LAB Report - Lecture notes 2-3 PDF

Preview

Summary

report...

Description

Properties of Enzymes Lab Report

Maria Maurant BIOL 1500 Sarah Koerner October 30th, 2018

I.

Abstract

The Properties of Enzymes lab was to discover the unique characteristics of enzymes and to also find their optimum conditions. In this lab, characteristics such as optimum pH, temperature and enzyme concentration were discovered. Different conditions in this lab were tested such as basic, neutral, and high pH concentration, low medium and high temperatures, and low medium and high enzyme concentrations. With the data that was collected, showed that enzymes reacts the fastest in warm temperatures, with a neutral pH and a high amount of enzymes and substrates. The reason why these enzymes react fastest in warm temperatures with a neutral pH is due to enzymes being proteins, and just like any protein they will denature if they are in a condition that is too extreme. In this lab it was shown that the catalytic reactions can increase as the amount of enzymes do.

II.

Introduction In this lab, when it came to testing there were three variables being tested against the catalytic rate of enzymes. These included pH, temperature, and enzyme concentration. Regarding enzymes, our lives revolve around them and life would be nonexistent without them. They range from helping digest food to helping diagnose a variety of diseases. Enzymes are used in medicine to expose certain elevations in enzymes that can be linked to diseases. In this case, enzymes called “marker enzymes” also help in diagnosing the diseases. An example would be the enzyme alkaline phosphatase is used for diagnosing conditions such as bone cancer. ALP will catalyze the hydrolysis of a

phosphate group that is attached to proteins. In this lab, the substrate pNPP was used (colorless). The solution that will make the substrate turn yellow is hydrolysis. In this case yellow indicates the rate of reaction. Enzymes reactions are controlled by things such as the properties of that specific enzyme, the kinetics of that enzyme, and the type of enzyme. The relation between enzymes and pH, in regards of enzymes being sensitive when it comes to pH, whether it’s be too high or too low. This is why the graph of pH versus enzyme activity is shaped as a negative parabola. The optimum pH for enzymes in the human body is approximately 7, and some enzymes pH can be high or low, such as stomach enzymes. Such as pH, enzymes have an optimal temperature where they work best. If the temperature ends up being too high or too low, the proteins in the enzymes will denature. The catalytic reaction of an enzyme will also depend on the enzyme concentration and also the substrate concentration. There is a relationship between the substrate or enzyme concentration with the rate of the reaction. If the substrate or enzyme concentration were to increase, so will the rate of the reaction. In this specific lab, the substrate concentration remained the same but the concentration of the enzyme varied. The first alternative hypothesis was; if the temperature increases then so will the rate of the reaction. The null hypothesis; if the temperature increases the rate of the reaction will stay the same. The second alternative hypothesis; if the concentration of the enzyme increases then the catalytic reaction will speed up. The hull hypothesis; if the concentration of enzymes will increase then the rate of the reaction. The third alternative hypothesis; as the pH gets closer to neutral then the enzyme activity will increase. The null hypothesis; as the pH gets closer to neutral then the enzyme activity will stay the same.

III.

Materials and methods Part I: pH vs. Enzyme Activity

In this part of the lab, the optimum pH of the enzyme was tested using ALP. First, 6.5mL of Solution B and 6.5mL of dH20 were added to Solution E. Four cuvettes were labeled and the spectrophotometer was set at 405 nm. In the first cuvette (control), 3mL of solution E were added, and 2mL OF dH20 to end with a volume of 5mL. In the second acidic cuevette, 3mL of solution E were added and 1.9mL of 0.2 M HCl, and 100 uL of high concentration enzyme for a volume of 5mL. in the third cuevette, 3mL of solution E were added and 1.9mL of dH20, and 100 uL of high concentration enzyme for a volume of 5 mL. In the fourth basic cuevette, 3mL of solution E were added, and 1.9mL of 0.1 Na2CO3 and 100 uL of high concentration enzyme for a volume of 5mL. Cuvette one (control) was used to zero out the spectrophotometer. After this, Solution D was added to cuvette two and it was then placed in the spectrophotometer using the parafil invert mix. Cuvettes two absorbance readings were recorded at time zero, and then every 30 seconds for 5 minutes in total. After, Solution D was added to cuvette three, and then it was placed in the spectrophotometer using the parafil invert mix. Cuvettes three absorbance readings were recorded at time zero, and then every 30 seconds for 5 minutes in total. Lastly, Solution D was added to cuvette four and it was then placed in the spectrophotometer using the parafilm invert mix. Cuvettes four absorbance readings were recorded at time zero, and then every 30 seconds for 5 minutes in total. (Wilson et al., 2018)

