Topic 2: Enzyme core practical

Question 1

(Enzyme core practical) What is the aim?

1 point

Answer 1

1. To investigate how ENZYME (trypsin) CONCENTRATION affects the rate of a reaction

NOTE: Trypsin is a digestive enzyme

Question 2

Explain the reaction of trypsin and casein

NOTE: casein is a protein found in milk

Answer 2

1. TRYPSIN is added to white, cloudy milk

2. As the casein breaks down (into amino acids) the suspension turns CLEAR AND COLOURESS

Question 3

(Enzyme core practical)

State the independent variable (1 point)

State the 5 different concentrations

How do you prepare them? (1 point)

Answer 3

1. Trypsin (enzyme) concentration (%)
2. 1, 0.2, 0.3, 0.4, 0.5%
3. Prepared by serial dilution from a stock solution of known concentration.

Question 4

(Enzyme core practical)

State the control (1 point)

Why do we need a control? (1 point)

Answer 4

1. {0% trypsin concentration / distilled water} (no enzyme)

1. for comparison as this would give the normal rate of breakdown without an enzyme

Question 5

(Enzyme core practical)

State the dependent variable (1 point)

How is it measured? (3 points)

Answer 5

1. % transmission (to allow calculation of rate of reaction)
2. Use a COLORIMETER to give QUANTITATIVE DATA (numbers)
3. Measures cloudiness of the solution
4. Measure every 10 seconds and used to calculate initial rate of reaction

Question 6

(Enzyme core practical)

State the 2 environmental controlled variables + how (1 point) for each controlled variable

Answer 6

1. Temperature - using a water bath set at 37 degrees C (body temperature, as trypsin works in the human body)
2. pH - using a buffer

Question 7

(Enzyme core practical)

State the 3 procedural controlled variables + how is it controlled (1 point) for each controlled variable

Answer 7

1. Substrate concentration - 1% casein solution by using the same stock solution of milk
2. Substrate volume - 2cm^3 with a graduated pipette
3. Volume of enzyme (trypsin) solution - 2cm^3 measured with a graduated pipette

Question 8

(Enzyme core practical)

Why is the volume of trypsin (enzyme) solution controlled?
1 point

Answer 8

1. Higher volume contains more enzyme molecules, so rate of reaction will increase

Question 9

(Enzyme core practical)

Why is the volume of substrate controlled?
1 point

Answer 9

1. Higher volume contains more substrate molecules, so rate of reaction may increase

Question 10

(Enzyme core practical)

Why is the substrate concentration controlled?
1 point

Answer 10

1. Higher concentration contains more substrate molecules, so rate of reaction may increase.

Question 11

(Enzyme core practical)

Why is the ph controlled?
4 points

Answer 11

1. pH could change
2. pH change denatures active site of enzyme
3. changes {ionic bonds/charger on R groups} in active site
4. changes rate of reaction

Question 12

(Enzyme core practical)

Why is the temperature controlled?
2 points

Answer 12

1. As temperature increases (up to optimum), enzyme and substrate molecules have more kinetic energy and collide more frequently, increasing reaction rate
2. Higher temperatures denature enzyme active sites

Question 13

(Enzyme core practical)

What needs to be done for reliability? (2 steps)

Answer 13

1. 5 REPEATS for each ENZYME CONCENTRATION

2. to identify anomalies, obtain 3 concordant results, calculate mean, to improve reliability

Question 14

(Enzyme core practical)

What needs to be done for validity? (3 things)

Answer 14

1. 5 ENZYME CONCENTRATIONS to allow a trend to be established and a valid conclusion made
2. CONTROL - 0% enzyme concentration - distilled water
3. CONTROLLED VARIABLES - same substrate concentration, same temperature, same pH etc.

Question 15

(Enzyme core practical)

State the risks along with how to minimise them
2 risks

Answer 15

1. Breakage of glassware can cause cuts in the skin – do not touch broken glass
2. Enzyme is a potential allergen in dry form which may damage lungs or eyes – wear gloves, lab coat, safety glasses, clear up enzyme solution spills to prevent dry enzyme powder forming

Question 16

(Enzyme core practical)

Using serial dilution create 5 concentrations of the enzyme trypsin (0.5, 0.4, 0.3, 0.2, 0.1)

What is the {purpose/justification} of this step? (1 point)

Answer 16

1. Serial dilution creates 5 different concentrations of trypsin to investigate

Question 17

(Enzyme core practical)

Using a colorimeter with a 440nm filter and set to % Transmission, zero the colorimeter with distilled water in a cuvette by pressing “R”. Remove the water from the cuvette and dry it.

What is the {purpose/justification} of this step? (1 point)

Answer 17

1. Calibrates the colorimeter using water as a reference point.

Question 18

(Enzyme core practical)

Mix enzyme and substrate thoroughly and immediately transfer all contents to the cuvette, place in the colorimeter and record % transmission every 10 seconds.

