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EXPERIMENT THREE

ASSAY OF ACTIVITY OF ALKALINE PHOSPHATASE

2012-09

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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.

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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

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△S P△• V= or

△t t△

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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).

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Total enzyme activity and specific activity

For example

Sample A BUnits of AKP activity 100 100

Volume (ml) 100 10

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?

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Total enzyme activity and specific activity

For example

Sample A BUnits of AKP activity 100 100

Volume (ml) 100 10

Specific activity (U/ml) 1 U/ml 10 U/ml

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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 K3

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

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

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Principle

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

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Protocol 1. Make the standard curve

Reagents blank 1 2 3 4 5 6

H2CO3-NaHCO3 buffer (pH

10)

1.0 1.0 1.0 1.0 1.0 1.0 1.0

Phenol standard solution 0 0.05 0.1 0.2 0.3 0.4 0.5

H2O 1.0 0.95 0.9 0.8 0.7 0.6 0.5

37℃, 5min

0.2mol/L NaOH solution 1.0 1.0 1.0 1.0 1.0 1.0 1.0

4-aminoantipyrene 1.0 1.0 1.0 1.0 1.0 1.0 1.0

K3Fe(CN)6 2.0 2.0 2.0 2.0 2.0 2.0 2.0

Mixing, waitting for ten minutes， read A510

A510

Phenol (μmol) 0 0.05 0.1 0.2 0.3 0.4 0.5

Correspond enzyme unit 0 2 4 8 12 16 20

ml

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Protocol

Draw the standard curve by using A510 as y-coordinate and correspond enzyme unit as x-coordinate

A510

Enzyme unit0

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Name of the curve

Operator

Date

Apparatus

A

Correspond enzyme unit

0

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【 Calculation】

Corresponding enzyme activity (mU/tube)

= corresponding µmol value × 125

× 1000

= corresponding µmol value × 40

25, is the time extent for water bath, or reaction time

× 1000, to change U to be mU

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Protocol 2. Assay of AKP activity

Reagent Blank Assay1 Assay2(Mg2+) Assay3(EDTA)

Substrate (0.02mol/L) (No Mg2+ and EDTA)

0 0.3 0.3 0.3

Mg2+(0.012mol/L) 0 0 0.1 0

EDTA(0.012mol/L) 0 0 0 0.1

H2O 1 0.7 0.6 0.6

Enzyme (0.5mg/ml) 0.1 0.1 0.1 0.1

H2CO3 buffer (pH10) 0.9 0.9 0.9 0.9

37℃, 25min

NaOH 1.0 1.0 1.0 1.0

4-aminoantipyrene 1.0 1.0 1.0 1.0

K3Fe(CN)6 2.0 2.0 2.0 2.0

ml

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Protocol 2. Assay of AKP activity

Reagent Blank Assay1 Assay2( Mg2+) Assay3(EDTA)

Mixing, let the test tubes stand on the table for ten minutes， then measure A510 each tube

A510 0

Total activity(mU/tube)

0

mU/ml 0

mU/mg 0

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

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X-coordinate

A

C0 2 4 6 8 10

Items Blank Assay1 Assay2( Mg2+) Assay3(EDTA)

A510 0 0.292

Total activity(mU/tube)

0 6

mU/ml 0 60

mU/mg 0 120

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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

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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?

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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 )