How to determine the optimal pH of salivary amylase

Salivary amylase (pancreatic amylase) is a hydrolase secreted by the salivary glands. Starch is hydrolyzed by amylase in saliva to produce dextrin and maltose. Knowing the optimal pH of salivary amylase is good for you, so what is the optimal pH of salivary amylase, and how to measure the optimal pH of salivary amylase?

Under the condition of excess substrate, in the same time, under the same temperature and enzyme concentration conditions, the amount of starch consumed in the reaction system close to the optimal pH is the largest. The lighter the color generated by the remaining starch and the excess iodine solution, the smaller the absorbance. After measuring the absorbance of each tube with a spectrophotometer, the pH value corresponding to the smaller absorbance range between the increasing and decreasing trends is the optimal pH of salivary amylase.

1. Materials and Methods Instruments: pipettes (5mL, 1mL), test tubes, test tube racks, small beakers, 722-type spectrophotometer, constant temperature box, ear suction bulbs Reagents: 0.01mol/L iodine solution, 0.2mol/L disodium hydrogen phosphate, 0.2mol/L sodium dihydrogen phosphate, starch solution, saliva, distilled water

2. Method: In the same time, under the same temperature, enzyme concentration and different pH conditions, the enzyme activity is different, the amount of starch hydrolyzed is different, and the color generated by the remaining starch and excess iodine solution is different. By measuring the absorbance at 660nm, the optimal pH range of the enzyme is obtained.

Steps:

1. Obtaining saliva: After gargling with distilled water, hold the distilled water in your mouth. After an appropriate time, place the distilled water and saliva in a pre-prepared container. During the experiment, attention should be paid to the inactivation of enzymes. To prevent this from happening, the liquid should be cooled in ice water.

2. Preparation of different pH buffer solutions: Prepare 10 ml of buffer solutions of different pH values ​​according to the table below

3. Determine the amount of starch (In order to better observe the color change, you can add an appropriate amount of distilled water to make the total volume the same. Mix well, observe the phenomenon, and select the one with moderate color change and record its amount.)

4. Determine the dilution multiple of the enzyme solution (In order to better observe the color change, you can add an appropriate amount of distilled water to make the total volume the same, mix well, observe the phenomenon, and select an appropriate one and record its dilution multiple.)

5. Determination of the optimum pH (adjust to zero with distilled water, measure the absorbance A at 660nm, and finally plot with A as the ordinate and pH as the abscissa. The pH value corresponding to the lowest point of the curve is the optimum pH value of salivary amylase.)

From the above experiment, we can conclude that 6.8 is the pH value corresponding to the lowest point of the curve, which is the optimal pH value of salivary amylase. However, it should be noted that the results of amylase determination are greatly affected by the method. How to determine the optimal pH of salivary amylase? Different methods have different reference values. There are also many methods used in clinical practice. Therefore, it is necessary to understand the determination method and its reference value to make a correct diagnosis.