Part II: Enzyme Concentration vs. Catalytic Rate In this second part, the effect of enzyme concentration on catalytic rate was tested. 15mL of solution A and 15 mL of solution B were mixed together in a 50mL beaker to make Solution F. Eight cuvettes were labeled as cuvette 1a, cuvette 2a, cuvette 3a, cuvette 4a, and cuvette 1b, cuvette 2b, cuvette 3b, and cuvette 4b. Cuvette 1 was the blank one used in order to zero out the spectrophotometer, which was set to 405 nm. 3mL of Solution F were placed into each cuvette using a micropipette. After, using a micropipette tip, 100 uL of solution C were added to cuvette 1a, it was quickly covered with parafilm, mixed, and then placed into the spectrophotometer. Absorbance level was recorded at time zero, and then every 30 seconds for 5 minutes in total. Using a micropipette, 400uL were added to solution C in cuvette 2a. Cuvette 2a was then quickly covered with parafilm, mixed, and then placed into the spectrophotometer. Absorbance level was recorded at time zero, and then every 30 seconds for 5 minutes in total. Using a micropipette, 500 μL of solution D was added to cuvette 4a. The parafilm was then quickly used to cover the cuvette. It was then placed into the spectrophotometer. The absorbance reading for 4a at time zero was recorded. The following absorbance readings were recorded every 30 seconds for 4a for 5 minutes. (Wilson et al., 2018)

Part III: temperature vs. enzyme activity

In part three, the effect of temperature on enzyme activity was tested. 1b was placed in the refrigerator (4 °C), 2b was placed out in room

temperature, 3b was placed in a 32 °C water bath, and 4b was placed in a 60 °C water bath. Cuvette 1b was taken from the refrigerator. Using the micropipette, 100 μL of solution C was added cuvette 1b. The cuvette was then quickly covered with parafilm and inverted. Next, the cuvette was cleaned, and then placed into the spectrometer. The absorbance level was then recorded at time zero. The absorbance was also recorded every thirty seconds for five minutes in total. Using a micropipette, 100 μL of solution C was added to cuvette 2b. Cuvette 2b was the quickly covered with parafilm and inverted. Cuvette 2b was then placed into the spectrophotometer, and time zero absorbance was recorded. Cuvette 2b’s absorbance was recorded every thirty seconds for five minutes in total. Cuvette 3b was then retrieved from the 32 °C water bath. The sides of the cuvette were cleaned. Using the micropipette, 100 μL of solution C were added to cuvette 3b. Cuvette 3b was then quickly covered with parafilm, inverted, and placed into the spectrophotometer. It was recorded at time zero, and every thirty seconds following for five minutes in total. Lastly, Cuvette 4b was obtained from the 60 °C water bath and cleaned. Using a micropipette, 100 μL of solution C was added to cuvette 4b. Cuvette 4b was then covered with parafilm, inverted, and placed into the spectrophotometer. Cuvette 4b’s absorbance readings were recorded at time zero, and every 30 seconds for five minutes in total. (Wilson et al., 2018)

IV.

Results

Figure 1: Enzyme Absorbance Levels at Various pH over time 0.350 0.300

Absorabance (A)

f(x) = 0 x + 0.23 0.250

Acidic Linear (Acidic) Neutral Linear (Neutral) Basic Linear (Basic)

0.200 f(x) = 0 x + 0.11 0.150 0.100 f(x) = 0 x + 0.05

0.050 0.000

0

50

100

150

200

250

300

350

Time (Seconds)

In figure 1, one can observe the absorbance level at various pH over time, in this case the neutral pH is higher, followed by the basic pH, and then the acidic pH. For all of them, throughout time their absorbance level increased.

Figure 2: Enzyme Activity at Varying pH 0

Enzyme Activity (A/sec)

0

f(x) = 0 x + 0

0 0 0 0 0 0 Acidic

Neutral

Basic

pH Enzyme activity Linear (Enzyme activity)

Linear (Enzyme activity)

In figure 2, the enzyme activity at varying pH is shown, in this graph we can see how the enzyme activity had a peak at neutral pH and then followed to be the same.

