Importance of ATP: WJEC A level Biology revision notes

Lesson 1 of 8

Why do cells need ATP?

These notes cover topic 3.1, Importance of , in WJEC A level Biology Unit 3: Energy, and the Environment. Each lesson follows the WJEC specification, and the exam tips are based on BioWelsh analysis of WJEC past papers.

Every living cell needs a steady supply of energy. It uses energy to move substances against their concentration gradients, to build large molecules such as proteins and DNA, and to divide. The energy comes from food molecules such as glucose, or from light in . But cells cannot use the energy in glucose directly. Instead, they transfer it to a small molecule called , and delivers it wherever it is needed.

You met the structure of (adenosine triphosphate) in the first year of the course. It is made of the base adenine, the sugar ribose and three phosphate groups joined in a row. The bonds between the phosphate groups are easily broken by , and this is what makes so useful.

The diagram below shows being to and .

Diagram of ATP as adenine and ribose with three phosphate groups, reacting with water to give ADP with two phosphate groups plus one inorganic phosphate and released energy. — ATP is hydrolysed to ADP and inorganic phosphate, releasing 30.6 kJ mol⁻¹. Original BioWelsh diagram.

On the left is : adenine and ribose with three phosphate groups attached.
A water molecule is used to break off the last phosphate group. This is a reaction, catalysed by an called ATPase.
The products are () and an ion, written .
About 30.6 kJ of energy is released for every mole of , a small and useful amount.
The reverse reaction joins and to make . Adding a phosphate group like this is called , and it needs an input of energy.

The two directions of the ATP reaction
Reaction	Word equation	Energy
	+ water → +	Releases 30.6 kJ mol⁻¹
(a condensation reaction)	+ → + water	Needs 30.6 kJ mol⁻¹, supplied by or by light in

is described as the . 'Universal' it is used in all organisms, from bacteria to plants and animals, and 'currency' it is used for almost all energy-requiring reactions in a cell. In the same way that money can be earned in one place and spent in another, energy is 'earned' when is made in or and 'spent' when is .

The diagram below shows how links the reactions that release energy with the reactions that use it.

Cycle diagram with ATP at the top and ADP plus inorganic phosphate at the bottom. The left arrow shows phosphorylation using energy from respiration or light; the right arrow shows hydrolysis releasing energy for active transport, protein synthesis, DNA replication and muscle contraction. — The ATP and ADP cycle links energy-releasing and energy-requiring reactions. Original BioWelsh diagram.

The left arrow shows being made from and . The energy comes from , or from light in the of photosynthesising cells.
The that makes most is . You will see how it works later in this topic.
The right arrow shows being back to and , releasing energy for work in the cell.
The same molecules go round and round the cycle. A cell holds only a small amount of at any moment, so it must be remade continuously.

Examples of processes that use ATP
Process	What the energy from ATP does
	Changes the shape of proteins so they move ions or molecules against a , for example the
Protein synthesis	Joins amino acids to tRNA and forms peptide bonds at the
DNA replication and cell division	Builds new DNA strands and moves chromosomes
Secretion by	Moves to the cell surface membrane
Activating molecules	Adds a phosphate to a molecule to make it more reactive, for example glucose at the start of
Muscle contraction (animals only)	Allows heads to detach from and re-cock

of is a single reaction, so energy is released instantly where it is needed.
Each releases a small, manageable amount of energy, so little is wasted as heat. Breaking down a whole glucose molecule at once would release far more energy than any one reaction needs.
is soluble and small, so it moves easily within the cell and out of into the cytoplasm.
is quickly remade from and , so the same molecules are used again and again.

Not all the energy released in ends up in . The rest is released as heat. In mammals and birds this heat helps to keep body temperature constant, which is especially important in small animals and newborn babies.

Lesson 2 of 8

How are electrons and hydrogen carried in cells?

Most is made using energy from electrons. To understand how, you first need two ideas: reactions and the that carry hydrogen.

In a one substance is and another is at the same time. A hydrogen atom is made of one and one electron, so when hydrogen atoms are passed from one molecule to another, electrons are passed too.

