Enzymes Bio Stage 2 sace PDF

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Bio enzymes assignment...

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Stage 2 Biology

TEMPERATURE ON ENZYMATIC ACTIVITY

Brooke McGann Urrbrae Agricultural High School

BROOKE MCGANN

STAGE 2 BIOLOGY

INTRODUCTION Enzymes refers to the class of proteins inhabiting living organisms which act as biological catalysts defined by their ability to increase the rate at which biochemical reactions occur. As a protein, enzymes are comprised of amino acids linked by many linear peptide chains, whose arrangement, encoded by the DNA sequence of the corresponding gene, governs its function. Enzymes are integral to living organisms as they effectively catalyse biological processes by accelerating the process by which chemical reactions occur within a metabolic pathway or an isolated component of a living cell (BBC, 2021). These processes are intrinsic to organism vitality which, without enzymatic facilitation, would occur at a rate slower than what is necessary in maintaining life. In their conversion of substrates to products, enzymes use lock and key models to lower the activation energy required for reactions by binding to reactant molecules securely and exactingly at the enzyme’s active site, structured to accommodate the substrate, lowering the energy expended by overactive molecules (Biology Online, 2020).

Figure 1: Enzyme lock & key model (Bio Ninja, 2017)

There are many factors which influence the dynamism of enzymatic activity, which, in disadvantageous instances, can act as a detriment to their functioning despite their significance. These factors include, but are not limited to, ionic conditions, pH, perinuclear space and the temperature of the compartmentalised area. Regarding enzymes, temperature plays the most impactful role. At low temperatures, enzymes will completely cease functioning as there is extremely limited molecular movement, preventing the collision of reacting elements. As temperature increases these molecular collisions will occur more frequently as the heat energy is converted into kinetic energy, however, enzymes do not work at very high temperatures as the molecules are exposed to an excess of kinetic energy, breaking down molecular bonds, changing the original characteristics of the enzyme. Such changes result in an altered structure which is no longer complementary to the chemical substrate. This is denaturing. Optimum enzyme temperature occurs at 37ºC, approximately human body temperature, regulated by homeostasis.

Naturally produced within the body, hydrogen peroxide, an oxidizing agent with disinfectant, antiviral and anti-bacterial activities, can be toxic in large quantities. Catalase is an enzyme found in the human liver which accelerates the natural reaction involving the decomposition of hydrogen peroxide, to produce water and oxygen via the resolution of oxygen bonds. Catalase production prevents hydrogen peroxide poisoning within the body (Bell-Young, 2021).

BROOKE MCGANN

STAGE 2 BIOLOGY

In this investigation, the temperature of a hydrogen peroxide solution will be varied significantly to assess its effects on the enzymatic activity of liver derived catalase in a reaction with hydrogen peroxide. With products water and oxygen, the reaction rate can be monitored through the bubble production of a dishwashing soap solution that is combined with the hydrogen peroxide solution.

Aim To investigate how differing temperatures of stock solution (H 2O2) and liver effects the rate of reaction during enzyme activity.

Hypothesis If the temperature of stock solution (H 2O2) and liver is increased (higher) to an optimum level, then a higher rate of reaction will occur until a reduce in temperature occurs, therefore more enzyme activity will occur at an optimum level.

Materials -

Stock solution pH5 of H2O2

-

1 250mL measuring cylinder

-

14 boiling tubes

-

Detergent

-

Stopwatch

-

Teat pipette

-

Fresh Liver

-

Distilled water

-

Thermometer

-

Water baths set for 37°C, 45°C, 60°C and

-

Forceps

-

1 10mL measuring cylinder

80°C -

Chilled/Ice/Water/Salt slurry bath

Safety risk

Danger

Precautions

Scalpel

Cuts or lacerations to the user

Careful cutting techniques on a chopping board

Hydrogen peroxide solution

Corrosive to the eyes, skin and respiratory system

Use of PPE and kept away from oral/nasal ingestion

250 mL and 100 mL measuring cylinders

Exposure to glass shards if shattered

Careful steady movements whilst handling

Test tubes

Exposure to glass shards if shattered

Careful steady movements whilst handling

Water baths

Exposure to water of extreme temperatures

Use of heat proof gloves whilst touching extreme temperatures

Table 1: Hazards and Safety Precautions

BROOKE MCGANN

S TAGE 2 BIOL OGY

Methods 1.

