Practical 6 Enzymes 1. Determination of Acid Phosphatase Activity

Practical 6 Enzymes 1.
Determination of Acid Phosphatase Activity
Introduction The analysis of enzyme kinetics refers to the study of enzymes by determining their reaction rates For many experimental and clinical purposes, it is necessary to determine the rate (activity) or the specific activity of an enzyme Activity: the number of moles of substrate changed per unit time the rate of reaction. Activity is determined by measuring the reaction rate over a relatively short time interval, and selecting conditions where reaction velocity is proportional to the amount of enzyme present. The time interval is kept relatively short so that there is not effect of product accumulation and/or subject depletion on reaction velocity Specific Activity: activity per unit mass of enzyme (usually per mg of protein) Working with Enzymes Enzymes are proteins and are susceptible to inactivation or inhibition from various sources, and also to destruction by proteolytic enzymes. Routine precautions should always be taken in handling enzymes (unless there is a specific reason to vary them). These include storing the enzyme preparation cold until use, avoid vigorous stirring, since foaming can cause inactivation by a process known as surface denaturation and using a separate pipette or tip for each reagent in the assay to keep them free from contamination (dont put them on the bench) Phosphatases This class of enzyme hydrolyses phosphate from phosphate esters Phosphatases are present in a wide variety of plant and animal tissues and in microorganisms These enzymes can be classified into two groups according to their pH optima o Acid phosphatases optimum about pH 5 o Alkaline phosphatases optimum about pH 9 You will assay acid phosphatase from potato The use of a natural substrate would require the determination of one of the products of the reaction, usually phosphate However, with some artificial substrates such as p-nitrophenyl phosphate, one of the products (p-nitrophenyl) is coloured in alkaline solution) and can be directly estimated spectrophotometrically Objectives 1. To construct a standard curve for p-nitrophenol (the coloured product of the enzyme catalyzed reaction) 2. To use a spectrophotometer to measure the increase in p-nitrophenol produced, as a function of time, for a known concentration of enzyme 3. To use the standard curve to convert the changes in absorbance measures to amounts (in mol) of p-nitrophenol produced
4. To plot the amount of p-nitrophenol produced versus time and to use this plot in calculating the specific activity of acid phosphatase under these conditions Part A the Standard Curve Equipment o 2 * 10 mL centrifuge tubes o 19 small test tubes Procedure o You must prepare a series of standard solutions containing different amounts of p-nitrophenol and measure their extinction at 400 nm in the spectrophotometer. You will then graph the extinction values against the amount of p-nitrophenol and later use this to determine the amounts of p-nitrophenol released by enzyme action o Reagents: 0.5 mM p-nitrophenol H2O 20 mM NaOH o 1. Label 10 clean test tubes 1-10 and an additional tube as a blank o 2. Prepare in duplicate 5 concentrations (of your own choice) or pnitrophenol as follows p-nitrophenol x uL (between 1 and 500 uL) H2O to 500 uL NaOH 4.5 mL o 3. In addition, prepare a blank which contains 500 uL H2O and 4.5 mL NaOH o 4. Mix well and measure the extinction of each tube against the blank. Record values o 5. Calculate the amount of p-nitrophenol in each tube and the final concentration of p-nitrophenol per tube. Record these values o 6. Plot a graph showing the amount of p-nitrophenol on the x-axis and E400 on the y-axis Part B Determination of Specific Activity In this part of the experiment you will set up a 10 mL centrifuge tube (A) containing only enzyme and buffer, and a second 10 mL tube (B) containing only the p-nitrophenol phosphate substrate. Both of these tubes will be held for several minutes in a water bath at 30C to equilibrate at this temperature before starting the enzyme reaction. The enzyme reaction will be started by adding 1 mL of enzyme solution from tube A to the substrate solution in tube B. the enzyme reaction starts immediately after this addition is made. Before starting this reaction you will have set up a series of 9 tubes containing NaOH. At intervals after the start of the reaction you will remove aliquots of the reaction mixture and add them in turn to each of the tubes containing NaOH. The NaOH will stop the reaction, and you will determine the amount of coloured p-nitrophenol product released as a function of time by measuring the extinction of the contents of these tubes in a spectrophotometer. Reagents o 25 mM p-nitrophenol phosphate
o Acid phosphatase (~1 mg/mL) o EDTA citrate buffer (pH 5.0) o 20 mM NaOH Procedure o 1. Label 9 clean test tubes C1-C9. Add 4.5 mL of 20 mM NaOH to each of these tubes. Each of these tubes will later (at different time points) receive 0.5 mL of enzyme reaction mixture o 2. Label two clean centrifuge tubes A and B and add to these tubes the amounts of enzyme/buffer/substrate detailed in the following table o 3. Read points 4-9 and complete exercise in figure 6.1 o 4. Place tubes A and B in a rack in a 30C bath for 3 min. ensure that tubes C1-C9 are nearby. Also ensure that you have a clean pipette ready to transfer from tube B to tubes C1-C9 o 5. To start the enzyme reaction, one student transfers 1 mL of diluted enzyme form tube A to tube B. Quickly mix the contents. Start the stopwatch. The second student must immediately transfer 0.5 mL from tube B to tube C1 and mix o 6. Continue the incubation at 30C. Take further .5 mL aliquots from tube B after 1, 2, 4, 6, 8, 10, 15 and 30 minutes, and add respectively to tubes C2, C3, C4, C5, C6, C7, C8 and C9 o 7. Determine the extinction of the tubes C1-C9 against a blank containing 500 uL of water and 4.5 mL 20 mM NaOH. Record the values o 8. Using the standard curve you prepared in part A, use your extinction data to interpolate the amounts of p-nitrophenol produced at each time point and record these values o 9. The amount of p-nitrophenol in each time point tells you how much product was produced in a 500 uL aliquot of the reaction tube at various time points. Using this value, determine the amount of p-nitrophenol that would have been produced in the original reaction tube B (ie 5 mL) o 10. Calculate the corresponding concentrations of p-nitrophenol produced in reaction tube B at the sampling time points o 11. Prepare a graph showing the amount of p-nitrophenol produced in reaction tube B (on y-axis) against time (on x-axis) o 12. Use this graph to calculate the initial rate of reaction and then calculate the specific activity of the enzyme (in units of umol product/min/mg of protein)

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Determination of Acid Phosphatase Activity

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