Biology 12 Peroxidase Experiment

Name: Yaman Asaf

Course: SBI4U-A

Date: December 6, 2007

Factors that Affect the Rate of Reaction of Peroxidase Purpose: To determine the effect of various factors on the rate of reaction between an enzyme and its substrate, and also to determine the optimal ranges under which the enzyme activity is maximized. Also to determine whether saline and alcohol are inhibitors or activators

Hypothesis: PH factor prediction: I predict that as the pH increases so the activity of the enzyme will increase until it reaches optimum pH range (pH 7) because the enzyme is less denatured when it reaches the preferred pH level, and after this it will decrease because the active site will change in shape and it will no longer accept substrates. Temperature factor prediction: I predict as the temperature increases, the enzyme activities will increase because there is more energy to speed up the reaction until it reaches the optimum temperature range (room temperature which is about 20 °C), and after that the enzyme activities will decrease because of denature of the enzymes (cause changes to active site that will no longer fit substrate) Concentration of enzymes prediction: I predict that as the concentration of enzyme increases, so the enzyme activities will increase because there is more enzyme to react with the substrates however when enzymes get saturated, the reaction will come to a plateau because eventually all the substrates will have enzymes to react with, and any extra will have no effect on the reaction whatsoever. I predict alcohol is an inhibitor of Peroxidase because alcohol when alcohol bind to the allosteric site it changes the active site shape of the enzymes thus deactivating enzymatic activities I predict salt is an activator of Peroxidase because salt contains Na ions which attaches to the allosteric site changing the shape of the enzyme to fit a substrate.

Materials: • • • • • •

Peroxidase (enzyme in potato) Hydrogen peroxide, 3% A strong acid, pH3 (lemon juice, or HCL) 0.5 A strong base, pH 10 (drain cleaner, NaOH) 0.5 mol/L A weak acid, pH 6 (vinegar, acetic acid( CH3COOH)) 0.5 mol/L A weak base, pH 8 (baking soda, sodium bicarbonate (NaHCO3)) 0.5 mol/L a

• • • • • • • • • • • •

A saline solution, pH 7 (table salt, NaCl) 0.5 mol/L Alcohol, pH 7 (rubbing or spirits (isopropyl or ethanol)) 1 mol/L Distilled water, pH 7 Hot plate, stove, or kettle (hot water bath) Cold water (ice water bath) Eye dropper or oral, needle-less syringe 10 cc (10 mL) Graduated cylinder or needle-less syringe 10 cc( 10 mL) Disposable plastic plates Disposable plastic cups Thermometer Timing device (with second hand) ice

Procedure: •

I placed a piece of raw potato in 10 mL of water at room temperature (20 °C) for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel)

•

I placed a piece of raw potato in 10 mL of cold water at temperature 10 °C for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel) to observe the effect of temperature on reaction activity

•

I placed a piece of raw potato in 10 mL of cold water at temperature 15 °C for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel) to observe the effect of temperature on reaction activity

•

I placed a piece of raw potato in 10 mL of hot water at room temperature 25 °C for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel) to observe the effect of temperature on reaction activity

•

I placed a piece of raw potato in 10 mL of hot water at temperature 30 °C for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel) to observe the effect of temperature on reaction activity

•

I placed a piece of raw potato in 10 mL of lemon juice 0.5 mol/L at room temperature (21 °C) for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel) to observe the effect of pH on reaction activity

•

I placed a piece of raw potato in 10 mL of drain cleaner, NaOH at room temperature (21 °C) for three minutes. Put three drops of hydrogen peroxide (3

%) on it (after dabbing dry with paper towel) to observe the effect of pH on reaction activity •

I placed a piece of raw potato in 10 mL of vinegar, acetic acid 0.5 mol/L at room temperature (21 °C) for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel) to observe the effect of pH on reaction activity

•

I placed a piece of raw potato in 10 mL of baking soda 0.5 mol/L at room temperature (21 °C) for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel) to observe the effect of pH on reaction activity

•

I placed a piece of raw potato in 10 mL of saline solution 0.5 mol/L at room temperature (21 °C) for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel) to determine if saline is an inhibitor or activator