Oxidation and reduction
Idea	Oxidation	Reduction
Electrons	Loss of electrons	Gain of electrons
Hydrogen	Loss of hydrogen	Gain of hydrogen
Oxygen	Gain of oxygen	Loss of oxygen
Energy	The molecule usually loses energy	The molecule usually gains energy

called remove pairs of hydrogen atoms from . The is . The hydrogen atoms are picked up by a , which is . A is a small organic molecule that helps an by carrying atoms or electrons from one reaction to another.

The three hydrogen-carrying coenzymes
Coenzyme	Where it is used	Reduced form
	: , the and the	()
	: the	(FADH₂)
	: the	()

What happens to hydrogen removed from a respiratory

1) A removes two hydrogen atoms from a , which is
2) or accepts the hydrogen atoms and is
3) The carries them to the inner mitochondrial membrane
4) The hydrogen atoms split into (H⁺) and electrons (e⁻)
5) The electrons enter the and the is , ready to be used again

Lesson 3 of 8

What is the electron transport chain?

The is a series of proteins in a membrane that pass electrons from one to the next. It is found in the inner membrane of and in the membranes of . Its job is to release the energy of electrons in small steps so that it can be used to make .

The electrons that enter the chain are at a high energy level. We call them , because they carry potential energy. In , the come from and , so their energy originally came from food molecules. In , light energy absorbed by chlorophyll raises electrons to a high energy level.

The diagram below shows what happens to the energy of the electrons as they move along the chain.

Energy level diagram. Excited electrons at the top pass down a staircase of three electron carriers to a final electron acceptor at the bottom. Proton pumps are shown at the steps between carriers. — Excited electrons lose energy in small steps as they pass along the carriers, and the energy drives proton pumps. Original BioWelsh diagram.

The vertical axis shows the energy level of the electrons, from high at the top to low at the bottom.
are passed to the first . Each is when it accepts electrons and when it passes them on, so every transfer is a .
At each downward step the electrons lose a little energy.
Some of this energy is used by to move across the membrane.
At the end of the chain the electrons, now at a low energy level, are passed to a .

The chain is made of two kinds of protein arranged alternately along the membrane. accept and pass on electrons. use the energy released by the electrons to actively transport (hydrogen ions, H⁺) across the membrane. You do not need to learn the names of the individual pumps or .

The parts of an electron transport chain
Part	What it does
	Accepts electrons (is ) and passes them to the next protein (is )
	Uses energy from electrons to move across the membrane against their
	Accepts the low-energy electrons at the end: oxygen in , in

In , oxygen is the . It combines with the electrons and with to form water: ½O₂ + 2H⁺ + 2e⁻ → H₂O. Without oxygen, the last cannot pass on its electrons. The whole chain backs up and stops, the pumps stop, and no more is made this way. This is why , which blocks the last protein in the chain, is so poisonous.

Lesson 4 of 8

How is a proton gradient built up and kept?

The in the move from one side of a membrane to the other. Because keep being pumped in one direction, a much higher concentration of builds up on one side. This difference is called a .

Pumping is : the move against their , and the energy comes from the passing along the chain. In , are pumped from the into the . In , they are pumped from the into the .

carry a positive charge. So the side with more has both a higher concentration of and a more positive charge. A difference in both concentration and charge is called an . Like water held behind a dam, it is a store of potential energy.

A gradient can only be kept if the cannot leak back. The inner mitochondrial membrane and the membrane are impermeable to , apart from special channels in the . The only way back for the is through , which is exactly where the energy is needed.

Because are hydrogen ions, the is also a pH gradient. The side with more has a lower pH.

What keeps the gradient going and what destroys it
Situation	Effect on the proton gradient
Electrons keep flowing and pumps keep working	Gradient maintained
A chemical blocks a	Fewer pumped, so the pH of the rises
No oxygen reaches the	Electron flow stops, pumps stop, gradient runs down
A chemical makes the membrane leaky to	leak back without passing through , so the gradient is lost
In : splits water in the	Releases extra into the , making the gradient steeper
In : is in the	Removes from the , making the gradient steeper

Lesson 5 of 8

How does ATP synthetase make ATP by chemiosmosis?

You now have a membrane with a steep across it. The final step is to use the energy stored in that gradient to make . This is : the flow of down their through , which is coupled to the synthesis of .

