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The Effect of Substrate Concentration on Enzyme Reactivity

Annelise Gress Brandon Eilberg Bryant Fisher

Hypothesis
As substrate concentration increases, the rate of reaction will also increase until it reaches a maximum value at which point it will proceed to decline. The data supports this hypothesis because the reaction proceeded most quickly at 1.5ml of substrate.

Background Information
Enzymes:
Proteins Catalyze reactions Substrate specific

Peroxidase
Enzyme Catalyzes decomposition of hydrogen peroxide

Background Information Contd


Peroxidase

2H202
Substrate Enzyme

2H2O + O2
Products

Background Information Contd


Substrate Concentration
Low: slow reaction Optimal: fastest reaction High: slower reaction

At Active Site
Low: substrate has low probability of binding Optimal: as products leave, substrate is available High: excess substrate interferes

Results

Table 2: The Effect of Substrate Concentration on the Initial Reaction Rate of Peroxidase
Substrate Volume 2.0 ml 0.02 ml 0.05 ml 0.1 ml 0.25 ml 0.50 ml 1.0 ml 4.0 ml Initial Reaction Rate 1.62 min-1 .69 min-1 1.11 min-1 1.35 min-1 1.56 min-1 1.68 min-1 1.68 min-1 1.11 min-1

Calculation: absorbance@ 20 seconds-absorbance@ 0 seconds/20 seconds-0 seconds Example: .54-0/20-0=.027 sec-1 x 60 seconds=1.62min-1

Figure 2: Effect of Substrate Concentration on the Initial Reaction Rate of Peroxidase


Substrate Volume (ml) vs Initial Reaction Rate (min-1)
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Initial Reaction Rate (min-1)

Initial Reaction Rate (min-1)

0.5

1.5

2 2.5 3 Substrate Volume (ml)

3.5

4.5

As Substrate concentration increases, the initial reaction rate also increases. An optimal substrate concentration is reached before the initial reaction rate begins to decline

Table 1: Average Absorbance at Indicated Substrate Concentrations vs. Time


Time 0 20 2.0 ml 0 .54 0.02 ml 0 .23 0.05 ml 0 .37 0.1 ml 0 .45 0.25 ml 0 .52 0.5 ml 0 .56 1.0 ml 0 .56 4.0 ml 0 .37

40
60 80 100 120

.91
1.09 1.26 1.37 1.47

.49
.70 .89 1.05 1.21

.68
.94 1.19 1.38 1.70

.83
1.18 1.40 1.70 1.78

.90
1.23 1.50 1.57 2.0

.98
1.30 1.52 1.80 2.0

.93
1.20 1.43 1.53 1.72

.57
.69 .80 .85 1.38

Figure 1: The Effect of Substrate Concentration on Peroxidase Activity


Absorbance vs. Time (s)
2 2.0 ml

0.02 ml
1.5 0.05 ml 0.1 ml 0.25 ml 1 0.5 ml 1.0 ml 0.5 4.0 ml Absorbance

0 0 20 40 60 80 Time (seconds) 100 120

The rate of reaction is greatest at the optimal substrate concentration, 0.5 ml. At higher and lower concentrations, the rate of reaction is slower.

Discussion
As substrate concentration increases, the rate of reaction will also increase until it reaches a maximum value at which point it will proceed to decline. Supported by data Referring to Table 1 and Figure 1 Concentrations: show a steady uniform increase in absorbance Larger concentrations=increased rate Larger concentrations: react with a steeper curve Exception: 4 ml of substrate Due to substrate interference Curve slightly less steep on graph and data shows decrease

Discussion Contd
Referring to Table 2 and Figure 2 Steady increase of initial reaction rate Increase due to increasing availability of substrate at onset More enzymes-substrate complexes at initial stage Levels off and decreases after optimal concentration All enzymes involved in enzyme-substrate complex Excess substrate in solution Also causes interference with enzyme: observed decrease

Conclusion
Initial reaction rate increased due to increased substrate concentration. Value over optimal concentration leads to noticeable decrease in rate. By using different concentrations of substrate, the abosrbance, rate, and initial rate of reaction also increase. Too much substrate, ie 4 ml, leads to interference and decrease in measured data.

References
2011. Foundations of Biology Laboratory 1. Haydeb-McNeil publishing. United States. Pg. 43-44 Freemen, Scott. Biological Science. 4th ed. San Francisco: Benjamin Cummings, 2011. Print.