Eilisha Joy Bryson

MISEP Chemistry 512 – Jacobs

Enzyme Catalyst Lab - Formal Report – August 8, 2007

 

 

 

Abstract

This investigation examined what would happen to the rate of an enzyme-catalyzed reaction if the concentration of substrate changed. We hypothesized that if the concentration increased, then the reaction rate would also increase. To test our question, we varied a combination of substrate and buffer, totaling 6mL, with a constant amount of 2 drops of catalyst. The enzyme catalyst, peroxidase, increased the rate of the reaction. The results of our experiment can be found by comparing the reaction rates for each trial. These rates are actually the slopes of the lines that were graphed during each reaction. The trend of data showed that the enzyme-catalyzed reaction rates gradually decreased as the amount of substrate decreased.

 

Materials and Methods

Materials:


2 - 10 mL Graduated cylinders

1 pipette

5 to 10 medium sized test tubes

10 drops of cold Chicken liver (Enzyme Catalyst (Peroxidase))

25 mL of buffer (H2O)

25 mL of substrate (H2O2)

Cold bath

LabPro Equipment


 

Changing Concentration Methods:

  1. Set-up LabPro equipment.
  2. Place the chicken liver enzyme into a cold bath to maintain a cold temperature.
  3. In separate graduated cylinders, measure out 2ml of substrate and 4ml of buffer and pour both into a test tube. Add 2 drops of catalyst. Cap immediately with LabPro stopper, and start the computer measurement program. Stop recording once the stopper pops off.
  4. Using the software, find the slope of the line, which will represent the reaction rate. Print the graph for your records.
  5. Repeat steps 3 and 4, increasing the substrate by 1 mL and decreasing the buffer by 1 mL, keeping the total volume at 6ml. Do this until you reach 6mL of substrate and 0mL of buffer. (See data table)
  6. Compare reaction rates using the different slopes from the varying concentrations.

Results

Data Table

Substrate

(H2O2)

Buffer

(H2O)

Enzyme Catalyst

(Peroxidase)

Reaction Rate (Slope)

6ml

0ml

2 drops

0.663 kPa/s

5ml

1ml

2 drops

0.545 kPa/s

4ml

2ml

2 drops

0.429 kPa/s

3ml

3ml

2 drops

0.295 kPa/s

2ml

4ml

2 drops

0.208 kPa/s

 

The table above shows the results of our experiment where we investigated what would happen to the rate of an enzyme-catalyzed reaction if we varied the amount of substrate. Our table shows the 5 different amounts of substrate that we used in combination with the buffer and the amount of enzyme used. The volume of the combination always totaled 6 mL. The results of our experiment can be found by comparing the reaction rates for each trial. These rates are actually the slopes of the lines that were graphed during each reaction (See graphs). As is shown in the table, the rates gradually decreased as the amount of substrate decreased.

 

Discussion

Our research question evolved into, “Is there a relationship between the amount of substrate and the rate of an enzyme-catalyzed reaction?” Since the results showed a decrease in rate as the amount if substrate decreased we can conclude that there is a relationship between the two. In other words, a higher concentration of substrate will yield an increase in the rate of the reaction. Since this reaction was between an enzyme, peroxidase, and a substrate some generalizations can be made.

            When an enzyme catalyzes a reaction a definite effect can be noted. The catalyst increases the rate of the reaction. In this case the catalyst was the chicken liver, which contained the enzyme peroxidase. Since an enzyme is involved, the reaction rate is increased. Every reaction has an activation energy, which is the amount of energy needed to make the reaction happen. Molecules with higher kinetic energy can match the activation energy and actually proceed with the reaction, but not all molecules have enough energy to do this. The enzyme lowers the activation energy required for the reaction by allowing the molecules to take its path as an alternate because it has a lower activation energy. If the reaction has a lower activation energy then more molecules will be able to have enough kinetic energy to proceed with the reaction.

            What happened during our experiment is that we kept the amount or concentration of catalyst constant and varied the concentration of the substrate. Our results showed that the reaction rate and concentration of substrate are proportional. Why this happens is interesting. A reaction is the change of the substrate into a new and different thing, called the product. The collision theory states that reactions happen as molecules collide, but they must collide at the correct orientation so that the activation sites on the molecules will match up. If you can increase the number of molecules in a reaction, you will also increase the chances of having the molecules collide. When we increased the concentration of substrate we increased the number of molecules, and thereby increasing the chances of the molecules colliding. So increasing the concentration of the substrate increased the rate of the reaction.

            Our results are based on theory, as stated above, and experimental evidence. Our evidence was not without flaws. Our group’s plan was to take four trials of every different concentration of substrate (see table). We had a difficult time manipulating the equipment and knew so when we kept getting drastically different data for the same concentration. We disregarded this data, realizing that the temperature of the enzyme had to be stabilized. Once we regulated this and got a handle on the equipment our data began to make more sense. Unfortunately, the amount of time we spent working out these problems was time away from our data collection. We were not able to take multiple samples like we planned. This always makes the data less reliable because we only have one trial for each concentration on which to base our conclusions.

            Our graphs provide us with proof that there was some error in our data collection methods. For two of the five trials, we noticed errors in our graphs. Those two trials had graphs that were not linear, which made the slopes of those lines contrary to the trend of the rest of the data.  We accounted for this by focusing on a linear portion of those lines and recalculated their slopes. Once we did that, the new data fit nicely into the trend.

            If we could repeat this experiment I am sure that we would have a more valid set of data. The computer and lab equipment would be more familiar to us, and we would have more time to take multiple trials of the data.