Satvika Bejawar 1

Dr. Stephen Thrash

General Biology Laboratory

30 September 2021

Enzymes Lab Report

An enzyme is a protein that helps catalyze chemical reactions in living organisms and it

can be affected by different biological factors such as temperature, pH level, and substrate

concentration. Enzymes can affect many biological processes in the body, especially viruses and

other diseases. Currently, we are all facing a pandemic of the Sars CoV-2 disease. The body has

to utilize many types of enzymes in order to allow the body to react faster to fight off this disease

when infected. According to the ScienceDirect Article “Trends in Immunology,” the enzymatic

reactions can help regulate the cells daily process of protecting its walls from allowing a virus

such as coronavirus to invade and kill it. Enzymes have many such functions, but sometimes

their reaction level and activity can change based on changes in different factors. In an article

from The Proceedings of the National Academy of Sciences, explains that the fluctuating

functions of enzymes have an electrostatic potential that can affect the bond formation and

polarity of molecules. We understand that in certain environments, enzymes can either form

stronger bonds or break bonds between molecules in order to catalyze a reaction.

In these series of experiments, we tested 3 factors that can affect enzyme activity. The

first factor was temperature. We measured how high the enzymatic reaction rate is based on

absorbance level using a spectrophotometer for all 3 experiments. For temperature, we chose 5

temperatures that we put the enzymatic solutions in and measured the absorbance level. Our

hypothesis was that if temperature increases, then the enzymatic reaction rate will increase. Our
Satvika Bejawar 2

Dr. Stephen Thrash

General Biology Laboratory

30 September 2021

results for the experiment, as well as the trend results are explained later on. For the second

experiment, we had a control, and we measured how the differing concentrations of the ONPG

substrate affected the absorbance level of the enzyme. Our hypothesis was that if the

concentration of ONPG increases, the temperature will increase. Furthermore, our reasoning was

based on the fact that a higher concentration of one solution will increase the concentration of the

other solution. Finally, for the last experiment, we measured the absorbance level based on

differing pH plus a blank pH cuvette. Our hypothesis was that if the pH level increases, then the

absorbance level will increase. Our reason for this was that high pH levels were basic, but acidic

solution can cause the protein to denature and lose shape.

This is the first graph that measures absorbance level based on differing temperatures.
Satvika Bejawar 3

Dr. Stephen Thrash

General Biology Laboratory

30 September 2021

This graph represents the data for experiment 1 from objective 3. We measured the

Absorbance of the Enzyme with the condition of different temperatures. The x-axis, as labeled in

the graph, shows the varying temperatures that we put the cuvettes in shown as degrees Celsius.

It is the independent variable of the experiment because we change the temperature while

everything else is controlled. The y-axis shows the average absorbance of the enzyme lactase.

The y-axis represents the dependent variable because it shows how the absorbance changes

based on temperature. The trend here shows us that as temperature increases, enzyme activity/

absorbance increases. However, we see a decrease in enzyme activity as the temperature reaches
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Dr. Stephen Thrash

General Biology Laboratory

30 September 2021

60 degrees celsius. The error bars here show us the range of error, or how much variability there

is in the data points depicted in the graph. This graph forms a slight bell curve where the highest

enzyme activity/ absorbance is around 37 degrees celsius.

In conclusion, according to the graph, the absorbance level reaches its maximum at 37

degrees celsius. We see the absorbance level increasing from 0, 4, 23, to 37. However, at 60

degrees, the absorbance drops back to less than 1. We can conclude that the absorbance level of

the lactase enzyme is greater at 37 degrees celsius because raising temperature can increase the

reaction rate and helps the enzyme catalyze the reaction of lactase with the other substances in

the cuvettes. However, at 60 degrees, the temperature is too high. The enzyme absorbance level

starts to drop because at high temperatures, the protein gets denatured. At 23 degrees celsius, we

can assume that the enzyme substance is around room temperature, so the enzyme activity is

around 1. At 37 degrees, the temperature starts to warm up and generally, as temperature

increases, the reaction speeds up. Proteins need to survive in a limited temperature range, so

when the temperature gets to boiling point, the protein breaks down and starts to lose its

function. We can assume that this is similar to when we get sick, our body temperature increases

and the proteins that generally serve to help us rid the body of toxins will slowly decrease in

activity, and we need time to fight off those toxins as body temperature decreases.
Satvika Bejawar 5

Dr. Stephen Thrash

General Biology Laboratory

30 September 2021
Satvika Bejawar 6

Dr. Stephen Thrash

General Biology Laboratory

30 September 2021

The method we used for this experiment involved 3 trials. We used 3 cuvettes for

each of the different concentrations, except the blank cuvette. First, we set the spectrometer to

420 nm and recorded the absorbency for the blank, which was 0 A. To the other 6 cuvettes, we

added 3.5 mL of pH 7 and 0.5 mL lactase as the controls. We measured the mL of pH and lactase

using the respective pipettes assigned to each bottle. Then to 3 of the cuvettes, we added 0.5 mL

of 1 mM ONPG, and to the other 3 cuvettes, we added 15 mM ONPG using the micropipette. To

the blank, we did not add lactase enzyme, and added 5 mM ONPG. We then covered each

cuvette with parafilm and inverted them to mix the solutions. The reaction took about 2 minutes

to settle, and then we measured the concentration/ absorbance of the enzyme in the spectrometer.

