Alkaline Phosphate Assay
Expt. BL 304
Kinetics of Alkaline Phosphatase
Objective
To study the kinetics of alkaline phosphatase.
Procedure
1. Sodium carbonate - sodium bicarbonate buffer: Dissolve 20 ml of 0.2 M solution of sodium carbonate (2.12 gm in 100 ml distilled water) and 230 ml of 0.2 M sodium bicarbonate (4.12 gm in 250 ml distilled water) to make up the volume 250 ml with pH 9 -9.2 2. 5 N sodium hydroxide solution (10 gm in 50 ml distilled water) 3. Substrate stock solution: Dissolve 0.1 gm of p-nitrophynyl phosphate in 25 ml of carbonate bicarbonate buffer (prepared as above). 4. Enzyme solution: Dissolve 10 mg of alkaline phosphatase enzyme in 10 ml carbonate -bicarbonate buffer to get 1 mg/ml stock solution. Pipette out 0.1 ml of stock A and make-up the volume to 10 ml with buffer to obtain 0.01-mg/ml solution, which is used for further dilution.
Theory
Chemical reactions in biological systems occur in the presence of highly specific protein catalysts called enzymes. Enzymes catalyse the reaction rapidly under mild conditions in contrast to inorganic catalysts. The rate of enzyme-catalyzed reaction is influenced by different environmental conditions like concentration of substrate, pH, temperature. Presence of inhibitors and concentration of the enzyme itself. The rate of an enzyme-catalysed reaction can be measured by: 1. The disappearance of substrate, or 2. The appearance of the product. In a typical experiment the enzyme and substrate are mixed and allowed to react for specific period of time; then amount of substrate disappeared or product formed is measured and this information will give the rate of activity of the enzyme per unit time. To study an enzyme, an assay is necessary. The assay is a measurement of a chemical reaction, which might involve measuring the formation of the product. The enzymes phosphatases catalyze hydrolysis of BL 304-1
phosphate esters to free inorganic phosphate in biological systems. There are two types of phosphatases depending on the
Kinetics of Alkaline Phosphatase
Objective
To study the kinetics of alkaline phosphatase.
Procedure
1. Sodium carbonate - sodium bicarbonate buffer: Dissolve 20 ml of 0.2 M solution of sodium carbonate (2.12 gm in 100 ml distilled water) and 230 ml of 0.2 M sodium bicarbonate (4.12 gm in 250 ml distilled water) to make up the volume 250 ml with pH 9 -9.2 2. 5 N sodium hydroxide solution (10 gm in 50 ml distilled water) 3. Substrate stock solution: Dissolve 0.1 gm of p-nitrophynyl phosphate in 25 ml of carbonate bicarbonate buffer (prepared as above). 4. Enzyme solution: Dissolve 10 mg of alkaline phosphatase enzyme in 10 ml carbonate -bicarbonate buffer to get 1 mg/ml stock solution. Pipette out 0.1 ml of stock A and make-up the volume to 10 ml with buffer to obtain 0.01-mg/ml solution, which is used for further dilution.
Theory
Chemical reactions in biological systems occur in the presence of highly specific protein catalysts called enzymes. Enzymes catalyse the reaction rapidly under mild conditions in contrast to inorganic catalysts. The rate of enzyme-catalyzed reaction is influenced by different environmental conditions like concentration of substrate, pH, temperature. Presence of inhibitors and concentration of the enzyme itself. The rate of an enzyme-catalysed reaction can be measured by: 1. The disappearance of substrate, or 2. The appearance of the product. In a typical experiment the enzyme and substrate are mixed and allowed to react for specific period of time; then amount of substrate disappeared or product formed is measured and this information will give the rate of activity of the enzyme per unit time. To study an enzyme, an assay is necessary. The assay is a measurement of a chemical reaction, which might involve measuring the formation of the product. The enzymes phosphatases catalyze hydrolysis of BL 304-1
phosphate esters to free inorganic phosphate in biological systems. There are two types of phosphatases depending on the
References: 1. Lubert stryer, Bio chemistry (1995) W.H. Freeman, New York 2. Wilson, K.and Walker, J (2000) Practical Biochemistry, Principles and Techniques. BL 304-3