Table 1. Solution concentrations, volumes and observations for Experiment 1: Observing the enzyme reaction.

Test TubedH20Potato ExtractCatecholObservations
15 ml + 500μl—–500μLSolution turned milky-white to clear. (High substrate)
25 ml500μl500μlSolution turned yellowish-brown. (High substrate)
35 ml + 500μl500μl—–Solution is clear, but cloudy-white at the bottom. (Zero substrate)

 

*The chemical reaction was observed with the introduction of catechol to the potato extract (tube 2).

 

Table 2. Solution concentrations, volumes and observations for Experiment 2: The effect of substrate concentration on enzyme activity.

Test TubedH20Potato ExtractCatecholObservations
15 ml + 500μl500μl500μLSolution turned light, yellowish-brown. (High substrate)
25 ml + 900μl500μl100μlSolution turned translucent peach. (Low substrate, diluted)

 

*The reaction time for tube 1 was the fastest due to the high substrate concentration and lower dH20 concentration.

 

Table 3. Solution concentrations, volumes and observations for Experiment 3: The effect of enzyme concentration on enzyme activity.

Test TubedH20Potato ExtractCatecholObservations
15 ml + 500μl500μl500μLSolution turned from bluish-green to light, yellowish brown. (High substrate)
25 ml + 900μl100μl500μlSlightly cloudier than the original clear solution; no real change in color. (High substrate, diluted, low enzyme)

 

*Lower Dh20 and higher potato extract concentrations allowed for a faster reaction time

for tube 1.

 

Table 4. Solution concentrations, Buffer pH, volumes, and observations for Experiment 4: The effect of pH on enzyme activity.

Buffer pHBuffer VolumedH20Potato ExtractCatecholObservations
42ml3ml500μl500μlCloudy white
62ml3ml500μl500μlDark yellow
82ml3ml500μl500μLOrangish-brown
102ml3ml100μl500μlTranslucent peach

 

*The reaction rate increased as the pH increased, with a pH of 6 being the best buffer for catechol oxidase activity. Increasing the pH past 6 showed a decrease in the reaction rate.

 

Table 5. Solution concentrations, temperatures, volumes and observations for Experiment 5: The effect of temperature on enzyme activity.

Test TubedH20Potato ExtractCatecholObservations
1 (0°C)5ml500μl500μlReally light yellowish-brown
2 (15°C)5ml500μl500μlYellowish-peach
3 (37°C)5ml500μl500μLOrange-peach
4 (100°C)5ml500μl500μlReally light peach

 

*The fastest reaction rate was observed at 37°C. The colder the temperature (0°C – 15°C), the slower the reaction rate. Enzyme denaturation was observed at 100°C.

 

Table 6. Solution concentrations, volumes and observations for Experiment 6: Inhibitor Effects – Inhibiting the Action of Catechol Oxidase

Test TubedH20Potato ExtractPTUCatecholObservations
15ml + 1ml500μl—–500μlYellowish-peach (Control)
25ml + 500μL500μl500μl500μlReally light peach
35ml500μl500μl500μLCloudy, clear-white

 

*The fastest, and most pronounced reaction was observed in tube 1 (the solution without phenylthiourea)

 

Enzyme Lab Discussion

For the first experiment, Observing the Enzyme Reaction, it was hypothesized that the enzyme reaction would only occur in the second test tube due to the fact that it was the only tube to contain both the enzyme and substrate. As expected, the solution in tube 2 was the only solution to show the characteristic yellow-brown pigment of benzoquinone production, which was caused by the potato extract converting its catechol into the new product.

In experiment 2, The Effect of Substrate Concentration on Enzyme activity, the hypothesis was that the tube with the higher substrate concentration would show a faster, and more pronounced chemical reaction than the tube with less catechol. The hypothesis was supported by the fact that the higher catechol concentration in tube 1 allowed for a similar result to tube 2 from experiment 1, the only difference being that the extra 5mL of dH20 diluted some of the yellowish-brown color observed in the first reaction. While there was a chemical reaction observed in tube 2 (experiment 2), it was much slower (with a translucent peach pigment) due to lower a catechol concentration, and a higher dH20 concentration. The higher the concentration of catechol, the more benzoquinone that can be produced.

