Biochemistry

Activity 3AEnzymes

Group 3 – BMLS-2A

To be able to know the properties of an enzyme

To be able to test for the presence of an enzyme

To be able to test the specificity of an enzyme

Objectives

Procedures

Preparation for Potato Extract:1. Peel and grate a potato

into 100 mL distilled water.2. Let it stand for 10 minutes.

Stir occasionally.3. After 10 minutes, strain

through cheese cloth.4. Filter the extract using

filter paper.Use the extract in the

following tests.

Procedures

Biuret Test1. To 1 mL of extract, add 2

mL of 10% NaOH Solution.

2. Mix thoroughly and add 2-3 drops of 1% Copper Sulfate solution.

Observe the appearance for violet color.

Result: Blue (negative)

Expected result: Violet (positive)

Results and Observations

Procedures

Test for Catalase Activity1. To 5mL of extract, mix it

with 1 mL of 3% H2O2.2. Observe whether the gas

evolve supports combustion, by way of holding a splinter over the mouth of the test tube.

3. Add 1 mL of 0.5% Benzidine.

Note the formation of the blue to green coloration.

Result: Brown (negative)

Expected Result: Blue to green coloration

Results and Observations

Procedures

Preparation of a dilute solution of Salivary Amylase1. Rinse mouth several times

with water.2. Collect 1 mL of saliva.3. Prepare a 1:8 solution of

saliva by adding 0.25 mL of saliva to 20 mL of distilled water.

Use prepared test in the following tests.

ProceduresTest for Specificity of Enzyme Action1. Prepare two test tubes, each containing

2 mL of 0.02M Phosphate buffer pH 6.7 and 1 mL of 0.9% Sodium Chloride solution.

2. Test tube no. 1: Add 1 mL cooked starch and 1 mL of salivary amylase solution.

3. Test tube no. 2: Add 1 mL 1% glycogen solution and 1 mL of salivary amylase solution.

4. Stand for 15 minutes at room temperature.

5. Stir the mixture in both test tubes. Place one drop of each solution in separate evaporating dish or spot plate.

6. Add a drop of Iodine solution. Observe the color produced.

7. Repeat the test at 5 minutes intervals for an hour. Tabulate results.

Result:

Results and Observations

Time(minutes)

Test Tube No. 1

Changes

Test Tube No. 2

Changes

5 positive As time passes, the color turns lighter.

Negative No change.

10 Positive Negative

15 Positive Negative

20 Positive Negative

25 Positive Negative

30 Positive Negative

35 Positive Negative

40 Positive Negative

45 Positive Negative

50 Positive Negative

55 Positive Negative

60 Positive Negative

1. Principle involved in the use of Biuret Test.

Biuret Test is based on the ability Cu (II) ions to form a violet-colored chelate complex with peptide bonds (-CONH-groups) in alkaline conditions.

2. How will you prove the presence of catalase? Show chemical equation.

A rapid appearance of sustained gas bubbles is an indication of the presence of a catalase.

2 H2O2 2 H2O + O2

3. How will you prove the specific action of salivary amylase?

Enzymes are normally very specific , catalyzing at most only a few types of chemical reactions. The enzyme amylase acts on the substrate starch catalyzing its break down into simple sugars. Amylase will not catalyze the break down of the complex sugar sucrose into simple sugars. Indicators can be used to test for the presence of these reactions like iodine solution in the iodine test, amylase in the starch (straight chain) forms helices where iodine molecules assemble and forming blue-black color. While amylopectin (branched portion) in glycogen forms much shorter helices and iodine are unable to assemble and only forming orange/yellow hue.

Analysis and Conclusion

Biochemistry

Activity 3BFactors Affecting Enzyme Action

Group 3 – BMLS-2A

Describe the effect of temperature and pH in enzyme action.

Explain how changes in pH and temperature affect enzyme action.

Objectives

Procedures

Effect of Temperature1. Prepare three test tubes and label

them respectively.2. Test tube no. 1: 5 mL 1% cooked

starch solution + 1mL saliva. Keep in water bath at 40ºC.

3. Test tube no. 2: 5 mL 1% cooked starch solution + 1 mL saliva. Keep in water bath at 60ºC.

4. Test tube no. 3: 5 mL 1% cooked starch solution + 1 mL saliva. Keep in water bath at 10ºC.

5. At 15 minutes interval, take a drop of the reaction mixture from each test tube, stir the mixture first before taking a drop, and test with iodine solution. Perform the iodine test for a period of an hour.

At 60ºC

At 10ºC

At 40ºC

Result

Results and Observations

Time (minutes)

Test tube no.1 @ 40ºC

Test tube no. 2 @ 60ºC

Test tube no. 3 @ 10ºC

15 White surrounded by black ring

Blue black or something dark in color

Light yellow with visible black ring formation in center

Orange or something light in color

Yellowish black*after 6 minutes – violet

Blue black or something dark in color

30 Light yellow surrounded by black ring

Blue black or something dark in color

White with visible black ring formation in the middle

Orange or something light in color

Yellowish black*after 40 minutes – black

Blue black or something dark in color

45 yellow surrounded by black ring

Blue black or something dark in color

Light yellow with visible scattered black particles

Orange or something light in color

(darker) yellowish black*after 10 mins. – Light Violet

Blue black or something dark in color

60 Dark yellow

Blue black or something dark in color

violet Orange or something light in color

Dark yellow Blue black or something dark in color

At40ºC

At60ºC

At10ºC

ProceduresEffect of pH1. Prepare four test tubes containing equal

amounts of egg white. Place the test tubes in hot water bath. When the egg white is coagulated, cool n=and mark the height of the solidified egg white.

2. Test tube no. 1: 5 mL 2% pepsin + 10 drops 0.4% HCl

3. Test tube no. 2: 5 mL 2% pepsin + 10 drops of 0.4% Na2CO3

4. Test tube no. 3: 5 mL 2% pancreatin + 10 drops of 0.4% HCl

5. Test tube no. 4: 5 mL pancreatin + 10 drops 0.4% Na2CO3

6. Keep the test tubes in a beaker of water controlled at 40ºC for one hour.

7. Use a pH paper to determine specific pH of each test tube.

8. Determine the extent of digestion in each test tube by noting the amount of protein dissolved or disintegrated.

9. Perform Biuret Test of 1 mL of supernatant liquid from each test tube.

10. Compare the colors obtained with 0.5% peptone standard.

Result

Results and Observations

Test tube

pH Color Expected color

Result

No 1. pepsin + HCl

Acidic

Purple

Purple

+

No. 2 pepsin + Na2CO3

Basis

Purple

Purple

+

No. 3 pancreatin + HCl

Acidic

Purple

Purple

+

No. 4 pancreatin + Na2CO3

Basic

Purple

Purple

+

1. Explain the mechanism of enzyme action at varying temperature. Give the principle involved.

Being a protein, an enzyme is denatured at high temperature and becomes inactive. Initially as the temperature increases, the rate of enzymatic reaction also increases but it has a limitation. When the temperature affects the molecular structure of the enzyme it gets inactivated.

2. How does different pH affect enzyme action? Explain your results using this principle. Discuss Isoelectric pH enzyme action.

Enzymes are picky with pH levels, as they are with everything else. They have an optimal level at which they work the best, and anything above or below that level, their activity begins to slow down until they shut down all together.

Analysis and Conclusion