Use the 10mL measuring cylinder and teat pipette to measure 9mL of hydrogen peroxide into the one of the boiling tubes.

2.

Add 2 drops of detergent and swirl to mix

3.

Cut liver into 7 cubes (approximately 1cm3)

4.

Using the forceps, take 1 cube of liver and place it in another boiling tube with 10 ml of distilled water.

5.

Place the two tubes (one with H2O2 and one with liver) into the ice/water/salt slurry bath. Leave them for 10 minutes to reach temperature.

6.

Pour the chilled H2O2 into the 250ml measuring cylinder, then add the chilled liver. Start the timer as soon as the liver enters the H2O2.

7.

Measure the bubble foam that has formed at 30s, 60s, 90s and 120s and fill out record in the table.

8.

Repeat this procedure 1 to 7 for each of the temperatures and record your results. Make sure you rinse your equipment thoroughly between procedures using distilled water.

9.

Clean all equipment and clean up laboratory.

Results 120s vol. -

Temp C°

30s

60s

90s

120s

-3°

130

150

190

210

190

95

17°

250

250

260

260

240

120

25°

100

150

200

210

190

95

39°

250

260

260

260

240

120

47°

260

270

260

260

240

120

62°

250

270

280

270

260

130

80°

20

20

20

20

0

0

20 (mL)

mL/min

Table 2: Overall individual results measurements of bubbles recorded for specific time lengths at varying temperatures

BROOKE MCGANN

S TAGE 2 BIOL OGY

Rate of Reaction of Catalase and Hydrogen Peroxide Solutions When Exposed to Varying Temperatures (mL/min) 140

Time (Seconds)

120 100 80 60 40 20 R² = 0.8927

0 -5

5

15

25

35 45 Temperature C°

Rate of Reaction (mL/min)

55

65

75

85

Trendline

Figure 2: Graphical overall individual results measurements of bubbles recorded for specific time lengths at varying temperatures

Av. Rate of Reaction of Catalase and Hydrogen Peroxide Solutions When Exposed to Varying Temperatures (mL/min) 140

Time (Seconds)

120 100 80 60

40 20 R² = 0.9826

0 -5

5

15

25

35 45 Temperature C°

Rate of Reaction (mL/min)

55

65

75

85

Trendline

Figure 3: Average graphical overall individual results measurements of bubbles recorded for specific time lengths at varying temperatures

BROOKE MCGANN

S TAGE 2 BIOL OGY

Analysis As observable in the scatter reflecting the results both individually achieved and averaged over a series of trials, the increase in temperature directly affects the reaction rate of the catalase and hydrogen peroxide solutions. Initially, as the temperature of the solution increases, a faster reaction rate is seen, as the activation energy is lowered due to the heat energy being converted into kinetic energy, increasing molecular collisions and hence, enzyme and substrate collisions. There is a decrease in reaction rate at 25 degrees in the individual results, however, it is clear the enzyme was still effective in the following results. This suggests a random error may have altered the results. The rate of reaction is optimised at 62 degrees in the individual results, with foam produced at 130mL/min. Proceeding this maximum, there is a steep decrease in the rate of reaction as the solutions are heated to 80 degrees, this temperature producing no foam. This drastic change suggests the enzymes have reached temperatures too high, resulting in a distorted protein structure, hence a distorted activation site, as seen during denaturing. Evidently, the averaged class graph depicts a different result, with the optimal temperature being reached at a much earlier temperature of 25 degrees, room temperature. Despite this difference, these results still reflect the same dramatic decrease in reaction rate at higher temperatures, however, the enzymatic denaturing seems to occur earlier, with enzymes heated to 62 degrees exhibiting a reduced efficiency.

The cubic polynomial trendlines pictured on both graphs fits the scatter well. The correlation between the results and the trend has high values of, approximately, 0.823 and 0.987. These high figures reflect the precision of the results and their predictability, however, as a cubic polynomial, even higher values should be achievable. This suggests that several random errors occurred throughout this experiment. Following the cubic polynomial which depicts the trend of the averaged class results, the trendline reaches its maximum at approximately 40 degrees. Although human enzymes function most efficiently at body temperature of 37 degrees, this investigation instead used sheep liver. Sheep have normal body temperature at around 38-39 degrees, supporting the accuracy of the trendline. After this optimum, the trendline descends, suggesting after reaching optimum temperatures, enzymes work less efficiently and eventually, in increasing temperatures, denature.

