IMPORTANT: Wear gloves at all times to avoid contamination or material and samples with ammonium from your hands (which will spoil your assay) and for personal protection!!
Taking leaf samples
Leaf samples may be taken in form of leaf discs using a cork borer. A size 4 borer (d = 8mm) produces discs that easily fit in 1.5 ml microfuge tubes. To take the sample, simply support the leaf with a sheet of plastic while using the borer to obtain the discs. Approximately 0.1 g of material is usually sufficient for a urease measurement (you might also get away with considerably less, depending on the plant).
Transfer the discs into a microfuge tube and store this on ice until you reach the lab. Do not freeze the samples (this often reduces or eliminates activity)!
Determine the weight
Determine the weight of your samples if you want to express activities on a fresh weight basis. The easiest way to do this is: Blank your balance with an empty microfuge tube. Transfer the disc from the tube they are in into the weighed tube by holding the openings together and flicking. Weigh the new tube with the discs and record the weight. Store the tube(s)on ice.
Extraction
The extraction procedure uses a little pestle that fits into 1.5 ml microfuge tubes (available e.g. from Sigma: Z35,994-7). This is attached to a power drill on a stand (see this for (a bad) illustration).
Add 0.5 ml extraction buffer [50 mM sodium phosphate buffer (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1.5% (w/v) polyvinylpolypyrollidone (PVPP)] to your sample. (NOTE: you need to swirl your extraction buffer shortly to resuspend the PVPP and cut the tips off from blue pipette tips for the pipetting of the 0.5 ml.) Then add 5 microliters from a 1 M dithiothreithol stock (final concentration: 10 mM) and 5 micrometer phenylmethylsulfonyl fluoride (PMSF) from a 10 mM stock (in EtOH; caution: toxic; final concentration 0.1 mM)
Before starting the drill, use the pestle attached to the drill to press your leaf material to the bottom of the microfuge tube. Then start the drill (set at low revolutions) and press the tube against the pestle until all trapped material is squeezed into solution (do it and you know what I mean). Keep on "drilling" for a minute or two, moving the tube up and down and trying to catch floating pieces. When you are happy with the degree of destruction you have caused store the tube on ice.
Centrifugation
After extraction, samples are centrifuged at 4°C for 10 minutes (14000 g). The supernatants are transferred into new tubes and centrifuged again under the same conditions for 20 minutes. The clarified protein solution is kept on ice.
Desalting by using spin columns
Why?
The desalting step is necessary to eliminate DTT included during extraction, because it is not compatible with the indophenol chemistry used in the subsequent assay. Some plant extracts (e.g. potato leaves) do contain further low molecular weight substances that interfere, but can be removed by desalting.
[NOTE: For some tissues/plants the inclusion of DTT during the extraction is not necessary (e.g. leaves from Arabidopsis thaliana cv. Columbia) - because no oxidative browning occurs during/after extraction. If this is the case with "your tissue/plant" you might want to try leaving DTT out during extraction and measuring urease directly without desalting]
Preparation of home-made spin columns
To 8 g Sephadex G25-fine (Pharmacia) add gel filtration buffer to make up a final volume of 45 ml. Gel filtration buffer is half strength extraction buffer without PVPP, DTT and PMSF. Place 0.5 ml of this slurry in each spin column (you can get empty spin columns e.g. from BioRad, cat. no. 732-6204).
Use of spin columns
Remove flow-stoppers from the columns and spin at 735 g for 1 min. Add 100 microliters clarified extract in the centre of the gel bed and transfer the column into a new microfuge tube. Spin for 2 min at 735 g. Approximately 100 microliters can be recovered. (NOTE: The protein recovery from the spin columns is normally between 83-90% of the protein present in the clarified crude extracts. For the calculation of urease activity per unit fresh weight, the protein loss during the spin column step may be considered with a factor 1.2)
The assay
Transfer 90 microliters prepared extract into 0.5 ml tubes (10-12 samples can be handled in parallel). Add 1 microliters of a 5 M urea stock, mix briefly by vortexing, pre-incubate tubes in a water bath at 50°C for 2-3 minutes.
Take the tubes out of the bath, spin briefly to collect liquid, open all tubes in a rack, start the a timer and immediately begin to collect samples. Samples of 20 microliters are pipetted into prepared microfuge tubes containing 980 microliters of water. After all samples are taken, close the 0.5 ml tubes and put them back into the water bath. Then start to process the samples. First add 100 microliters phenol nitroprusside reagent (toxic!), followed by 200 microliters hypochloride reagent (in case you wonder: the order is important!). Close each tube directly after adding the hypochloride and mix shortly by inverting. These samples can now be placed in a 50°C water bath for a minimum of 15 min (after which the endpoint of colour development should be reached). Repeat this sampling protocol at 15 min, 30 min and 45 min. With some practice, samples may be taken out of the water bath about 1 min (for 10 samples) before the respective time point is reached, spun down and opened. Start sampling when the exact time is reached - maintaining the same order of samples for each time point.
Spec measurements + analysis
The colour is read at 636 nm. The better the spec, the larger the linear range (The colour development itself is very linear). Plot the readings against assay time and determine the slope by regression analysis (NOTE: The last data point is sometimes a bit low. In that case you might want to eliminate this point from your analysis. The reasons for this phenomenon? I have no clue.) From the slope you can work out the activity.
If you want to express the activity per unit protein, you have to determine the protein concentration in your sample. You should have 10 microliters of sample left from the spin column, which is plenty for the protein determination with e.g. Bradford reagent in a microtiter format. We use commercial Bradford reagent from BioRad. For a leaf sample from potato, 20 microliters of a 25-50 times diluted sample in 300 microliters Bradford (prepared following BioRads' instructions) gives a reasonable reading. The total volume of 320 microliters is good for flat bottom microtiter plates.
For the expression of activity per unit fresh weight, you have to consider the protein loss in the spin column (see above in "Use of spin columns")!!
Claus-Peter Witte, 2001 HOME
