Determination of alkaline phosphatase (AKP) meter constant (km).

principle:
When the temperature of the environment, pH and enzyme concentration and other conditions, the initial speed (V) of the enzymatic reaction is accelerated with the substrate concentration (S) increases until a limit is reached, i.e. the maximum reaction speed (V). This relationship between substrate concentration and the initial speed of the enzyme reaction, Michaelis-Menten's derivation of the following formula, called the Mi-Man equation: km in formulais called the meter constant. Km is the concentration of the substrate when the reaction speed is equal to half the maximum reaction speed (v-V/2). Km is the characteristic constant of the enzyme. The determination of Km is one of the main methods for studying enzyme dynamics. Lineweaver-Burk's equation follows the following equation:with a different substrate concentration of 1/S as the ordinate coordinates 1/v, calculated by a straight-line regression equation, graphed, and extended in the direction of the vertical axis, which is intercepted at a negative value of 1/Km on the horizontal axis.
This experiment took alkaline
phosphatase
as an example, selected different concentrations of sodium phenyl phosphate as its substrate, phenol and phosphoric acid were hydrolyzed by AKP, phenol in alkaline solution with 4-aminoantebilin action, oxidation by iron cyanide to produce derivatives of red arginine, according to the depth of red can be measured enzyme vitality High and low, its reaction type is as follows instruments:
medium
test tube
18
redration temperature
water bath
tick pipe 8 721
ternary photonometercolor cup
reagents
:1. Substrate fluid (10mmol/l, sodium benzodia phosphate)
called sodium benzodia phosphate (C6H5Na2PO4.2H2O) 2. 54 g or sodium benzodia phosphate (no crystalline water) 2. 18g, dissolved and diluted to 1000ml with boiling cooling distilled water, plus 4ml chloroform anti-corrosion, brown bottle 4c save, used within a week.
2. Buffer (0. 1mol/L phosphate buffer, pHl0. 0)
waterless sodium carbonate 6. 36 grams and sodium phosphate 3. 36 g, dissolved in distilled water 800ml, plus distilled water quantified to 1000ml.
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