Finding out the optimum environment; may it be temperature, pH levels, for enzymes to work is important because then reactions can be made more efficient, and we then can understand more about plant life with respects to the chemical reactions happening within. Due to this I will be conducting an experiment, in which I will try to find the optimum pH value for the enzyme phosphatase.
Phosphatase is found in many plant and animals tissues. It releases phosphate ions from many phosphate substrates, the ions are then components of different molecules such as DNA, RNA, phospholipids.
For the experiment I will use an artificial substrate Phenolphthalein phosphate (PPP) when made available to the enzyme it will produce a product that I can observe and measure (phenolphthalein).
Phosphatase
Phenolphthalein phosphate phenolphthalein + phosphate
Depending on how efficiently the enzyme is working, will be shown be the colouration of the phenolphthalein. The deeper the colour, the more phenolphthalein produced, this shows that the enzyme has broken the PPP up into its substrates.
Method
- grind 50 mung beans into a paste ( which have been left for 4 days to start sprouting)
- Add 20 ml distilled water to dilute
- pipette liquid into a teat tube (leave debris)
- centrifuge the mixture for 5 min at 1400 rpm
- clear supernatant is the enzyme preparation
- add 2.5 ml buffer then 0.5 ml PPP
- incubate in a water bath at 35 °C for 35 min
- stop reaction by adding 2.5 ml of 10% sodium carbonate,
- measure colouration using calibrated cards, and colorimeter
Equipment
Test tubes
Pestle and mortar
Substrate
PPP
Mung beans
Buffer- of different concentrations, ranging from 2.2-9.2
Sodium carbonate
Pipettes
By adding sodium carbonate, this it will cause the reaction to stop, and any phenolphthalein to turn pink. It is the colouration of the pink that I will be observing and distinguishing the results.
I have chosen to use 8 different buffers of different ph levels; 2.2, 3, 4.2, 5, 6, 7, 8 and 9.2. I tested the pH level of each of the buffers and these are the results I got, that is why same have a reading of .2, however this will make no difference to the experiment.
The level of pH is my only variable. As with all experiments, you should only change one thing at a time, keeping all other aspects that may affect the results the same. Because the enzyme mixture will be used for all the experiments, the concentration will be the same. If the concentration were to be different, the experiment with greater enzyme concentration would have a result that would show a greater colour change. Due to there being more enzymes, a greater rate of reaction can be reached. The PPP mixture is of all the same concentration. It will be hard to monitor temperature; however there are ways in which I can prevent this factor affecting my results. I will perform my experiment in the same incubator, so that all the test tubes are exposed to exactly the same amount of heat. Another thing that I will have to monitor closely is the time each reaction takes place for. I plan to start all the reactions at the same time and stop them at the same time as well. There will be a slight delay between each one starting, as I will have to fill my pipette up with PPP each time. However I will remember the order in which they start, and end them in the same order, this will hopefully even out the possibility of the difference in time affecting the results greatly. I will also do all the experiments in the same area of the lab; this is because light can affect the rate of reaction.
Throughout the experiment I will make sure that I am wearing goggles. Sodium carbonate is corrosive. Phenolphthalein is harmful if swallowed and has suspect cancer hazards, for this reason I will not touch the PPP or any of the mixtures at any time, if I do accidentally come into contact with either of the substances, I will wash it under cold water.
I will test the colouration by using a colorimeter, with a 550 nm filter. The colorimeter will be calibrated, set at zero using distilled water. A reading should be given indicating the amount of light absorbed, they are as follow and are in absorbance units.
Results
From the results given by the colorimeter, the enzyme phosphatase works most effectively in an environment with a pH level of around 6. The difference between pH 6 and 7 is quite large; 0.94 to 0.31, with resects to the other way; pH 6 to 5 - 0.94 to 0.57. You can tell from my results that the enzyme is very inefficient in alkali environments; there is a great decline in production of phenolphthalein when you start getting below pH 7. Whereas the production of phenolphthalein in an acidic environment as a much less rapid decline. The ionic boning between the amino acids in the proteins which make up the enzyme must break quicker in an alkali environment. The breaking of the bonds allows the enzymes’ active site to become misshaped; this means that the rate of reaction is reduced because it is taking longer for the enzyme to join with the substrate to form a substrate-complex and break them up into the products; phosphate and phenolphthalein.
There are very few anomalies, the main one which is most faulty is the pH 6 experiment, were there is a difference of 12, however this is quite small, and doesn’t really affect the overall result of the experiment. As I said before, this might be due to many reasons; a misread on the colorimeter, experiment having longer time, exposed to different temperature/amount of light.
To expand on the investigation I would re-do the experiment, but my range of buffers would be less. I would probably use buffers ranging from pH 5 to pH 7 to get a more exact reading on what the most effective pH level is.
