In this experiment, sucrose was used as the substrate to determine the time course curve of the reaction between sucrase and the substrate, i.e., under the optimum conditions of the enzyme reaction, the amount of product generated was measured at certain intervals, and then the time course curve of the enzyme reaction was plotted with the enzyme reaction time as the horizontal coordinate and the amount of product generated as the vertical coordinate.
Operation method
Dinitrosalicylic acid method
Principle
The starting part of the time course curve of an enzyme reaction is straight within a certain period of time, and its slope represents the initial rate of the enzyme reaction. With the prolongation of the reaction time, the slope of the curve decreases, indicating a gradual decrease in the speed of reaction, which may be due to the decrease in the concentration of the substrate and the increase in the concentration of the product to make the inverse reaction strengthened, etc. Therefore, the determination of the accurate enzyme activity must be carried out in the process of the curve within the time range of the initial velocity, the determination of the curve and the time range of the initial velocity, is the enzyme kinetic nature analysis of the component parts of the analysis and the experimental basis.
Materials and Instruments
Sucrose Move 1 . Prepare 12 test tubes for the assay according to Table 2. The first tube with a reaction time of 0 was used as a blank control, and this tube was spiked with 0.1 mol/L NaOH before adding the enzyme. The 10th tube was corrected for acid hydrolysis of sucrose. The 11th tube was used as a control, and the light absorption value of the glucose standard in the 12th tube was measured to calculate the μmole of reducing sugar produced in each of the tubes 2 to 9. For more product details, please visit Aladdin Scientific website.
Acetic acid buffer Glucose Dinitrosalicylic acid solution
Spectrophotometer Water bath Test tubes Cling film
2 . The amount of substrate sucrose in Table 2 is 0.25 μmol per tube. 0.5 μmol of reducing sugar is produced after all the reactions, and all the concentrations of sucrose and enzyme should be such that the substrate is essentially reacted within 20 min.
3 . Draw a curve of the number of μmol of reducing sugar produced (i.e., product concentration μmol/ ml) versus the reaction time, i.e., the time course of the reaction, and find the initial rate of the reaction. 