The effect of pH on the speed of enzymatic reaction

related topicsObjectives and Requirements: Learn
pH on the speed of enzymatic response.
2. Learn the most appropriate pH method for determining enzymes.
principle: The
reaction is very significantly affected by environmental pH. Usually a variety of enzymes only in a certain pH range to show its vitality, an enzyme to show its highest activity when the pH, called the enzyme's most suitable pH. When the enzyme is below or above the most suitable pH, the activity of the enzyme decreases gradually. The most appropriate pH for different enzymes is different. In this experiment, the method of determining the
pH of
was studied by taking the catalytic reaction of lactic acid and dehydrogenase as an example.


equipment
(i) Reagents:
1. Buffers of different pH:
100ml 0.33M citric acid, 100ml 0.33M phosphoric acid, 3.54g boric acid, 343ml 1M NaOH, add water until 1 litre. Use HCl to titrate into pH 3.0, 3.5, 4.0, 4.5 to 12 different pH buffers.
2. 0.01M NADH
3. 0.1M Sodium acetone
4. LDH
(Experimental Reagents and Equipment)
(i) Reagents: 0.1M Potassium Phosphate Buffer (pH 7.0); 10M sodium acetoneate; LDH
(ii) equipment: ultraviolet
phosphorescometer
, pipe gun, suction head, 1.5 ml small
centrifugal tube
, centrifugal tube rack
(experimental method)
(i) enzyme activity determination: method with the same step 1 in experiment 7.
(ii) pH on the speed of enzymatic response:
1. Add: (total volume of 1 ml reaction fluid)




2. Mix the above reagents, pour into
cuvette
, and then put the cuvette into the hydrometer after reading 340nm OD value, add 0.02 ml LDH use liquid, quickly mix, so that the reaction starts, determine the reaction 1 minute OD value change.
3. Based on the change in the measured OD value, the initial speed of the reaction is calculated and the following table is filled in:







3. With pH as the horizontal coordinate, the reaction speed makes a v-pH graph for the ordinate to find out the most appropriate pH value of LDH.