We then recorded in our data table. We recorded the blank cuvette before we switched to a

different concentration of ONPG. We recorded the first 3 trials of 1 mM ONPG first, then we

recorded 15mM ONPG cuvettes next. After we finished the experiment, we poured the liquids

into the liquid waste container and cleaned each cuvette.

In this graph, we measured the absorbance level of enzyme activity where the

independent variable is the concentration of ONPG in mM and the dependent variable is the

absorbance level (A). According to the graph, we see an increase in absorbance level at 15 mM

of ONPG, while the absorbance level at 1mM ONPG is fairly low. We had a blank to serve as a

control. The standard error of the standard deviation represents the variability in the range of
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Dr. Stephen Thrash

General Biology Laboratory

30 September 2021

absorbance level data points depicted in the graph. We measured concentration using the

absorbance level of the spectrometer.

In conclusion, the absorbance level of the enzyme lactase increases as the concentration

of ONPG, measured in mM, increases. At 1 mM ONPG, the absorbance level is around 0.5 A. At

15 mM ONPG, the absorbance level is around 3.5 A. In this experiment, the ONPG

concentration is the substrate. We can assume that the absorbance level increases as the

substrate, ONPG, concentration increases because more substrate molecules will collide with the

enzyme molecules, so more products will be formed. In this case, product means the absorbance

level, or how yellow the liquid in the cuvette is. The more yellow it is, the higher the

concentration. This is similar to how our body if we take a catalyst, it would act as a substrate

and increase the concentration of the enzyme because more enzymes are needed to speed up the

reaction.
Satvika Bejawar 8

Dr. Stephen Thrash

General Biology Laboratory

30 September 2021

For the second experiment, we measured the different concentrations of pH and how it

affected the absorbance level of the enzyme. The method we used is similar to the previous

experiment with the ONPG. We set the spectrometer to 420, and we added 0.5 mL DI water to

the blank, 0.5 mL of 5 mM ONPG to the blank using the pipette assigned to it. Then we set up 3

cuvettes for pH 2 and 2 cuvettes for pH 10. To each cuvette, we added 0.5 mL lactase, 0.5 mL of

5 mM ONPG stock as controls using the specific pipettes for them. Then to 3 of the cuvettes, we

added 3.5 mL of pH 2 buffer, and we added pH 10 buffer to the other cuvettes using a

micropipette. We covered each cuvette in parafilm and mixed the solutions. After 2 minutes, the

reaction settled, and we set the spectrometer to the blank at 0 A. Then we measured the pH 2

cuvettes first, recording the Absorbance level. Then we measured the pH 10 cuvettes and
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Dr. Stephen Thrash

General Biology Laboratory

30 September 2021

recorded the absorbance level. Again, we cleaned up by pouring the liquids into the waste

container and cleaning each cuvette.

This graph shows the different pH levels and the absorbance level measured in A. For pH

2, we see a high absorbance of 0.45 A, while the absorbance is almost at 0 for pH 10. We had a

blank to serve as a control. The standard error of the standard deviation represents the variability

in the range of absorbance level data points depicted in the graph. We measured concentration

using the absorbance level of the spectrometer.

In conclusion, the absorbance level of the enzyme is high at a lower pH, and very low at a

higher pH. The most favorable pH value for the enzyme, which is called the optimum pH,

appears to be at pH 2. pH levels that are around the same as water tend to be most favorable for

enzymes. Here, we see that at an extremely high pH, the enzyme activity decreases because the

protein in denatures at a basic pH. Even though 2 is acidic, the enzyme seems to work well

because it can work in acidic conditions, as we know from our human body. However, the

enzyme works best at the body’s optimum pH, which is around 7. At extremely high pH values,

this interference causes the protein to unfold, the shape of the active site is no longer

complementary to the substrate molecule and the reaction can no longer be catalysed by the

enzyme.

Citations
Satvika Bejawar

10

Dr. Stephen Thrash

General Biology Laboratory

30 September 2021

Salmi M, Jalkanen S. 2001. VAP-1: an adhesin and an enzyme. Trends in Immunology.

22(4):211–216. doi:10.1016/S1471-4906(01)01870-1. [accessed 2021 Sep 30].

https://www.sciencedirect.com/science/article/pii/S1471490601018701.

Warshel A. 1984. Dynamics of enzymatic reactions. Proceedings of the National

Academy of Sciences. 81(2):444–448. doi:10.1073/pnas.81.2.444. [accessed 2021 Sep 30].

https://www.pnas.org/content/81/2/444.