock solution in distilled water and prepare a dilution series containing 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5 mM NH4 all to a final volume of 1 ml [however, make 3 x 1 ml volumes for each dilution].
  • To 1 ml sample add 40 μl phenolic alcohol, 40 μl Na-nitroprusside and 100 μl oxidising solution.
  • Mix well and allow colour to develop in the dark at room temperature for 1-3 hours [but be consistent in the development time]. One hour is usually sufficient.
  • The resultant colour spectrum will range from a very pale straw [zero NH4] through pale blue/green [middle of the range] to deep blue [max NH4].
  • Use the 620 nm filter on the ELISA platereader. Dispense 100 μl into ELISA wells.

2. Enzyme assays

  • Depending on the enzyme concentration, you may need to dilute several times to remain within the standard curve range.
  • From the NH4 standard curve, you will notice that 0.5 mM NH4 gives a very dark product which is starting to go beyond the absorbance limits of the spectrophotometer. Therefore, if you wish to remain in the range, one needs to ensure that no more than 0.5 mM product is produced during the incubation time. Again, as with the picric acid method, rather dilute your sample and re-assay than change the incubation times, temperature or volumes – this will ensure that all your assays done at each step in the purification table are comparable. This also avoids any confusion later on when trying to remember which assay parameter was changed at various steps.