(also called ) is a large that spans the membrane. It has a channel that can pass through, a rotor and stalk that can turn, and a head where and are joined. As diffuse through the channel, they make the rotor and stalk turn. The turning changes the shape of the head, and this joins and to form .

The diagram below brings the whole process together for a .

Inner mitochondrial membrane with the intermembrane space above and the matrix below. Three proton pumps alternate with two electron carriers. Electrons from reduced NAD enter at the first pump and electrons from reduced FAD enter at the first carrier. Each pump moves protons up into the intermembrane space. Oxygen accepts electrons to form water. Protons flow down through ATP synthetase, which converts ADP and inorganic phosphate to ATP in the matrix. — The electron transport chain and ATP synthetase in the inner mitochondrial membrane. Orange arrows show electrons and blue arrows show protons. Original BioWelsh diagram.

The top region is the , with a high concentration of . The bottom region is the , with a low concentration.
delivers hydrogen atoms at the left. They split into and electrons, and the electrons enter the chain at the first .
The orange arrows follow the electrons from pump to to pump. Each of the three pumps moves up into the .
passes its electrons to the first , so they skip the first pump. This is why leads to less than .
At the end of the chain, oxygen accepts the electrons and combines with to make water.
On the right, flow back down their gradient through into the , and and are joined to make .

Scientists have worked out the real three-dimensional structure of . The image below is a computer model of the from cow .

Ribbon model of a large protein complex. A gold ring of protein helices is at the top, joined by a long thin central stalk to a large round head made of many orange and green protein chains at the bottom, with a thin side arm on the right. — Computer model of the structure of ATP synthetase (ATP synthase) from cow mitochondria, built from protein data bank entry 5ARA. Photo: Zhou A, Rohou A, Schep DG, Bason JV, Montgomery MG, CC0.

Each coloured ribbon is a protein chain. The whole is built from many protein subunits.
The gold ring at the top sits in the membrane. passing through beside it make this ring turn.
The long, thin stalk links the ring to the large head at the bottom, which sits in the .
As the stalk turns inside the head, the head changes shape and joins and to make .
You do not need to learn the names of the subunits. The model shows that really is a tiny rotating machine.

Two ways of making ATP by chemiosmosis
Feature	Oxidative phosphorylation	Photophosphorylation
Where	Inner membrane of ()	membranes of
Energy for exciting electrons	of food molecules, delivered by and	Light absorbed by chlorophyll
	Oxygen	(non-cyclic) or chlorophyll again (cyclic)

Not all is made by . A small amount is made in and the by , where a phosphate group is transferred directly from a molecule to . You will study these stages in the topic.

How do we know happens? The idea was proposed by Peter Mitchell in 1961. In 1966, scientists tested it with isolated from . They soaked the in an acidic solution at pH 4 in the dark, so the filled with . They then moved the into a solution at pH 8 containing and , still in the dark. A burst of was made, with no light and no electron transport. was made only while a existed. This showed that a on its own can drive synthesis.

What happens when parts of the system are blocked
Chemical	What it does	Result
	Blocks the last protein in the chain, so electrons cannot pass to oxygen	Electron flow stops, no pumping, no made by , oxygen is not used
Oligomycin	Blocks the channel in	cannot flow back, the gradient becomes so steep that pumping stops, electron flow stops and builds up
DNP (dinitrophenol)	Makes the inner membrane leaky to , so electron transport is '' from synthesis	Electrons keep flowing and oxygen is still used, but little is made. The energy is released as heat and body temperature can rise dangerously

Lesson 6 of 8

How are mitochondria and chloroplasts similar in making ATP?

and do different jobs, but they make in the same way. Both use an to pump into a small enclosed space, and both let the flow back through .

First, look at the structure of a in the diagram below.

Labelled cut-away illustration of a mitochondrion showing the outer membrane, intermembrane space, inner membrane folded into cristae, matrix, ATP synthase particles, ribosomes and mitochondrial DNA. — The structure of a mitochondrion. Photo: Kelvinsong, CC0.

A has two membranes. The outer membrane is smooth.
The inner membrane is folded into , which gives a large surface area for , and .
The narrow gap between the two membranes is the . This is where are pumped to.
The fluid inside the inner membrane is the . is made on the side, where the heads of the molecules are found.
The diagram also labels structures such as and mitochondrial DNA, which you do not need for this topic.

