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Enzyme lab report from bio 101...

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Angie Awad Tina Harrison Section W5

The Effect of Temperature and pH on Enzyme Activity and Product Generation (alkaline phosphatase) Abstract: The enzyme alkaline phosphatase was used on the substrate pNPP to assay for effects of pH and temperature on rxn rate. We wanted to determine the amount of product formed for pH values that ranged from 4-12 and temperatures that ranged from 0° C- 100° C. It was predicted that as the pH increased, the amount of product formed decreased; also as the temperature

increased the amount of product will increase as well, until you reach the boiling temperature. The enzyme showed to have an optimum pH value of 4 and an optimum temperature value of 100° C. This tells us that under these conditions there will be the highest amount of product formed, which is pNP and Pi. From the data we can see that increasing the pH of the buffer

decreases the amount of product formed. We can see that if the temperature is too high or too low the amount of product formed will decrease. At unfavorable pH and temperature the enzyme alkaline phosphatase becomes denatured, which is the reason that less product formed.

Introduction: Chemical reactions are regulated through the use of enzymes, which are biological catalysts that increase the speed of a chemical reaction without being consumed by the reaction (Solomon et al., 2008). Enzymes have an optimal temperature where the rate of reaction is the fastest (Solomon et al., 2008). Denaturation is the process in which physical properties are altered by effects of temperature and pH. High temperatures can denature the enzyme and low

temperatures slow down the reaction. We hypothesize that a low temperature there will be a

small amount of product formed and if temperature is too high the enzyme will be denatured and no product will be formed. Along with having an optimal temperature, enzymes also have an optimal pH, where the rate of the reaction is fastest. Most enzymes are active only over a narrow pH range (Solomon et al., 2008). We predict that an acidic pH will be the optimum pH so the most amount of product will be formed, and at a basic pH there will be less products formed. For the assay for temperature in our lab, we are testing how the effect of the pH of our buffer affects the enzyme’s activity. The enzyme being used is a type of phosphatase, which is a group of enzymes that catalyze the removal of phosphate groups from other molecules. Specifically, we are using alkaline phosphatase (purifies from pig intestines), which is hypothesized to be a phosphatase that is able to convert the substrate, pNPP to pNP and inorganic phosphate (Davis et al,. 2011). The amount of product formed after ten minutes will give us this information. Our temperature assay helped us determine the effect of temperature on enzyme activity by looking at the amount of product formed. I predicted that the lower the pH the greater amount of product formed. The pH and amount of product formed are indirectly proportional. Also, if you increase the temperature the greater the amount of product will be formed, but the boiling temperature will denature the enzyme. Without regard to the 100° C, temperature and amount of product formed are directionally proportional.

Materials and Methods The importance of assay number one was to determine the effect of pH on the enzyme alkaline phosphatase, Sus scrofa, which is purified from pig intestines (Davis et al,. 2011). We did this by measuring the amount of product formed from the reaction after 10 minutes. In each of our test tubes we put water and substrate and a buffer of a different pH level in each tube, except for the blank. We added alkaline phosphatase and mixed to begin the reaction and we took our measurements when color developed in at least one tube, which took our group ten minutes.

To determine the amount of product formed we then put each tube in the Spec 20, which we set to a wavelength of 415 nm, and recorded the absorbance (Davis et al,. 2011). Our second assay was for determining how temperature affected the enzyme alkaline phosphatase. We began by adding buffer and the substrate pNPP into each tube, except for the blank, and then placed the tubes into ice, room temperature, water bath, and boiling temperature environments. We allowed the tubes to stay in these environments for five minutes, and then removed them and added alkaline phosphatase in four of the tubes, and in the other four we added water. After doing so we placed the tubes back into their appropriate temperatures and allowed them to sit until color had formed in tube three with the enzyme (3+), which took our group three minutes. The tubes without the enzyme were used as our control so that we have a standard of measurement. We then placed the tubes in the Spec 20 and recorded the absorbance value, which was the amount of product formed. To calculate the rate for 10 minutes in each experiment we used the equation, ΔA / 10 min = (A10 – A0) / (t10 – t0); where Δ is the change in absorbance, A is absorbance and t is time. For

