Depth Study

Bio notes – Depth Study

Enzymes are protein molecules that act as biological catalysts that speed up chemical reactions in living organisms without being used up themselves by lowering activation energy required. They are made of proteins and are specific—each enzyme only works with one type of substrate, like a key fitting into a specific lock. This means that for each and every reaction in a living organism, there is a unique enzyme.

Enzymes are made up of protein molecules that create a shape, the shape’s surface is the active site where substrates temporarily bind to create the substrate enzyme complex. Once the reaction takes place, the substrates are released and the unchanged enzyme is available for the same process again.

There are two main models to explain how enzymes work:

• The lock and key model: the enzyme’s active site has a rigid shape that exactly fits the substrate.

• The induced fit model: the enzyme’s active site slightly changes shape to better fit the substrate when it binds. (accurate because proteins are not rigid).

Enzyme activity is affected by several factors:

• Temperature: Activity increases with temperature up to an optimal point (the organism’s body temperature, 37°C in humans), but too much heat causes the enzyme to denature (bend and flex, active site can no longer accommodate substrate), irreversible.

• pH: Each enzyme has an optimal pH and a narrow range of efficiency. Too acidic or too alkaline conditions can denature an enzyme.

• Substrate concentration: As substrate concentration increases, so does enzyme activity until the saturation point. Once all enzymes are all being used: working at their maximum turnover rate (saturation), adding more substrate doesn’t increase the reaction rate. Concentration would have to increase.

Enzymes are essential for life because they allow all metabolic reactions in cells to happen quickly and efficiently. Without them, the reactions would be so slow they would barely take place.

Catalase is an enzyme found in many cells of the body, especially in the liver, kidneys, and red blood cells. Its job is to protect the body by breaking down hydrogen peroxide (the substrate), which is a harmful waste product made during normal cell activity. Catalase speeds up the reaction that turns hydrogen peroxide into harmless products: water and oxygen. This reaction is important because too much hydrogen peroxide can damage cells. Catalase works best in slightly alkaline conditions (around pH 7–8), which matches the natural environment of the liver where it’s most active.

Reliability

The repeatability or reproducibility of measurements. Reliable measures are similar when repeated over multiple experiments (x5) (under similar conditions). Refers to the consistency of measurements within an experiment and the consistency of measurements over multiple, independent experiments.

Validity

Whether all procedures accurately measure what was intended to be measured, all variables are identified, only the independent variable is changed. All other variables are controlled, there is a control.

Accuracy

Considering evident sources of error and the limitations of instruments used.

What is the purpose of:

Substrate concentration on enzyme activity

Hydrogen peroxide is a toxic waste product of chemical reactions in our cells. Our cells produce catalase to decompose hydrogen peroxide into water and oxygen.

Aim

To observe how substrate (hydrogen peroxide) concentration affects the relative activity of the enzyme catalase.

Hypothesis

The relative activity of the enzyme catalase increases as substrate concentration increases until all the active sites of the enzyme are occupied by the substrate.

Materials

Test tube rack, 8 large test tubes, stopwatch, marker pen, ruler, 2x10mL syringes, 2x5mL syringes, 1 glass dropper for the detergent, 2 plastic pipettes for the liver homogenate distilled water, dishwashing detergent, 5% hydrogen peroxide, liver homogenate (50g sheep liver, made up to 200ML then homogenized using a Barmix).

Safety

Equipment should be safely left, hydrogen peroxide is hazardous, contact on skin should be washed immediately, goggles should be worn. Apron for chemical spills, enclosed shoes to protect against chemical splashes and grip.

Variables

Dilution

Method

1.        Label 8 test tubes (1E, 1C, 2E, 2C …)

2.        Place hydrogen peroxide/water solutions into each of the beakers.

3.        Add detergent to each tube.

4.        Add five drops of distilled water to each control tube while starting a stopwatch, giving the tube a gentle swirl. Record height of bubbles from meniscus at 1 min.

5.        Add five drops of liver to the experiment tubes, swirl, start stopwatch. Record height of bubbles after 1 min.

6.        Repeat for every tube.

Results

Conclusion

As the concentration increased, the activity of the enzyme increased.

Discussion

No bubbles formed in the control tubes, having a control confirmed that any reaction is only a result of the liver.

The bubbles provide a measure of enzyme activity because oxygen is the product and it reacts with the dishwashing detergent to make bubbles.

If the substrate concentration continued to increase, the activity would eventually reach an equilibrium.

Reliable: there were inconsistent results but a similar trend.

Validity: there were at least three controls, there was only one dependent (height of bubbles) and independent variable (concentration)

Accuracy: Timer was used, but ruler was improper, and droppers/pipettes were inaccurate.

This experiment is best undertaken by a team to do all the roles.

The effect of pH on enzyme activity

Hydrogen peroxide is a toxic waste product of chemical reactions in our cells. Our cells produce catalase to decompose hydrogen peroxide into water and oxygen.

Aim

To observe how pH affects the relative activity of the enzyme catalase.

Hypothesis

The relative activity of the enzyme works best at an optimum pH. A change in pH (lower or higher) will decrease activity because it will change the shape of the active site, affecting the enzyme’s ability to bind with a substrate.