Real look less tidy. The electron micrograph below shows two in a lung cell.

Black and white electron micrograph of two rounded mitochondria, each with a dark double membrane around the edge and dark folded lines of cristae crossing the grainy matrix. — Transmission electron micrograph of two mitochondria in a mammalian lung cell. Photo: Louisa Howard, Public domain.

Each is surrounded by a double membrane, seen as a dark edge.
The dark lines running across the inside are the , the folds of the inner membrane.
The grainy material between the is the .
The look round here because they have been cut across. are often sausage shaped, so their outline in a section depends on the angle of the cut.

Now compare the structure of a , shown in the diagram below.

Labelled cut-away illustration of a chloroplast showing the outer and inner membranes of the envelope, stroma with high pH, grana made of stacked thylakoids, thylakoid lumen with low pH, starch granules and ribosomes. An enlarged circle shows water being split and hydrogen ions passing through ATP synthase to make ATP. — The structure of a chloroplast, with an enlarged view of a thylakoid. Photo: Kelvinsong, CC0.

A is also surrounded by two membranes, called the envelope.
Inside is a third system of membranes, the . are flattened sacs, often stacked into piles called .
The space inside each is the , also called the . are pumped into it, so it has a low pH.
The fluid around the is the . It has a high pH, and is made on the side.
The enlarged circle shows water being split and flowing through to make .

The electron micrograph below shows part of a single-celled green alga, Chlamydomonas. It contains both a and .

Black and white electron micrograph with many long, dark, parallel membranes curving across the image, an oval mitochondrion with pale folds in the lower left, another mitochondrion in the upper left and a fuzzy cell wall on the right edge. — Transmission electron micrograph of part of a *Chlamydomonas* cell, showing stacked thylakoid membranes of the chloroplast and mitochondria. Photo: Dartmouth Electron Microscope Facility, Dartmouth College, Public domain.

The long, dark lines running across the middle of the image are stacks of membranes inside the .
In the lower left is an oval . The pale folds inside it are its .
Another can be seen in the upper left.
The fuzzy layer at the far right is the cell wall.
Both have large areas of folded or stacked membrane, because this is where their and are found.

The diagram below shows the in a membrane. Compare it with the mitochondrial diagram in the previous lesson.

Thylakoid membrane with the stroma above and the thylakoid space below. Light excites electrons in photosystem II, which receives electrons from photolysis of water in the thylakoid space. Electrons pass through a carrier, a proton pump, another carrier and photosystem I, then reduce NADP in the stroma. The pump moves protons into the thylakoid space. Protons flow up through ATP synthetase, making ATP in the stroma. — The electron transport chain and ATP synthetase in a thylakoid membrane. Original BioWelsh diagram.

The is at the top, with a low concentration of . The is at the bottom, with a high concentration.
Light excites electrons in (). The electrons it loses are replaced by splitting water, called , which also releases into the .
The pass along to a , which moves from the into the .
Light excites the electrons again in (). They then pass to , which is in the .
flow back into the through , and and are joined to make .

The diagram below puts the two side by side, so you can see the matching parts.

Two side-by-side panels. In the mitochondrion panel, a proton pump moves protons from the matrix into the intermembrane space and ATP synthetase lets them flow back. In the chloroplast panel, a proton pump moves protons from the stroma into the thylakoid space and ATP synthetase lets them flow back. — Where the proton gradient is in a mitochondrion and in a chloroplast. Original BioWelsh diagram.

In both , a moves into a small space: the in and the in .
The low region is the in and the in .
In both, flow back by through .
is made on the low side: in the of a and in the of a .

Mitochondria and chloroplasts compared
Feature	Mitochondria	Chloroplasts
in the membrane	Yes	Yes
of and	Yes	Yes
made by	Yes:	Yes:
Membrane containing the chain	Inner membrane ()	membrane
pumped from
pumped into
Source of energy for exciting electrons	Chemical energy in food, carried by and	Light
	and
	Oxygen, forming water	(non-cyclic)
Other contributions to the gradient	None	adds to the ; reducing removes from the

In , the number of pumps used explains why the give different amounts of . Electrons from pass through three , so enough are pumped to make three . Electrons from enter the chain further along and pass through only two pumps, so they make two . WJEC mark schemes use these values of three and two.