both assays we had a control which was our blank, it was a test tube with distilled water. Results The highest rate of product formation after 10 minutes occurred in tube 1, which had a buffer pH of 4, that we see in table 1. As the pH became more basic, the amount of product formed decreased. This allows us to conclude that optimum pH of the enzyme alkaline phosphatase is 4, which is acidic. Figure one shows that as the pH of the buffer increases, the amount of product formed after 10 minutes decreases. Table 1: Effect of pH Tube Number 1 2 3 4

pH of Buffer 4 6 7 8

Product Formed: After 10 minutes 0.82 0.604 0.466 0.247

Rate A/10 minutes 0.82 0.604 0.466 0.247

5 6

10 12

0.057 0.029

0.057 0.029

Figure 1

Product Formed Afer 10 Minutes (A/10 Minutes)

Effect of pH on Product Formation 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 pH

b tu

e1

4

b tu

pH e2

6

pH

be tu

3

7

b tu

pH e4

8

b tu

pH e5

10

b tu

pH

12

e6

Tube Number and pH Value

Legend: This graph shows the relation between pH and the amount of product formed after 10 minutes of a reaction. Table two shows that the highest rate of product formation after 3 minutes occurred in tube 4-, which did not have enzymes. The temperature of tube 4- was 100 degrees C, which was boiling. The highest rate of product formation for the tubes with enzyme occurred in tube 4+. The temperature of tube 4+ was 100 degrees C, which was boiling. Figure two shows that Table 2: Effect of Temperature Temperature

Tube Number

0° C 25 °C (room temp) 37° C (water bath) 100° C (boiling)

1+ 2+

Product Formed with Enzyme 0.112 0.066

3+ 4+

12-

Product Formed without Enzyme 0.012 0.017

0.152

3-

0.009

0.248

4-

0.283

Tube Number

Effect of Temperature on Product Formation 0.2 0.15 0.1 With Enzyme Without Enzyme

0.05 10 0 e4 Tu b

e3

37

25 Tu b

e2 Tu b

e1

0

0

Tu b

Product Formed Afer 3 Minutes (A/10 Minutes)

0.3 0.25

Tube Number and Temperature (In °C) .

Legend: This graph shows the relation between temperature and amount of product formed after 3 minutes of a reaction. The blue bars represent the tubes with enzyme, and the red bars represent the tubes without enzyme.

Discussion Ph effects the enzyme by allowing it to catalyze either quickly or slowly depending on whether the pH is more acidic or more basic. The activity of an enzyme changes if the pH changes because there would be an alternation in electric charges on the enzyme. The changes in charge affect the ionic bonds that contribute to tertiary and quaternary structure, which changes the conformation and activity. Many enzymes become inactive and usually irreversibly denatured depending on certain pH values (Solomon et al., 2008). I predicted that the lower the pH, the greater the amount of

product formation. I thought that pH and product formation are indirectly proportional to one another. We found that the optimum pH for the enzyme alkaline phosphatase was 4. Based on the data that my group obtained we can conclude that alkaline phosphatase is an enzyme that works best when the pH of the buffer is 4, making this pH the optimum pH for the enzyme. This means

that a pH of 4 will have the fastest reaction rate. The data agreed with the prediction that as the pH increased, the amount of product formed decreased. I predicted that as temperature increased, the amount of product formed would also increase. We know that enzymes have an optimal temperature. We also know that at low temperatures reactions occur slowly or not at all (Solomon et al., 2008). The rate of enzyme controlled reactions increases as the temperature increases, up until you reach a certain temperature. High temperatures denature most enzymes and they are unable to continue the reaction (Solomon et al., 2008). I predicted that as the temperature increased the amount of product will increase as well, until you reach the boiling temperature. At this temperature I said that the enzyme will be denatured. However, our data does not agree with this hypothesis. In both the tubes with enzymes and without enzymes, the amount of product formed was the greatest in the 100° C, boiling temperature. It also disagreed with the prediction that temperature and the amount of product formed is directly proportional.

Davis, B., Martin, D. and associates. 2011. General Biology 101 A Laboratory Manual.

Division of Life Sciences, Rutgers University. New Brunswick, NJ. Solomon, Eldra P., Berg, Linda R., Martin, Diana. 2008. Biology, 8th ed. Thomson Brooks/Cole....