Prof. Dr. D. P. Wejesinghe

Daiva Ahire

SCB 201

Prof. Dr. D. P. Wejesinghe

10/13/2009

Week IV

Title: Enzymes

Objectives: using spectrophometer predict the effect that the enzyme and substrate concentrations will have on the rate of an enzyme-catalyzed reaction. Determine the effects of ph and temperature will have on the rate of an enzyme-catalyzed reaction.

Introduction: Enzymes are made from proteins. It is a biological catalyst that speeds up the rate of chemical reaction by lowering the activation energy of the reaction. Enzymes are specific and catalyze only specific reaction. Specificity is a result of active site of enzyme that acts on the substrate. The enzyme activity is influenced by ph and temperature. Change in ph will have impact on hydrogen bonding that can change the shape of protein. Increase in the temperature will increase the reactions rate; however, if the temperature is too high the enzymes will denatures. For this experiment we use spectrophometer which measures the absorbance of the solution for particular wavelength.

Experiment I- prediction of the effect of the enzyme concentration to the rate of reaction. Hypothesis: Higher concentration of the enzyme will increase the rate of the reaction; therefore the absorbance also will increase.

Procedure: first we calibrate the spectrophometer with blank solution. We measured 1ml of Catecholase in the test tube using 1 ml graduate pipette and added 5 ml of distilled water. Covered the test tube with Parafilm and mixed the content. On the spectrophometer we set the wavelength to 540 nm, because this wavelength is best absorbed by the solution. Using the left knob on the machine we set absorbance reading to infinity. We inserted the test tube into sample holder. Using the knob on the right we set the absorbance reading to 0. In the mean time we prepared three test tubes in each added 2 ml of substrate Catechol with 3 ml, 3.5 ml, and 4 ml of water respectively. The last is added enzyme, because the reaction will take place as soon as we add the enzyme. The amount of the substrate Catechol is constant. We are changing the concentration of the enzyme Catecholase ( 1ml, 0.5ml, 0 ml) by diluting it with water. Therefore, the concentration of enzyme is independent variable. The dependent variable is absorbance, which will be changing according to concentration. We mixed the contents of the test tubes. After 3 min. we used blank to rezero the spectrophometeer, inserted the first test tube and noted the absorbance. Same procedure we conducted for the rest of the test tubes.

Results:

Tube / Water (ml) / Catechol (ml) -substrate / Catecholase (ml)- enzyme / Absorbance and color intensity
1 / 3 / 2 / 1 / 0.9 – intensive brown color.
2 / 3.5 / 2 / 0.5 / 0.6 – brownish color
3 / 4 / 2 / 0 / 0 – off white color

Conclusions: The results of the experiment showed that lowering the concentration of enzyme and adding more water, the absorbance of the solution decreases. Therefore, our experiment supports our hypothesis that higher concentration of enzyme will increase the rate of reaction. Higher concentration of enzyme - higher absorbance and color intensity. Graphic relationship:

Experiment II – effect of pH to the enzyme activity.

Hypothesis: different concentration of hydrogen ions, therefore different ph, will have some effect on the enzyme activity and the reaction rate. The normal ph for living organisms cells is neutral (about 7), therefore we expect that the absorbance and the highest rate of the reaction should occur at the neutral ph.

Procedure: We prepared three test tubes by adding constant amount of 2 ml of substrate Catechol, 3ml of different ph solution (pH 4, pH 7 and pH 9) and 1ml of enzyme Catecholase. Independent variable in this experiment is ph value and dependent variable is absorbamce.We mixed the contents of the test tubes. After 3 min. we used blank to rezero the spectrophometeer, inserted the first test tube and noted the absorbance. Same procedure we conducted for the rest of the test tubes.

Results:

Test tube / Ph solution and amount taken (ml) / Catechol (ml) -substrate / Catecholase (ml)- enzyme / Absorbance and color intensity
1 / pH 4 - 3 / 2 / 1 / 0.4
2 / pH 7 - 3 / 2 / 1 / 1.3 –intensive brown color
3 / pH 9 - 3 / 2 / 1 / 0.3

Conclusions: The results of the experiment showed that the optimal enzyme activity occurs in ph 7 solution. In supports our prediction that most enzyme activity occur in the neutral solution. The alkaline solution with pH 4 showed higher absorbance compared with acidic solution with pH 9. Graphic illustration of the relationship ph vs. absorbance:

Experiment III – effect of the temperature to the enzyme activity.

