1 EXPERIMENT THREE ASSAY OF ACTIVITY OF ALKALINE PHOSPHATASE

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 = ? katal1 IU=16.67×10 -9 kat

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 activity and specific activity For example SampleAB Units of AKP activity100 Volume (ml)10010 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 activity and specific activity For example SampleAB Units of AKP activity100 Volume (ml)10010 Specific activity (U/ml)1 U/ml10 U/ml

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 K 3 Fe(CN) 6 to generate red complex which has the highest absorbance at 510 nm. K 3 Fe(CN) 6

9 Mg 2+ Mg 2+ is the activator for AKP, EDTA is the inhibitorEDTA

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

11 Protocol 1. Make the standard curve Reagentsblank H 2 CO 3 -NaHCO 3 buffer (pH10) 1.0 Phenol standard solution H2OH2O ℃, 5min 0.2mol/L NaOH solution1.0 4-aminoantipyrene1.0 K 3 Fe(CN) Mixing, waitting for ten minutes ， read A 510 A 510 Phenol (μmol) Correspond enzyme unit ml

12 Protocol Draw the standard curve by using A 510 as y- coordinate and correspond enzyme unit as x- coordinate A 510 Enzyme unit 0

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

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

15 Protocol 2. Assay of AKP activity Reagent BlankAssay1Assay2(Mg 2+ )Assay3(EDTA) Substrate (0.02mol/L) (No Mg 2+ and EDTA) 00.3 Mg 2+ (0.012mol/L) EDTA(0.012mol/L) H2OH2O Enzyme (0.5mg/ml)0.1 H 2 CO 3 buffer (pH10) ℃, 25min NaOH1.0 4-aminoantipyrene1.0 K 3 Fe(CN) ml

16 Protocol 2. Assay of AKP activity Reagent BlankAssay1Assay2( Mg 2+ )Assay3(EDTA) Mixing, let the test tubes stand on the table for ten minutes ， then measure A 510 each tube A Total activity(mU/tube)0 mU/ml0 mU/mg0 According to the data of your results, calculate the activity units of AKP and its specific activity of AKP, filling the following table, please:

17 X-coordinate A C Items BlankAssay1Assay2( Mg 2+ )Assay3(EDTA) A Total activity(mU/tube)0 6 mU/ml0 60 mU/mg0 120

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 Mg 2+ 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 mol/L pH10 H 2 CO 3 -Na 2 CO 3 buffer ( Carbonate buffer ) mol/L C 6 H 5 Na 2 PO 4 ( Disodium phenyl phosphate ) 3. 1μmol/ml C 6 H 5 OH ( Phenol standard solution ) mol/L MgCl 2 ( Magnesium chloride )Magnesium mol/L EDTA ( Disodium ethylene diamine tetraacetate )EDTA mol/L NaOH (Sodium hydroxide ) mg/ml snake venom % 4-aminoantipyrene % K 3 Fe(CN) 6 ( Potassium ferricyanide )Potassium ferricyanide