BIOCHEMISTRY

LAB: PRACTICAL Total mark: [10]

Effect of optimum temperature and pH on activity of alkaline phosphatase.

Introduction

1. Definition of enzyme

2. The importance of enzyme in cells

3. Factors that affects the activity of enzyme

4. What does denaturation of enzyme means?

Materials

Reagents Unit Amount Total amount (20 groups)

1 p-Nitrophenol phosphate mL 2 40
2 Alkaline phosphatase on ice mL 1.5 30
3 Water bath (5 °C) unit 1 20
4 Water bath (30 °C) unit 1 20
5 Water bath (37 °C) unit 1 20
6 Water bath (55 °C) unit 1 20
7 Water bath (70 °C) unit 1 20
8 Distilled water mL 1 20
9 Glove box 1 20
10 Acetate buffer, pH 5 mL 5 100
11 Citrate buffer, pH 6 mL 5 100
12 Phosphate buffer, pH 7 mL 5 100
13 TRIS buffer, pH 8 mL 5 100
14 TRIS buffer, pH 9 mL 5 100
15 Ice box unit 1 20
16 Spectrophotometer unit 2 40

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 17 Cuvette unit 2 40

*p-nitrophenol (product) is NOT the same as p-nitrophenol phosphate (substrate)

Method

Part A

1. Set up a series of test tubes, each containing the following materials (refer to table
below).

0 1 2 3 4 5
Test tube
Control (ice) (30 °C) (37 °C) (55 °C) (70 °C)

TRIS buffer, pH 8.5 (mL) 1.0 1.0 1.0 1.0 1.0 1.0

p-nitrophenol phosphate 0.2 0.2 0.2 0.2

0.2 0.2
(substrate) (mL)

Alkaline phosphatase 0.2 0.2 0.2 0.2

- 0.2
(mL)

Water (mL) 0.2 - - - - -

2. Place test tube 1 in an ice bath for 5 °C and test tubes 2-5 into water baths at respective
temperature: 30 °C, 37 °C, 55 °C and 70 °C.

3. Prepare a control test tube by mixing 1.0 mL glycine, 0.2 mL substrate and 0.2 mL water.

4. Add 0.2 mL alkaline phosphatase to all tubes except the control tube. Control tube will
have 0.2 mL of distilled water instead of alkaline phosphatase. Mix all tubes thoroughly
and incubate again at the respective temperature for another 15 minutes.

5. After 15 minutes, add 8 mL of 0.02 M NaOH to stop the reactions.

6. Read the absorbance (A) using the spectrophotometer adjusted to 410 nm wavelength.

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 7. Plot a graph of absorbance vs temperature to determine the optimum condition for the
enzyme activity.

Part B

1. Set up a series of test tubes, each containing different amount of substances and chemical.

0 1 2 3 4 5
Test tube
Control (pH 5) (pH 6) (pH 7) (pH 8) (pH 9)

Acetate Citrate Phosphate TRIS TRIS

Distilled Buffer, Buffer, Buffer, Buffer,
1 mL of Buffer,
water pH 5 pH 6 pH 7 pH 8 pH 9

p-nitrophenol phosphate 0.2 0.2 0.2 0.2

0.2 0.2
(substrate) (mL)

Alkaline phosphatase 0.2 0.2 0.2 0.2

- 0.2
(mL)

Distilled water (mL) 0.2 - - - - -

2. Place all test tubes in 37 °C water bath for 5 minutes.

3. Take the test tubes out and add 0.2 mL alkaline phosphatase to all tubes except the
control tube. Control tube will have 0.2 mL of distilled water instead of alkaline
phosphatase.

4. Mix all tubes thoroughly and incubate again for another 15 minutes.

5. After 15 minutes, add 8 mL of 0.02 M NaOH to stop the reactions.

6. Read the absorbance (A) using the spectrophotometer adjusted to 410 nm wavelength.

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 7. Plot a graph of absorbance vs pH to determine the optimum condition for the enzyme
activity.

Result

1. Plot a graph of absorbance vs temperature. [2]

2. Plot a graph of absorbance vs pH. [2]

Discussion

1. What makes enzyme different from other proteins [1]

2. Why does enzyme activity change with temperature? State the optimum [2]
temperature for enzyme to work in the human body.

3. Why does enzyme activity change with pH? State the optimum pH for [2]
enzyme to work in the human blood and gaster fluid.

4. What precautions must you take when handling enzymes? [1]

Conclusion

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