Hypothesis: Since the increase of the temperature will increase the number of collisions between enzyme and substrate we expect the increase in the rate of reaction. However, if the temperature will be too high, we expect that enzyme will change its structure, perhaps even be denatured and enzyme will be not able to catalyze the reaction.

Procedure: We took first test tube and determined the absorbance at about 0 C (ice). We added 3 ml of water and 2 ml of substrate and kept in the ice bucket. As the solution in the test tube cool down we added 1 ml of enzyme. Mixed the content of the solution, left the test tube for the rest 3 min. in the ice bucket and then checked the absorbance. The second test tube we prepared in room temperature. We added 3ml of water, 2 ml of substrate and 1 ml of enzyme, all substances were at room temperature. Mixed the content of the solution, left the test tube for 3 min. at room temperature and then checked the absorbance. The third test tube we prepared at high temperature. We took 3 ml of water and 2 ml of substrate and placed in the hot water bath. After the temperature of solution increased we added 1ml of enzyme solution, which also was kept at the high temperature. We mixed the content of the solution, left the test tube for 3 min. in the water bath and then checked the absorbance. In this experiment temperature is independent variable and absorbance is a dependent variable.

Results:

Test tube / condition / Water (ml) / Catechol (ml) -substrate / Catecholase (ml)- enzyme / Absorbance and color intensity
1 / Ice (0 C) / 3 / 2 / 1 / 1.0
2 / Room temperature (24 C) / 3 / 2 / 1 / 0.9
3 / Hot water bath (40 C) / 3 / 2 / 1 / 0.6

Conclusions: The results of the experiment showed that ice and room temperature substances had the highest absorbance. Test tube kept in the ice bucket showed the highest absorbance. Therefore, our experiment result contradicts our hypothesis. At very high and very low temperatures we expected the absorbance or enzyme activity be low. Te highest absorbance should have appeared at room temperature, because most human enzyme activity occurs at room temperature. I believe that mistake occur in preparing the test tube in ice conditions. The absorbance result is very close to room temperature test tube, which indicates, that we probably didn’t cool the solution very well. Therefore, after adding enzymes the reaction proceeded as if it is in room temperature. The test tube prepared in the hot water bath also showed some of the enzyme activity. I suggest that next time conducting this experiment it is useful to use thermometer in order to check the accuracy of the temperatures. Graphic representation of the relationship between temperature and absorbance of this experiment:

Homework:

1. A freshly cut potato turns brown when left standing. Why do mashed potatoes stay white?

Mashed potatoes are cooked. The heat from cooking them denatures the enzyme Catecholase which causes raw potatoes to turn brown through oxidation when they are exposed to air.

2. The three graphs in Figure 11 represent three different enzymes that are found in a unicellular pond organism. Assuming that these enzymes must function in order for the organism to grow, what water pH is best for this organism? Explain your answer.

Water pH 5 is the best for this organism to survive. In all three graphs the highest activity of the enzyme is at the pH 5 - the highest point of the curve.

3. Lemon juice, which has a pH of about 3, can be sprinkled on freshly cut fruit to keep it from turning brown. Propose a hypothesis to explain this observation.

The lemon juice with pH3 will denatures the polyphenol oxidaze enzyme presented in most of the fruits. Therefore the fruit will not turn brow.

How could you test your hypothesis?

We can cut one fruit and leave it open, which will expose enzyme to oxygen and the fruit will turn brown fast. On the other fruit we can sprinkle the lemon juice and observe the changes.

Propose an alternative hypothesis. (Hint: Lemon juice is a complex substance.)

4. You hypothesize that reaction rate is proportional to enzyme concentration and design an experiment to test your hypothesis. Fill in the blanks below to complete the design.

Test tube / Water / Substrate / Enzyme
Tube A / 20 drops / 10 drops / 0 drops
Tube B / 15 drops / 10 drops / 5 drops
10 drops / 10 drops / 10 drops
Tube D / 0 drops / 10 drops / 20 drops

Explain your answers:

Water: Water is used to dilute the concentration of the enzyme; therefore we minimize the amount of water to 0 in the test tube D, where the concentration of enzyme is the highest.

Substrate: Substrate concentration is constant.

Enzyme: Enzyme concentration we decreased from 20 drops to 0 drops, because it is our independent variable and we measured how it’s concentration will affect the rate of the reaction.

5. A student team is studying the effect of substrate concentration on the rate of an enzymatic reaction. They supply each reaction mixture with the same amount of enzyme. Predict what the results will be and explain why.

The rate of reaction will be the same, because the students didn’t change the enzyme concentration. The enzyme concentration is directly proportional to reaction rate. If there is no increase in concentration, the reaction rate will remain the same.