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EXPERIMENT THREE

EXPERIMENT THREE. ASSAY OF ACTIVITY OF ALKALINE PHOSPHATASE. 2012-09. Aims. To master the basic method of the assay of the AKP (Alkaline phosphatase ). To strengthen the comprehension of enzyme activity and its inhibition.

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EXPERIMENT THREE

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  1. EXPERIMENT THREE ASSAY OF ACTIVITY OF ALKALINE PHOSPHATASE 2012-09

  2. Aims • To master the basic method of the assay of the AKP (Alkaline phosphatase ). To strengthen the comprehension of enzyme activity and its inhibition. • To be familiar in the meaning of the activity unit, total activity , ratio activity and how to do the calculation of AKP activity.

  3. Some important concepts • An enzyme unit is that amount of enzyme which can catalyze the transformation of 1μmol of substrate per minute at 37℃ under optimal conditions for that enzyme. • International unit (IU): 1µmol/min • Katal unit:1mol/sec 1 IU = ? katal 1 IU=16.67×10-9kat

  4. △S △P • V= or △t △t

  5. Some important concepts • Total activity refers to the total unit of enzyme units of enzyme in the sample • Specific activity is the number of enzyme units per milligram of protein(unit/mg).

  6. Total enzyme activityand specific activity For example Do you think whether the AKP activity of this two samples are same or not? How to compare the activity of AKP in A sample with in B sample?

  7. Total enzyme activityand specific activity For example

  8. Principle • Alkaline phosphatase (AKP ) can catalyze disodium phenyl phosphate decomposition in basic condition and produce phenol and disodium hydrogen phosphate. • Phenol can react with 4-aminoantipyrene and K3Fe(CN)6to generate red complex which has the highest absorbance at 510 nm.

  9. Mg2+is the activator for AKP, EDTA is the inhibitor

  10. Principle • This red compound has the most absorbance atλ510nm. According to this we can calculate AKP enzyme unit.

  11. Protocol ml 1. Make the standard curve

  12. A510 0 Enzyme unit Protocol Draw the standard curve by using A510 as y-coordinate and correspond enzyme unit as x-coordinate

  13. A Correspond enzyme unit 0 Operator Date Apparatus Name of the curve

  14. = corresponding µmol value × × 1000 1 25 = corresponding µmol value × 40 【Calculation】 Corresponding enzyme activity (mU/tube) 25, is the time extent for water bath, or reaction time × 1000, to change U to be mU

  15. Protocol ml 2. Assay of AKP activity

  16. Protocol According to the data of your results, calculate the activity units of AKP and its specific activity of AKP, filling the following table, please: 2. Assay of AKP activity

  17. A C X-coordinate 2 4 6 8 10 0

  18. Caution 1. The AKP come from cobra snake venom, so carefully to handle the enzyme solution 2. To add the basic solution (NaOH) quickly to stop the enzyme-catalyzed reaction when the time is due, maintaining the time extent of reaction identical in each test tube

  19. Discussion 1.Why is it needed to add NaOH solution to stop the reactions? 2. What are the action of Mg2+ and EDTA in this AKP enzyme-catalyzed reaction, respectively? 3.Do you know anything about AKP and the relationship between AKP and some clinic diseases?

  20. Reagents 1. 0.1mol/L pH10 H2CO3-Na2CO3 buffer ( Carbonate buffer ) 2. 0.02mol/L C6H5Na2PO4 ( Disodium phenyl phosphate ) 3. 1μmol/ml C6H5OH ( Phenol standard solution ) 4. 0.012mol/L MgCl2 ( Magnesium chloride ) 5. 0.012mol/L EDTA ( Disodium ethylene diamine tetraacetate ) 6. 0.2mol/L NaOH (Sodium hydroxide ) 7. 0.5mg/ml snake venom 8. 0.3% 4-aminoantipyrene 9. 0.5% K3Fe(CN)6 ( Potassium ferricyanide )

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