The Kinetics of Alkali Phosphatase Inhibition
This practical builds on the enzymology lab skills you learned in the Acid Phosphatase practical. Again, you will measure the initial reaction velocity (V 0) of an enzyme reaction, but this time in the absence and then presence of an inhibitor.
Last time you used Acid Phosphatase (Prac 1), but this time you will use the enzyme Alkali Phosphatase. These enzymes have different primary (and hence tertiary) structures, pH optima and cofactors, so are more unlike than alike. However, Alkali Phosphatase can also catalyse the hydrolysis of the artificial substrate PNPP to PNP + P i, so PNPP will again be the substrate in this prac.
The inhibitor you will test will be the yellow-coloured product PNP – so this is an experiment to investigate product inhibition. What do you think will happen to V0 if the [PNP] were significantly increased? Again, you will be marked on the quality of your data and understanding of the experimental details. This prac also provides some nice enzyme inhibition data for you to use in later workshops.
Please observe meticulous good lab practice: no eating or drinking; wear lab coats at all times; handle chemicals carefully and with gloves; read, understand and think about what you are doing; cooperate as a pair and use common sense.
The enzyme you will use is bovine Alkali Phosphatase (EC 22.214.171.124), bought from Sigma Life Sciences (www.sigmaaldrich.com). As the name suggests, it has a pH optima above 7.0, requires divalent Mg2+ ions for activity, and can catalyse the hydrolysis of PNPP to p-nitrophenol (PNP) and inorganic phosphate (see prac 1 schedule).
Remember that PNP is yellow at alkaline pH, and its appearance can be detected at 405 nm using a spectrophotometer. Because this enzyme works best at alkali pH, we do not need to use a timepoint-quench method (as we did with Acid P), but can monitor the ΔA 405 directly in real time. Experimenters always like to follow real -time kinetics if possible, because as you’ll see, the rate and accuracy of data collection are greatly improved. In fact, you’ll only collect a fraction of the data available today.
You will need to work efficiently together in pairs. First, you will run a timecourse T1 to get the hang of the procedure (which is more straightforward than last time since no quench step is required). Then, as you get more confident, you can divide up who is going to prepare the reactions, and who will record the live data from T2-T10. Good luck!
3. REACTION TIMECOURSES
Please read several times before doing anything!
3.1) Description of a continuous timecourse.
First of all, switch on your spectrophotometer (why?) and set to 405 nm. A timecourse requires timepoints. You’ll read the absorbance directly from the spectrophotometer every minute during each 5 min reaction, so teamwork is crucial. Label a polystyrene cuvette rack from T1 to T10. Then identify (but don’t yet add) the four reagents you use for each timecourse: ‘Buffer’ (1 M Tris-HCl and 2.5 mM MgCl2 at pH 8.0)
‘PNPP’ (1.5 mM in buffer)
‘PNP’ (1.0 mM in buffer)
‘Enzyme’ (the alkali phosphatase, at 0.2 U/ml with 1 mg/ml BSA) on ice.
You’ll also need a stopclock, P1000, P200, and P20 pipettes. Keep your pipetting slow and accurate, and mix reactions thoroughly. Change tips only when changing solution. 3.2) Timecourses T1-T10.
First, have a really good look at Table 1 below, and use it as follows. Do one timecourse at a time, working vertically down each column. Place a clean cuvette in your rack and add Buffer, PNPP and PNP according to the top of Table 1. (Note that PNP inhibitor is only present in reactions T6-T10). Do not add enzyme yet! As you know, a good habit is to tick the table as you make each addition. Now mix gently by pipetting, then blank the spec with this solution (set ref). Now three things happen in quick succession (your pipetting should remain smooth throughout):...