•

I placed a piece of raw potato in 10 mL of alcohol solution 1 mol/L at room temperature (21 °C) for three minutes. Put three drops of hydrogen peroxide (3 %) on it (after dabbing dry with paper towel) to determine if alcohol is an inhibitor or activator

•

I put three drops of hydrogen peroxide (3 %) on large pieces of potato to observe the effect of concentration (large pieces have smaller surface area which have less enzymes)

•

I put three drops of hydrogen peroxide (3 %) on medium pieces of potato to observe the effect of concentration (large pieces have smaller surface area which have less enzymes)

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I put three drops of hydrogen peroxide (3 %) on small pieces of potato to observe the effect of concentration (smaller pieces have larger surface area which have more enzymes, the more the enzymes the greater the reaction activity)

Analysis: Inquiry skills (scientific Method): The dependant variable is time The independent variable is Peroxidase enzyme The controlled variables are PH, temperature, and concentration The reason to create a datum is so that we could make a comparison. Without creating this datum, it would be hard to see the effect of enzymes on the decomposition of

peroxide. It’s to create this reference point to see how it decomposes before any enzymatic reaction and after. Inquiry skills (data management):

Figure 1: Qualitative observation scale of Peroxidase-catalyzed peroxide decomposition Analysis for temperature factor (10 °C): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average:

Qualitative Datum (extent of bubbling) 0 1 1 2 1 1 1

Analysis for temperature factor (15 °C): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 1 1 2 2 2 2 2

Analysis for temperature factor (20 °C): Quantitative Datum (time in seconds) 10 20 30 40 50

Qualitative Datum (extent of bubbling) 2 2 3 3 3

60 Average

2 3

Analysis for temperature factor (25 °C): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 3 3 4 4 2 2 3

Analysis for temperature factor (30 °C): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 3 2 2 2 1 0 2

Analysis for pH factor (pH 3): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 0 1 1 1 1 2 1

Analysis for pH factor (pH 6): Quantitative Datum (time in seconds) 10 20 30 40 50

Qualitative Datum (extent of bubbling) 1 2 2 3 4

60 Average

4 3

Analysis for pH factor (pH 7): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 2 3 3 3 4 4 3

Analysis for pH factor (pH 8): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 3 2 2 2 2 1 2

Analysis for pH factor (pH 10): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 2 1 1 1 0 0 1

Analysis for concentration factor (large pieces): Quantitative Datum (time in seconds) 10 20 30 40 50

Qualitative Datum (extent of bubbling) 0 1 1 1 2

60 Average

2 1

Analysis for concentration factor (medium pieces): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 4 4 3 3 3 2 3

Analysis for concentration factor (small pieces): Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 4 4 3 3 3 3 3

Analysis for saline inhibitor/activator factor: Quantitative Datum (time in seconds) 10 20 30 40 50 60 Average

Qualitative Datum (extent of bubbling) 4 4 3 3 3 2 3

Analysis for alcohol inhibitor/activator factor: Quantitative Datum (time in seconds) 10 20 30 40 50

Qualitative Datum (extent of bubbling) 1 1 1 1 0

60 Average

0 1

Knowledge and understanding (Data Analysis): The optimal range of temperature and pH of Peroxidase is about 20°C to 25° C at a pH of 6.0 to 7.0 It seems to be that Peroxidase has a different temperature range than Catalase however both have similar pH range. Knowledge and Understanding (Concept Analysis): Enzymes are made of protein, depending on the structure of the amino acid, and the hydrogen and ionic bonds is what makes the difference between the two enzymes (Catalase and Peroxidase). It seems to be that Catalase has stronger hydrogen and ionic bonds than Peroxidase and that’s why it can withstand more temperature before it’s denatured.

Conclusion: My experiment results conform to my hypothesis. According to the data tables I have created, you could notice that the enzymatic reaction (amount of bubbles) first increases starting from 15°C then it starts to go down when it reaches over 25°C (this conforms with my first prediction on the effect of temperature on Peroxidase) Starting from pH 3 to pH 7, the reaction increases then it decreases after pH 7 (this conforms with second prediction) Starting from low concentration, we get less reaction then it increases gradually (this conforms with my third prediction)