Evaluation With the inevitable occurrence of several random and systematic errors throughout any practical, the accuracy of the obtained results is often impaired. To combat the negative effects of these errors and minimise their influence, many precautions should be implemented, permitting the execution of the most precise experiment possible.

BROOKE MCGANN

S TAGE 2 BIOL OGY

Highly unpredictable and not easily identified, random errors describe the flaw in measurement caused by factors that can vary from one measurement to another. Throughout this investigation, several random errors were recognised, however, one of the most prevalent was the size of the sheep liver cubes. This practical implemented the use of 1cm3 pieces of sheep liver as the enzyme vessel. The liver was measured using metal rulers and was cut using scalpels, which proved an issue. By measuring each piece with a ruler, there was ample room for error as a result of inaccuracies. The liver was also a very thick substance which the scalpels struggled to slice cleanly through. Ultimately, this left some pieces larger than others which is detrimental to the accuracy of the results as larger cubes may contain more enzymes, meaning they could produce bubbles at a faster rate. By using some article of precise technology that could ensure total accuracy when measuring and cutting the liver pieces, the effects of this random error could be eliminated, and the results would be of much higher reliability.

Reactant agitation is another random error with ample potential to throw off the accuracy of the results. Reactants may be agitated while being poured into the measuring cylinder too harshly. As reactants are agitated, reaction time is decreased as there is more collision between substrate particles and the catalase enzyme. More caution can minimise the effects of this error.

Systematic errors, a flaw in the design of a practical investigation that affects every measurement in the same capacity, also had a negative influence on the reliability of this investigation. These errors are often observed in the equipment used: faulty timers, inaccurate thermometers, unreliable pH detectors. One of these systematic errors may have been the concentration of hydrogen peroxide. Despite the solution reading a 6% concentration, the bottle provided was previously used, no test was conducted to determine its concentration and the solution naturally denatures, especially when exposed to sunlight. These factors suggest the solution may have decomposed past the claimed concentration, which has the potential to lower the reaction between the reactants due to a lower amount of hydrogen peroxide. This error can be reduced or minimised drastically in several ways, including titrations which may be implemented to ensure consistent molar concentrations, the reduction of exposure to light and heat or even just the use of a new bottle.

Other systematic errors include the state of the sheep liver used and the temperature of the solutions. Prior to its use, the sheep liver was frozen. Enzymatic exposure to such low temperatures has ample potential to affect enzyme efficiency, hence lowering the reaction due to a lower concentration of effective enzymes. This can be minimised by using fresh liver. The solutions were also not exposed to the radical temperatures for adequate periods, meaning the influence of the extreme temperatures was not realised, combatted via longer exposure.

BROOKE MCGANN

S TAGE 2 BIOL OGY

This investigation could be improved by correcting these errors, replication and increasing sample size. A larger sample size permits a more reliable average to be calculated and reduces the consequence of errors throughout the investigation while replication confirms the validity of this practical as more results can be considered and outliers can be accounted for and, hence, diminished. For this reason, the class obtained results are more credible, as they represent a repeated practical.

Conclusion The result of this investigation infers that the optimum temperature for the catalase enzyme to function occurs at 25 degrees, at which the greatest amount of hydrogen peroxide decomposition occurs. With this, the hypothesis stating 39 degrees is the optimum temperature for enzymatic activity is not supported. Executed within a semi-controlled environment, many errors, random and systematic, had ample potential to alter the precision achieved and hence, the accuracy of the final results. Many facets could be improved upon in future replications of this experiment, including liver cube sizes, reactant agitation, the determination of reactant concentration and other thermal factors. Further replication and more comprehensive analysis would also achieve more accurate and, hence, more reliable results.

1486 words

BROOKE MCGANN

S TAGE 2 BIOL OGY

References •

Lock-and-key model 2020, Biology Online, viewed 8 May 2021, .

•

Bell-Young, L 2021, The Decomposition of Hydrogen peroxide, Reagent Chemicals, viewed 30 April 2021, .

•

BBC Bitesize 2021, What are enzymes? BBC, viewed 8 May 2021, .

•

Models of action n.d., Bio Ninja, viewed 8 May 2021, ....