STANDARDCURVE

In the reaction ofphosphatase enzyme on p-nitrophenol phosphate(p-NPP), the product that is formed i.e.p-nitrophenol (p-NP) isdetermined based on its absorbance at wavelength 410 nm. This isbecause this chemical is yellow in colour under alkaline conditionswhereas the substrate p-NPP is colourless.

To convert theabsorbance value of p-NPP at 410 nm into itsconcentration, a standard curve is used. The data for standardcurve as follows:

p-NP (mg)	Abs 410 nm
0	0
5	0.098
10	0.194
15	0.280
20	0.374
25	0.464

The contents of the reaction for thestandard curve as follows:

Content	Volume (ml)
p-NP	1.0
Buffer, pH 10.0	1.0
0.04 M NaOH	1.0
Total	3.0

Plot the absorbanceat 410 nm versus weight of p-NP. Determine theweight of p-NP for 1 unit of absorbance. Based onthis value, you will be able to convert all the absorbance valuesat 410 nm to values of weight p-NP.

The molecular weight ofp-NP is 139.1. This value is used to convertweight of p-NP to umol unit.

1. Effect of time on phosphataseactivity

1. Prepare 6 test tubes as follows:

Test Tubes 1 2 3 4 5 6

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Buffer, pH 10.0(ml)	1	1	1	1	1	1
p-NPP (ml)	0.5	0.5	0.5	0.5	0.5	0.5
dH2O (ml)	0.4	0.4	0.4	0.4	0.4	0.4
Incubation time (min)	0	5	10	15	20	25

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2. Label the test tube 1 till 6 and incubate for 5 minutes at roomtemperature.

3. In test tube 1, place 1.0 ml 0.04 MNaOH and followed by 0.1 mlenzyme. This test tube is the control for zerotime.

4. In test tubes 2 till 6, place 0.1 mlenzyme and incubate according to the table above.After 5 minutes, place1.0 ml 0.04 M NaOH into test tube2. After 10 minutes incubation,place 1.0 ml 0.04 M NaOH intotest tube 3 and so on.
5. Determine the absorbance of all the test tubes at410 nm. Use distilled water to blank the spectrophotometer.

   Buffer (pH 10)	1.0 ml
   p-NPP	0.5 ml
   dH2O	0.4 ml

EXPERIMENTA:

1. Complete the table below

T/tubes	Incubation time (minutes)	Absorbance at 410nm	mmolp-NPper ml enzyme
1	0
2	5
3	10
4	15
5	20
6	25

2. Show the calculation below:

3. Plot the product formed (in unit mmol p-NPper ml enzyme; axis-y) versus time (axis-x). (GRAPH 1)

4. Is the activity linear for all the incubation times? Explainyour answer.