What is the {purpose/justification} of this step? (1 point)

Answer 18

1. Measuring % transmission every 10 seconds allows the calculation of the initial rate of reaction

Question 19

(Enzyme core practical)

Discard the solution from the cuvette. Wash both cuvette and 10cm^3 beaker with distilled water and dry

What is the {purpose/justification} of this step? (1 point)

Answer 19

1. Prevents contamination or dilution of the next enzyme concentration.

Question 20

(Enzyme core practical)

Repeat for 0.4, 0.3, 0.2 and 0.1%

What is the {purpose/justification} of this step? (1 point)

Answer 20

1. Gives data to determine the effect of enzyme concentration on the initial rate of reaction.

Question 21

(Enzyme core practical)
Key Summary: Describe the method for the trypsin and {casein/milk} practical
(6 steps)

Answer 21

1. Use 5 different {enzyme/trypsin} concentrations for validity - independent variable - 0% (control) 0.1, 0.2, 0.3, 0.4, 0.5%
2. Place enzyme + substrate into a water bath (37 degrees C)
3. Mix enzyme and substrate
4. Measure the % transmission in a COLORIMETER every 10 seconds until the solution becomes clear + plot graphs of time against transmission to CALCULATE THE INITIAL RATE OF THE REACTION (dependent variable)
5. Repeat 5 times FOR EACH TRYPSIN CONCENTRATION to obtain 3 concordant results, calculate mean rate, improves reliability
6. Controlled variables for validity – SAME temperature 37 degrees C using water bath, SAME pH using buffer, same VOLUME of substrate and enzyme using graduated pipette - 2cm^3 , SAME substrate concentration - 1%

Question 22

For the enzyme core practical, if it was changed to measure the gas produced, how would you measure it?
(2 points)

Answer 22

1. Measure gas using a gas syringe

2. Measure gas produced every 10 seconds for 5 minutes

Question 23

For the enzyme core practical, if it was changed to measure the pH change, how would you measure it?
(2 points)

Answer 23

1. use a pH probe

2. measure the pH every 10 seconds for 5 minutes

Question 24

(Once the results of the enzyme experiment have been acquired)
For each enzyme concentration, plot curves on separate axes. (X is time in seconds, Y is percentage transmission in %)

How do you calculate the initial rate of reaction using the plotted curve? (2 steps)

Answer 24

1. Calculate the initial rate of reaction by drawing a tangent to the first or steepest part of the curve

THEN

2. Rate = change in y / change in x

Question 25

Key Summary: Why measure the initial rate (rate at the start) of an enzyme reaction? (2 points)

Answer 25

1. there needs to be enough substrate molecules to saturate the enzyme, to ensure that substrate is not a limiting factor 2. the fastest rate is at the start (initial rate) then decreases, due to the substrate concentration decreasing as the substrate gets used up by enzyme in the reaction

Question 26

Provided that there is excess substrate and other conditions are kept constant, the rate of reaction is _________ _________ to the enzyme concentration (linear relationship)

Answer 26

directly proportional

Question 27

Key Summary: Explain why increasing enzyme concentration increases the initial rate of reaction As the enzyme concentration increases, the initial rate of reaction increases because... (5 points) Eventually graph {levels off/reaches a plateau} and {there is no further increase in rate/rate becomes constant}, as the __________ __________ becomes the _______ _______.

Answer 27

1. number of enzymes (trypsin) increases 2. so number of trypsin active sites increases 3. MORE FREQUENT, SUCCESSFUL COLLISIONS between enzyme and substrate 4. MORE ENZYME-SUBSTRATE COMPLEXES form in a given time 5. more product forms in a given time substrate concentration limiting factor

Question 28

(For a fixed concentration of enzyme) as the substrate concentration increases, the ___ __ _______ increases as more substrate molecules are present {they collide with the enzyme more frequently /more successful collisions} , more enzyme-substrate complexes form, so more _______ forms in a given time eventually a _______ _____ is reached - all the enzymes are involved in enzyme-substrate complexes (all active sites are {saturated with substrate/occupied}) if the substrate concentration carries on increasing, there is no further increase in ______ ___ ________

Answer 28

rate of reaction product maximum rate rate of reaction

Question 29

(Adapting the {trypsin/casein} practical to investigate the effect of substrate concentration on the rate of reaction) The substrate is the _______ ________ The enzymes are in the _______ ________. The rate of reaction needs to be calculated from the rate at which the _________ is produced. Most of the controlled variables are the same, except we can now control enzyme concentration too. Some method steps stay the same e.g. 5 different substrate concentrations, mix enzyme and substrate etc.

Answer 29

hydrogen peroxide mussel tissue oxygen