F.6B Leung Tsz Hang (29)Biology Laboratory Report 17 November 2004

Investigation of the effect of substrate concentration on enzyme activities

Aim

To investigate the effect of urea (substrate) concentration on urease (enzyme) activity.

Biological Principle

Urease is found in bacteria, moulds, higher plants and in some loweranimals. It is virtuallysubstrate-specific. It acts on urea and converts it intoammonia and carbon dioxide by hydrolysis.

/ + H2O  2NH3 + CO2

The ammonia liberated can be detected by using theindicator bromothymol blue. Once enough amount of ammonia is evolved, the solution will become alkaline and a complete blue colour is observed. By noting the time needed to cause the complete change in colouration, the rate of enzymatic activity in the corresponding solution can be evaluated.

Procedure

  1. 1.0 gram of soya bean was weighed
  2. The softened beans were pouted into a mortar and were grinded into a milky paste
  3. 25 cm3 of distilled water was added to the mortar to dilute the paste
  4. The suspension was filtered through a layer of muslin cloth to remove cell debris. The filtrate was then centrifuged for 5minutes.
  5. 6 test tubes were labelled from 1 to 6.
  6. Graduated pipettes were used to add the followingvolumes ofbromothymol blue solution, 2% ureasolution and distilled water in tubes 1 to 6 asfollow:

Tube / 1 / 2 / 3 / 4 / 5 / 6
Bromothymol blue (cm3) / 1.0 / 1.0 / 1.0 / 1.0 / 1.0 / 1.0
2% urea solution (cm3) / 2.0 / 1.5 / 1.0 / 0.8 / 0.5 / 0.2
Distilled water (cm3) / 0 / 0.5 / 1.0 / 1.2 . / 1.5 / 1.8
  1. The contents of each of the 6 test tubes were well mixed.
  2. 0.5 cm3 of the enzyme extract was added into each of thetubes. Time was noted immediately.
  3. The tube was shakento mix the contents thoroughly.
  4. The time taken for the bromothymol blue tochange from yellow/green to blue in the solution was record.
  5. Steps 8-10 were repeated for all test tubes

Results

Tube / 1 / 2 / 3 / 4 / 5 / 6
Bromothymol Blue (cm3) / 1.0 / 1.0 / 1.0 / 1.0 / 1.0 / 1.0 / Independent Variables
2% Urea Solution (cm3) / 2.0 / 1.5 / 1.0 / 0.8 / 0.5 / 0.2
Distilled Water (cm3) / 0.0 / 0.5 / 1.0 / 1.2 / 1.5 / 1.8
Urea Concentration (%) / 2.0 / 1.5 / 1.0 / 0.8 / 0.5 / 0.2 / By Calculation
(Graphed)
Time Used for Complete Reaction (s) / 38 / 32 / 25 / 18 / 13 / 7 / Logged Data
Rate of Reaction
(% urea s-1) / 0.0526 / 0.0469 / 0.0400 / 0.0444 / 0.0385 / 0.0286 / By Calculation
(Graphed)

Note: The graph was attached on a new page.

Discussion

1)Trend of results

As the urea concentration increases, the rate of reaction increases as well. It is because the more substrate molecules available for the formation of the enzyme-substrate complex, the faster the reaction will be.

However, if the substrate concentration continues to increase, the increase in reaction rate gradually slows down because the reaction rate limited by enzyme concentration. When the substrate concentration increases to a certain extent, the active sites of the enzyme are fully utilized and the reaction rate levels off.

2)Source of errors

A. Inconsistency of Enzyme Concentration

The amount of urease contained in the extract might not be uniformly distributed. That meant the amount of ureasein the 0.5 cm3 solution added to each test tube may not be equal. It leaded to the inconsistency of the amount urease in the experiment. The tube containing more urease would have a higher rate of reaction since more active sites were available for the formation of enzyme-substrate complex.

B.Heat change during the reaction

Heat might be released during the reaction. The increased heat might raise the rate of reaction in the sense that it provided higher kinetic energy to the molecules in the solution. With more kinetic energy, substrate and enzyme molecules moved faster and collided with each other more frequently. The enzyme-substrate complexes were formed more rapidly and hence the reaction went on faster.

3)Assumptions

The concentration of enzyme in the extract is assumed to be uniform; and

The increase in temperature during the reaction was assumed to be negligible.

Conclusion

The enzyme activity increases with substrate concentration in this experiment.

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