Lesson 7 of 8

How can dehydrogenase activity be measured?

cannot be seen working, but you can detect their activity with a dye that changes colour when it is . This is the specified practical for this topic.

In respiring cells such as yeast, remove hydrogen atoms from and normally pass them to and . If an is added, it picks up some of these hydrogen atoms and electrons instead, and is . changes its colour. The faster the work, the sooner the colour changes.

Artificial hydrogen acceptors you may use
Acceptor	Colour when oxidised	Colour when reduced	Notes
	Blue	Colourless (the yeast looks cream)	Oxygen in the air re-oxidises it, turning it blue again
	Blue	Colourless	Also used to show electrons released from in the light
, a compound	Colourless	Red	The red product is not re- by air, so it suits a

Specified practical

Investigating dehydrogenase activity in yeast using methylene blue

Unit 3, section 1, specified practical 1 (BioWelsh entry number)

Aim To investigate the effect of temperature on the activity of in yeast, using as an and timing how long it takes to lose its colour.

Five panels in order: a beaker of yeast in glucose; a water bath to equilibrate the tubes; a syringe to add 1 cm³ of methylene blue; a stop clock to time the colour change; a bunged tube of cream yeast used as a colour standard. — The dehydrogenase practical at a glance. Original BioWelsh diagram.

Equipment

Yeast suspension
Yeast suspension10 g dried yeast per 100 cm³ of 0.2 mol dm⁻³ glucoseLiving yeast cells supply the ; the glucose is their .
Methylene blue solution
solution0.05 g per 100 cm³: blue when , colourless when .
Thermostatically controlled water baths
Thermostatically controlled water baths20, 30, 40, 50 and 60 °CHold each tube at a set temperature.
Thermometer
ThermometerChecks the actual temperature of each water bath.
Test tubes with bungs and a rack
Test tubes with bungs and a rackHold the mixtures; bungs limit air reaching them.
Syringes
Syringes5 cm³ and 1 cm³Measure volumes accurately.
Glass rod
Glass rodStirs the yeast so every sample has the same number of cells.
Stop clock
Stop clockTimes the colour change.
Colour standard tube
Colour standard tube5 cm³ yeast + 1 cm³ waterShows the colour when all the dye is .
Boiled and cooled yeast suspension
Boiled and cooled yeast suspensionFor the tubes: boiling denatures the .
Gloves, eye protection and a test tube holder
Gloves, eye protection and a test tube holderProtect against and hot water.

Safety

is an irritantRiskIrritation of the skin or eyes if it is splashed or transferred from hands to eyes while using a syringe.ControlWear eye protection and gloves; wash off any splashes immediately.
may cause an allergic reactionRiskSkin reaction if it touches the skin while being drawn into or pushed out of a syringe.ControlWear gloves and cover any exposed skin.
Hot water in the 50 and 60 °C water bathsRiskScalding when placing or removing tubes.ControlUse a test tube holder and do not reach into the water bath.

Variables

Independent variableTemperature

Water baths at 20, 30, 40, 50 and 60 °C, checked with a thermometer.

Temperature affects the kinetic energy of and molecules and, at high temperatures, the shape of the .

Example: At 40 °C the time was 164.3 s; at 20 °C it was 613.3 s.

Dependent variableRate of dehydrogenase activity

Time for the to lose its colour, converted to rate = 1000 ÷ time.

The faster the remove hydrogen, the sooner all the is .

Controlled variablesConcentration and volume of yeast suspension, Concentration of glucose, Volume and concentration of methylene blue, pH

Concentration and volume of yeast suspension

5 cm³ of the same stirred suspension in every tube.

More yeast cells more molecules and a faster colour change.

Concentration of glucose

All yeast was suspended in the same 0.2 mol dm⁻³ glucose solution.

Glucose is the . More could increase the rate.

Volume and concentration of

1 cm³ of 0.05 g per 100 cm³ solution in every tube.

More dye would take longer to reduce completely, even with the same activity.

Make the suspension in a pH 7 solution.

, like all , work at different rates at different pH values.

