Introduction
Crude acid phosphatase from wheat germ can be isolated by gentle aqueous extraction of pulped wheat germ to provide an enzyme-rich and vitamin-rich liquor, containing amongst other things Vit E, Vit B1 (thiamine), Vit B6 (pyridoxine), Vit B2 (riboflavin) and Vit B3 (niacin). This is generally spray dried at low temperatures and the dried product (fine powder) is sold as wheat germ extract.
The enzyme acid phosphatase is ubiquitous in nature and wheat germ provides a convenient source. It is a non-specific esterase utilising a variety of phosphate esters. It exists in three isoforms (I, II and Ill), each having pH optima around pH 5. In plants, its physiological role is to provide inorganic phosphate to the growing wheat seedlings during germination. Many different phosphate esters of sugars and substrates are stored in wheat seed and these need to be hydrolysed during germination to make the carbohydrates available as an energy source. The phosphates that are released are in part used to make building blocks for making new cells (e.g. recall that new RNA and DNA molecules need phosphates in their backbones).
However, the highest concentrations of acid phosphatases are found in the human prostate gland. Clinical chemists can measure serum enzyme levels as an index of prostatic cancer.
Other laboratory uses of acid phosphatase include reactions where phosphate group transfers are required.
Although not specifically requiring acid phosphatase, molecular biologists use wheat germ extract as a basis for in vitro translation systems as this contains virtually all the components for translating mRNAs into proteins.
Acid phosphatase hydrolyses phosphate esters at a pH of about 5. In this experiment the substrate will be p-nitrophenyl phosphate and the phosphatase catalyses the reaction below:
The p-nitrophenol liberated can be conveniently measured because it is yellow at alkaline pH. The absorbance of the colour that develops can be measured in a spectrophotometer at a light wavelength of 405 nm (the extinction coefficient is 18800 AU M-1 cm-1).
The aim of this Practical is to determine graphically Km and Vmax of the enzyme
Reagents
0.3 mM and 2 mM disodium p-nitrophenyl phosphate (substrate)
Diluted wheat germ extract (enzyme extract)
1.0 M sodium hydroxide
Citrate buffer, pH 5
The following enzyme extraction has been carried out for you. 5 g of wheat germ were weighed out and suspended in 100 ml of distilled water. After stirring the mixture for 20 minutes the suspension was centrifuged at 2000 g for 10 minutes at 40 C. The supernatant was decanted and diluted to 1/10 with distilled water.
Procedure
Clearly label a series of eleven test tubes and add the solutions as detailed in the following table. For tubes 2 -9 use the 0.3 mM substrate but for tubes 10 and 11 use the 2 mM substrate. Tube no. Use 2 mM substrate solution. Mix each tube thoroughly.
Add 0.5 ml of enzyme extract to tube 1 quickly mix and start a stop clock. Similarly, at 1 minute intervals add 0.5 ml enzyme extract to tubes 2 to 11. Set up another series of 11 labelled tubes each containing 1 ml of NaOH to stop the reaction.
Allow each of the enzyme-containing tubes to incubate for exactly 20 minutes by removing 1 ml from tube 1 20 minutes after the enzyme was added and adding it to the NaOH and repeat for the other tubes at 1 minute intervals. Thus each of your tubes, 1 to 11 should now have had a 20 minute incubation. The NaOH should stop the reaction and make the p-nitrophenol released by the acid phosphatase absorb light of wavelengths around 400 nm.
