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acidic phosphate enzyme dynamics analysis
< p-style-text-align: left; "> temperature on enzyme vitality - the most temperature-appropriate measurementthe most suitable temperature for various enzymes is different, general animalstissue> The most suitable temperature of the various enzymes in the plant is 35 to 40 degrees C, and the temperature range of the various enzymes of plants andmicroorganisms is large, about 32 to 60 degrees C. However, the most suitable temperature is not a characteristic constant of the enzyme, the most suitable temperature of an enzyme is not a constant value, it is related to the reaction conditions. If the reaction time is extended, the most suitable temperature is generally reduced. Therefore, for the same enzyme, it should be stated under what conditions the most suitable temperature.
< p style is "text-align: left;" > temperature is one of the important factors affecting the speed of enzymatic reaction. At lower temperatures, the effect of absolute temperature on Vmax follows the > Arrnenius formula: the active energy of the Ea-enzymatic reaction in the formula < (J'mol-1); mol-1-K-1;T-absolute temperature (K);
Vmax" - the speed of reaction measured when the enzyme is completely saturated by an excess substrate.
experiment, the maximum reaction speed of enzyme-based Vmax at different temperatures is determined, and the absolute temperature T is masted with log10Vmax, resulting in a straight line with a slope equal to -Ea/2.3R, from which the active energy Ea can be found.
"Methods and steps"
left; "> take 8test tube 0 to 7 number, 0 is blank. Each tube is added with a 0.5mL 5mmol/L sodium benzodiaxate solution (pH5.6). Tubes 1 to 7 are placed in turn in awater bath at 20 degrees C, 30 degrees C, 35 degrees C, 40 degrees C, 50 degrees C, 60 degrees C and 70 degrees C, and warm up for 2 minutes.
then each tube timed to add a diluted enzyme solution 0.5mL, shake well, accurately react 15min, add 1mol/L sodium carbonate solution 2mL, then add 0.5mL Folin-phenol dilute solution, continue to warm 10min. The amount added to < tube is the same > reagent,the enzyme fluid is finally added. After taking out the cooling, take tube No. 0 as blank and read the absorbent A680 value of each tube at wavelength 680nm in"> dicing photonometer".
the entire operation is as follows:
reagents | 1 | 2 | . 3 | 4 | 5 |
6
7
0"
reaction temperature/C
20
. 0
35
40
50.
60
50
5mmol/L sodium benzodiaxate
0.5 mL
diluted enzyme solution (35 degrees C warm-up)
each 0.5 mL
reacts accurately at different temperatures 15min
1mol/L sodium carbonate solution
2.0 mL each
Folin-phenol solution
0.5 mL
35 degrees C insulation color 10min
A680
"data processing";
1, with the reaction temperature as the horizontal coordinates, A680 (representing the reaction velocity) as the ordinate, drawing the temperature-enzyme vitality curve, to find the most suitable temperature of acid phosphatease under the experimental conditions.
2, with the inverse (1/T) of the absolute temperature reaction as the horizontal coordinates, log10A405 as the ordinate to map, find out the slope of the straight line part, calculate the active energy of the enzymatic reaction.
.