Materials

Test tube rack, 8 large test tubes, stopwatch, marker pen, ruler, 4x5mL syringes, 1 glass dropper for the detergent, 2 plastic pipettes for the liver homogenate distilled water, dishwashing detergent, 5% hydrogen peroxide, liver homogenate (50g sheep liver, made up to 200ML then homogenized using a Barmix), pH buffers at 2,4,7.

Safety

Equipment should be safely left, hydrogen peroxide is hazardous, contact on skin should be washed immediately, goggles should be worn. Apron for chemical spills, enclosed shoes to protect against chemical splashes and grip.

Variables

Method

1.        Label 6 test tubes (2E, 2C, 4E, 4C, 7E, 7C).

2.        Add 5ml of 5% H₂O₂ to each tube.

3.        Add 5ml of pH2 buffer to the ‘2’ tubes, add pH4 to the ‘4’ and pH 7 to the ‘7’.

4.        Add detergent to each tube.

5.        Add five drops of distilled water to each control tube while starting a stopwatch, giving the tube a gentle swirl. Record height of bubbles from meniscus at 1 min.

6.        Add five drops of liver to the experiment tubes, swirl, start stopwatch. Record height of bubbles after 1 min.

7.        Repeat for every tube.

Results

Conclusion

The higher the pH, the higher the bubbles, optimum pH of which you have maximum activity of the enzyme catalyse.

Discussion

The pH at which the enzyme was most active was 7 – it had the highest bubbles.

the activity of the catalase is determined by the height of the bubbles due to oxygen from the breakdown of hydrogen peroxide.

To avoid cross contamination, different syringes pipettes, and beakers were used.

If the pH increased more, the activity would decrease, and the enzyme would become denatured.

Accuracy:

Reliability:

Validity: it tests the hypothesis, it has one independent variable (pH), controlled variables and there was a control.

The effect of substrate temperature on enzyme activity

Aim

To observe how substrate (hydrogen peroxide) temperature affects the relative activity of the enzyme catalase.  

Hypothesis

There will be an optimum temperature at 37°C at which the activity of the enzyme is the highest.  

Materials

Test tube rack, 9 large test tubes, Stopwatch, Marker pen, Ruler, 2x 10mL syringes, Thermometer, 1 glass dropper (for the detergent), 2 plastic pipettes (for the liver homogenate and distilled water), Dishwashing detergent (Morning Fresh Lemon), 5% hydrogen peroxide, Liver homogenate, Test tube tongs.

Safety

Equipment should be safely left, hydrogen peroxide is hazardous, contact on skin should be washed immediately, goggles should be worn. Apron for chemical spills, enclosed shoes to protect against chemical splashes and grip.

Variables

Method

1.        Label 9 tubes, 1C, 1E, 1E2, 2C, 2E, 2E2, 3C, 3E, 3E2

2.        Add 5mL of hydrogen peroxide to each tube.

3.        Add two drops of detergent to each beaker using the glass dropper.

4.        Add all tubes marked with ‘1’ into a beaker and place that beaker into an ice bath.

5.        Keep all tubes marked with ‘2’ into the test tube rack.

6.        Add all tubes marked with ‘3’ into a beaker and place that beaker into a boiling water bath.

7.        Add 5 drops of distilled water using the plastic pipette the tube marked ‘1C’ while simultaneously starting the stopwatch. Give the tube a gentle stir and record the height of bubbles, if any, from the meniscus after one minute. 

8.        Add 5 drops of liver homogenate to the tube marked ‘1E’ while simultaneously starting the stopwatch, give the tube a gentle stir. After one minute, use the test tube tongs to take the tube out of the bath and record the height of bubbles, if any, from the meniscus. 

9.        Repeat sequentially for all other tubes, ensuring distilled water is placed the tubes labelled ‘C’ and the liver homogenate is placed in tubes marked ‘E’. 

Results

Discussion

The enzyme activity was most optimal at 37, and the highest temperature of 85 being the least optimal.

the activity of the catalase is determined by the height of the bubbles due to oxygen from the breakdown of hydrogen peroxide.

The hypothesis was supported that the optimal temperature was at 37 degrees celsius, the body’s natural temperature.

Accuracy: Precise measurements were used with measuring equipment like the beakers and syringes, however a syringe should have been used instead of a pipette to ensure exact and consistent quantities of liver.

Reliability: The experiment was repeated and the results were consistent across both trials.

Validity: There was only one independent variable, the temperature, only the dependent variable, the height of the bubbles and there were consistent controlled variables of the amount of detergent, the time measured and the amount of liver across all experiments. There was also a control that determined that it was the liver causing the reaction.

To improve the experiment, pipettes should be replaced by syringes and the two temperatures chosen should have had an equal distribution from 37 degrees to best represent the bell curve shape. This would better represent the gradual increase and decrease in enzyme activity, creating a more symmetrical bell curve.

Conclusion

The aim of this experiment was to observe how substrate temperature affects the relative activity of the enzyme catalase. Three different temperatures (10, 37, 85) were measured and it was concluded that the optimal temperature was 37 degrees Celsius, supporting the hypothesis. This experiment was mostly valid, accurate and reliable but further trials and more precise measurements could have enhanced results.