Design your own experimental program to determine the effects of pH, temperature and inhibitors on sucrase activity.
(Source: Laboratory instruction of biochemistry and molecular biology, Shao Xueling, etc., Wuhan University Press, 2003).
Operation method
Dinitrosalicylic acid method
Principle
The activity and stability of enzymes are easily affected by the pH of the environment. Usually, all kinds of enzymes only show activity in a certain pH range, and all kinds of enzymes have their own optimal pH under specific conditions.Temperature has a double effect on the action of enzymes, in the lower temperature range, the speed of enzyme reaction increases with the increase of temperature, but after exceeding a certain temperature, the speed of reaction decreases instead. Inhibitors bind to the active site of an enzyme, changing the structure or nature of the active site and causing a decrease in enzyme activity. The binding of inhibitors to enzymes is categorized into reversible and irreversible inhibitors. Reversible inhibitors bind to the enzyme through covalent bonds and cannot be released by physical methods such as dialysis.
Materials and Instruments
Sucrose Move [Experimental Methods] 2. For experiments without inhibitor (urea), data from experiment (VII) can be used, but the same dilution of enzyme must be used, or the experiment can be redone, taking care that the enzyme concentration should be larger. For more product details, please visit Aladdin Scientific website.
Disodium hydrogen phosphate Sodium dihydrogen phosphate Citric acid Sodium acetate Urea Acetic acid buffer Glucose Dinitrosalicylic acid
Spectrophotometer Water bath Test tubes Cling film
Self-design.
[Program example]
Experimental method 1.
1. Prepare 12 buffer solutions (for public use) according to the table below.
The total volume of the two buffer reagents after mixing is 10 ml, and the pH value of the solution is based on the value measured by an acidimeter.
2. Prepare two groups of 12 test tubes, the first group of 12 test tubes are added to each 0.2 ml of the corresponding buffer in the following table, and then add a certain amount of sucrase (at this time, sucrase can only be diluted with H2O, the enzyme dilution and the amount of the addition of the appropriate choice, so that in the prevailing experimental conditions can be obtained in the light absorption value of 0.6 ~ 1.0 ( A650 )). Another group of 12 test tubes were also added with 0.2 ml of the corresponding buffer in the following table, but no enzyme was added and an equal amount of deionized water was added to serve as blank control tubes for the assay. All tubes were topped up to 0.8 ml with water.
3. Add 0.2 ml sucrose (0.2 mol/L) to all test tubes at certain time intervals to start the reaction, and after 5 min of reaction, add 1.0 ml 0.2 mol/L NaOH to terminate the reaction, and then add 1 ml dinitrosalicylic acid reagent, wrap the mouth of the test tubes with plastic wrap and pierce a small hole, cook it for 5 min in a boiling water bath, take it out to cool it quickly, and then add 5.0 ml of water to the test tubes. Then add 5.0 ml of water and shake well to determine A520.
4. Prepare two more test tubes for this experiment, one with water as blank control; the other as glucose standard tube.
5. Draw the curves of sucrase activity (μmol/min) versus pH at different pH, and pay attention to the two points where pH is the same but ions are different, so as to observe the influence of different ions on the enzyme activity.
Experimental Method 2.
This experiment is to determine the reaction rate of sucrase catalysis and acid catalysis at different temperatures between 0 and 100 ℃. Including ice water bath at 0 ℃, room temperature (about 20 ℃), boiling water bath at 100 ℃ and different water bath temperatures: 10 ℃, 30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃.
For each temperature, 2 test tubes were prepared, one with enzyme to measure enzyme catalysis, and one without, using acetate buffer as acid to measure acid catalysis.
1. Determine the dilution of the enzyme, add 0.2 ml 0.2 mol/L acetate buffer, pH 4.9, 0.2 ml diluted enzyme, add water to 0.8 ml, add 0.2 ml 0.2 mol/L sucrose to start the clock, and the reaction was performed at room temperature for 5 min. 1.0 ml 0.2 mol/L NaOH was added to terminate the reaction respectively, followed by the addition of 1 ml dinitro Then 1 ml of dinitrosalicylic acid reagent was added, the mouth of the test tube was wrapped with plastic wrap and a small hole was pierced, and the test tube was boiled in a boiling water bath for 5 min, then taken out and cooled quickly, then 5.0 ml of water was added, and A520 was determined by shaking well.
2. To determine the reaction rate at each of the above temperatures, use 2 test tubes each time, both add 0.2 ml of acetate buffer, one with 0.2 ml of enzyme, and the other without enzyme, both adjusted to 0.8 ml with water, put into the water bath temperature to equilibrate the reactants for 30 s, add 0.2 mol/L sucrose 0.2 ml, and accurately react for 10 min, and then immediately add 1.0 ml of 0.2 mol/ LNaOH to terminate the reaction, the operation was carried out as specified, the A520 value of each tube was measured, and the exact temperature of each water bath was recorded.
3. The A520 values of each tube for enzyme catalysis were corrected for acid catalysis. The reaction rate versus temperature and lnk-1/T curves were plotted for enzyme-catalyzed and acid-catalyzed reactions, respectively, and the linear parts of the two lnk-1/T curves were used to calculate the two activation energies.
Experimental Methods 3 .
1. An optional experiment to determine reversible and irreversible inhibition.
3. Experiments containing urea inhibitors can refer to Table 2 to design the experimental program. A total of three kinds of inhibitor concentration [I] experiments, that is, adding 4 mol/L of urea 0.10 ml, 0.20 ml and 0.30 ml (note: at this time to add less H2O 0.1 ml, 0.2 ml and 0.3 ml, respectively), is still 12 test tubes, each tube should be added to the urea, the 9th, 10th two tubes are still corrected for acid hydrolysis. The 11th and 12th standard tubes were also spiked with urea to eliminate the effect of urea on color development.
4. Draw a graph of reaction rate versus substrate concentration, and a graph of I/V-I/[S], calculate Km, Vmax, KI and the corresponding apparent values, and discuss the effect of urea on sucrase activity. 