Control experimentBoiled and cooled yeast

5 cm³ boiled yeast suspension with 1 cm³ at each temperature.

It should stay blue, showing that living yeast cause the colour change.

Control experiment or controlled variable?

A is kept the same in every tube, such as the volume of . A is a separate tube, here boiled yeast, that shows what happens without active . Examiners do not accept one term for the other.

Method, step by step

1)
Set up water baths at 20, 30, 40, 50 and 60 °C and check each with a thermometer.
Why?
A of at least five temperatures shows the trend and where activity is highest.
2)
Stir the yeast suspension, then use a 5 cm³ syringe to put 5 cm³ into each of three test tubes for every temperature.
Why?
Stirring stops the yeast settling, so every tube contains the same concentration of cells.
3)
Put the yeast tubes, and a tube of , in the water bath for 10 minutes.
Why?
This lets both solutions reach the temperature of the water bath before they are mixed, so the reaction starts at the correct temperature.
4)
Add 1 cm³ of to a tube, put in the bung, turn the tube upside down once to mix, and start the stop clock immediately. Put the tube back in the water bath.
Why?
Mixing once spreads the dye evenly. Adding the same volume of the same concentration keeps the amount of acceptor the same in every tube.
5)
Do not shake the tube again while timing.
Why?
Shaking mixes in air, and oxygen re-oxidises the , making the colour change take longer.
6)
Stop the clock when the mixture matches the colour standard, ignoring the thin blue layer at the surface. If there is no change after 900 s, record 'no change'.
Why?
Comparing with a colour standard makes the end point less subjective. The surface stays blue because it is in contact with air.
What you see when methylene blue is used to show dehydrogenase activity in yeast. Original BioWelsh diagram.
Open full size
7)
At each temperature, set up a tube using 5 cm³ of boiled and cooled yeast suspension with 1 cm³ of .
Why?
The should stay blue. This shows that the colour change is caused by in living yeast and not by glucose or temperature alone.
8)
Repeat so there are three readings at each temperature, calculate the time, and calculate the rate as 1000 ÷ time.
Why?
Repeats allow anomalous results to be spotted and give a more reliable . A shorter time a faster rate, so the time must be converted to a rate.

Results and analysis

Example data

Time for to lose its colour in yeast suspension at each temperature

Temperature / °C	Time 1 / s	Time 2 / s	Time 3 / s	time / s	Rate (1000 ÷ time) / s⁻¹
20	612	598	630	613.3	1.63
30	318	305	331	318.0	3.14
40	164	171	158	164.3	6.09
50	236	251	244	243.7	4.10
60	no change in 900 s	no change in 900 s	no change in 900 s	not reached	0 (recorded as zero)

Line graph of rate against temperature. Rate rises from 1.63 at 20 °C to 3.14 at 30 °C and a peak of 6.09 at 40 °C, then falls to 4.10 at 50 °C and 0 at 60 °C. — Rate of dehydrogenase activity in yeast at each temperature. Original BioWelsh graph.

What the results show

The rate rose from 1.63 s⁻¹ at 20 °C to its highest value, 6.09 s⁻¹, at 40 °C. It fell to 4.10 s⁻¹ at 50 °C, and at 60 °C there was no colour change within 900 s.

From 20 to 40 °C, molecules gain kinetic energy, so - complexes form more often.

Above 40 °C, hydrogen bonds in the break, the change shape and the are denatured.

The optimum lies between 30 and 50 °C. Testing more temperatures in this would find it more precisely. The boiled yeast controls stayed blue at every temperature.

Worked calculations

time at 40 °C = (164 + 171 + 158) ÷ 3 = 493 ÷ 3 = 164.3 s

Rate at 40 °C = 1000 ÷ 164.3 = 6.09 s⁻¹ (to 3 figures)

Rate at 20 °C = 1000 ÷ 613.3 = 1.63 s⁻¹, so the rate at 40 °C is about 3.7 times faster

Evaluation

Judging when the blue colour has gone is subjective.

Effect on resultsDifferent people may stop the clock at different times, making results less accurate and less repeatable.

ImprovementCompare with a colour standard, or use and a to measure the colour change at times.

Oxygen from the air re-oxidises methylene blue at the surface and if the tube is disturbed.