Figure 3: Absorbance Levels of Varying Concentrations of Enzymes over Time 1.800 1.600 f(x) = 0 x + 0.59 1.400

Low Linear (Low) Med Linear (Med) Med-High Linear (Med-High) High Linear (High)

Absorbance Level (A)

1.200 1.000 0.800 f(x) = 0 x + 0.28 0.600 f(x) = 0 x + 0.39 0.400 0.200 0.000 0

f(x) = 0 x + 0.1 50

100

150

200

250

300

350

Time (Seconds)

in Figure 3, the absorbance levels of varying concentrations of enzymes over time was shown. As the hypothesis stated, the higher the temperature, the higher the absorbance over time, and we can observe that in the scatter plot.

Figure 4: Enzyme Activity at Varying Concentrations 0

Enzyme Activity (A/sec)

0 0

f(x) = 0 x − 0

0 0 0 0 0 0 Low

Med

Med-High

High

Concentration Enzyme activity

Linear (Enzyme activity)

In Figure 4, the Enzyme Activity at varying concentrations was shown. When it came to the medium and medium-high temperatures there were slight changed in the enzyme activity. Also with the low concentration, it was higher than expected as well as the high concentration.

Figure 5: Absorbance Levels of Varying Temperatures of Enzymes over Time 0.45 0.4

f(x) = 0 x + 0.29

0.35 4°C Linear (4°C) 20°C Linear (20°C) 32°C Linear (32°C) 60°C Linear (60°C)

Absorbance (A)

0.3 0.25 f(x) = 0 x + 0.19 f(x) = 0 x + 0.2

0.2

f(x) = 0 x + 0.12

0.15 0.1 0.05 0

0

50

100

150

200

250

300

350

Time (Seconds)

In Figure 5, The absorbance levels of varying temperatures of enzymes over time were shown. In this case the 32 C was the one with the highest absorbance level. And the 4 C was the one with the smallest absorbance level.

Figure 6: Enzyme Activity at Varying Temperatures 0

Enzyme Activity (A/Sec)

0 0 0 0

f(x) = − 0 x + 0

0 0 0 0 0 4°C

20°C

32°C

60°C

Temperature (Deg C) Enzyme activity

Linear (Enzyme activity)

In Figure 6, the activity at varying temperature was shown. As the linear line was expected to be, the actual enzyme activity was very different and had a peak at 0.0004.

V.

Discussion

In this lab, the pH vs. enzyme activity part, the alternative hypothesis was supported. As the pH approached neutral, the enzyme reaction increased. In correlation, as the pH approached basic or more acidic pH values, the rate of the reaction decreased. In the academic journal "Ligand-Induced Variations in Structural and Dynamical Properties Within an Enzyme Superfamily" it states “Enzyme catalysis accelerates reaction rates of chemical reactions up to 20 orders of magnitude relative to uncatalyzed reactions (Wolfenden, 2006)”. In this case this relates back to enzymes being affected with high and low temperatures. These high temperatures can cause the protein to lose its shape, called denaturation. In the temperature vs. enzyme activity part of the experiment, the hypothesis was also supported by the data collected. As the temperature increased so did the rate of the reaction. The four temperatures we tested we 4, 20, 32, and 60 degrees C. The end absorbance for 4 °C was .1549 nm, the absorbance for 20 °C was .2318 nm, the absorbance for 32 °C was .3469 nm, and the absorbance for 60 °C was .2104 nm. The last hypothesis tested was enzyme concentration vs. catalytic rate. The alternative hypothesis was also supported throughout this experiment. As the enzyme concentration increased so did the rate of our reaction. The levels of enzyme concentration that were tested were low, medium, medium-high, and high. As a result, we can conclude that enzymes work best at certain environments, in this case warm environments with a neutral pH and a high concentration of substrates and enzymes. There are some exceptions were it comes to enzymes such as acidic conditions in regards to stomach enzymes.

Works Cited

Chitra Narayanan, et al. “Ligand-Induced Variations in Structural and Dynamical Properties Within an Enzyme Superfamily.” Frontiers in Molecular Biosciences, Vol 5 (2018), 2018. EBSCOhost, doi:10.3389/fmolb.2018.00054/full....