It was hypothesized in experiment 3, The Effect of Enzyme Concentration on Enzyme Activity, that the higher the concentration of enzyme in the solution, the faster and more pronounced the chemical reaction would be. This hypothesis was able to be accepted based on the rate at which the tube with the higher potato extract concentration reacted. Tube 1 had 400μL more potato extract and 400μL less dH20 than tube 2. Because enzymes are biological catalysts that speed up chemical reaction time, the solution in tube 1 quickly changed from a bluish-green pigment, to the yellowish-brown color associated with benzoquinone. The lower concentration of potato extract and higher concentration of dH20 in tube 2 showed no change in color, other than the cloudiness of the potato extract itself.

In experiment 4, The Effect of pH on Enzyme Activity, the initial hypothesis was that the lower the pH level of the buffer added to the solution, the quicker the reaction rate would be. This hypothesis was not supported by the data observed because higher acidity levels actually slowed the production of benzoquinone – which was the opposite of what was predicted. The solution with a pH buffer of 4 remained cloudy white, while the solution with a 6 pH buffer turned yellowish-brown. As the pH increased, the benzoquinone production rate increased. While lower pH buffers proved to be too acidic, more neutral buffers allowed for the best environment for catechol oxidase activity. Buffer pH levels higher than 6 showed a slower and less pronounced chemical reaction as well – illustrating the enzyme reaction’s need for neutrality.

The hypothesis for experiment 5, The Effect on Temperature on Enzyme Activity, was that extremely low temperatures would slow the rate of benzoquinone production, while extremely high temperatures would cause the enzymes to denature. This hypothesis was supported by the rate at which the solutions at 0°C – 15°C slowly reacted, and the rate at which the solution at 37°C quickly produced benzoquinone. After five minutes at each solution’s designated temperature, the colder solutions barely started to change color, while the warmer temperatures quickly reacted – so much so that at 100°C, the enzymes denatured and the solution began to pale in pigment. Colder temperatures slowed the movement of molecules in the solutions, while warmer temperatures (not including 100°C) allowed for a better environment for catechol oxidase activity.

For experiment 6, Inhibitor Effects – Inhibiting the Action of Catechol Oxidase, it was hypothesized that the addition of phenylthiourea (PTU) would keep the enzyme reaction from occurring. The hypothesis was able to be accepted due to the fact that the tubes which contained the PTU showed very little change in pigment. Tube 1 served as the control, which showed the production of benzoquinone (yellowish-brown color) and allowed for comparison between the three solutions. Considering PTU is a noncompetitive inhibitor, tubes 2 and 3 contained solutions that prevented the enzyme from catalyzing the reaction, regardless of whether or not the substrate was bound to the active site.

The only real issue with any of the 6 experiments was the unsupported hypothesis for the Effect of pH on Enzyme Activity experiment. I must have tied the preservative nature of benzoquinone with how acidic lemon juice keeps apples from turning brown, so I assumed a low pH would increase the reaction rate. In reality, acidity slows the reaction rate – which is why the apples don’t change color.

In conclusion, these experiments have shown that benzoquinone production can only occur with the presence of both an enzyme and substrate. Factors such as substrate and enzyme concentration, pH, temperature, and the presence of noncompetitive inhibitors can affect enzyme reaction. High substrate concentration will allow for greater benzoquinone production, while high enzyme concentration will speed up the reaction rate – and vise versa. In order for enzyme reaction to rapidly occur, it must be done in an environment where the pH is as close to neutral as possible, with the reaction rate slowing in both highly acidic or basic solutions. The same goes for temperature – extremely high or extremely cold temperatures can decrease enzyme reaction rates, or cause the enzymes to denature altogether. The introduction of a noncompetitive inhibitor (such as phenylthiourea) allows it to bind to the allosteric site on the enzyme, which keeps the reaction from occurring (regardless of the enzyme or substrate concentration).

Leave a Reply

Be the First to Comment!

avatar