Effect on resultsThe colour change takes longer, so the rate is underestimated.

ImprovementUse a bung, mix only once and avoid moving the tube while timing.

Yeast cells settle out of suspension.

Effect on resultsTubes filled later may contain different numbers of cells, so the rates are not comparable.

ImprovementStir the suspension thoroughly before taking each 5 cm³ sample.

The temperature intervals are 10 °C wide.

Effect on resultsThe true optimum temperature cannot be identified precisely.

ImprovementRepeat with temperatures at 2 °C intervals between 30 and 50 °C.

Going further

Other ways to carry out this practical

Change the glucose concentration instead of temperature, making a such as 0, 0.05, 0.1, 0.2 and 0.4 mol dm⁻³ by diluting a stock solution with water.
Test the effect of a : if the dye stays blue with the inhibitor but not without it, the inhibitor has stopped activity.
Compare different strains of yeast, keeping the mass of yeast per cm³ the same.
Use instead of ; it also turns from blue to colourless.
Use , which turns from colourless to red. The red colour is not reversed by oxygen, so a can measure the absorbance after a time. A higher absorbance more has been .

Why yeast without glucose may still change colour

Yeast cells store some carbohydrate, such as . Even with no added glucose, they respire this store slowly, so is still , but it takes much longer.

Model answer: describe the method

Describe how you would use to investigate the effect of temperature on activity in yeast.

Original practice response · 6 indicative marks

Set up thermostatically controlled water baths at a of at least five temperatures, for example 20, 30, 40, 50 and 60 °C, and check each with a thermometer.
Stir a yeast suspension in glucose solution, and use a syringe to add the same volume, for example 5 cm³, to three test tubes for each temperature. Keep the concentration of yeast and glucose, and the pH, the same in every tube.
Leave the tubes and the in the water bath for 10 minutes so they reach the required temperature.
Add the same volume and concentration of , for example 1 cm³, to each tube, mix gently once, put in a bung and start the stop clock. Do not shake the tube again, because oxygen would re-oxidise the .
Record the time taken for the blue colour to disappear, comparing with a colour standard of yeast and water and ignoring the blue layer at the surface.
Set up a with boiled and cooled yeast at each temperature, which should stay blue. Calculate the of the three times at each temperature and the rate as 1000 ÷ time, then plot rate against temperature.

What the answer needs to cover

A of temperatures (at least five) controlled with water baths
Same volume and concentration of yeast suspension and glucose; same pH
Solutions equilibrated at the temperature before mixing
Same volume and concentration of added; not shaken so that oxygen does not re-oxidise it
Time recorded until the blue colour disappears, using a colour standard
Boiled yeast , repeats and a , with rate calculated as 1 ÷ time

Exam practice

Explain why a tube of boiled and cooled yeast with methylene blue was set up.Show answer

Boiling denatures the in the yeast. If this tube stays blue while the tube with living yeast turns colourless, it shows that the colour change is caused by () activity in living cells and not by the glucose, the temperature or the itself.

Boiling denatures the
Shows the colour change is caused by activity
Rules out a reaction without active

Suggest why a thin layer at the top of the tube stayed blue after the rest of the mixture had lost its colour.Show answer

The surface of the mixture is in contact with air. Oxygen in the air is an oxidising agent, so it re-oxidises the at the surface, turning it blue again.

Surface is in contact with air or oxygen
Oxygen re-oxidises the

Explain why no colour change was seen at 60 °C.Show answer

At 60 °C the hydrogen bonds holding the tertiary structure of the break. The change shape, so no longer fit and no - complexes form. No hydrogen is removed from the , so the is not and stays blue.

Hydrogen bonds break and the is denatured
changes shape, so no - complexes form
No hydrogen passed to , so it is not

Related learning

Nucleic acids
The structure of and its role as an energy are covered in Unit 1.
Cell structure & organisation
You need the structure of and .
Membranes & transport
carry out , and the membrane must be impermeable to .
Enzymes
Temperature affects activity in the same way as other .
Photosynthesis
uses the and .
Respiration
and from , the and the supply electrons for .
Experimental skills
Timing colour changes, calculating rates and evaluating methods are practical skills tested in Unit 5.

Specification & sources