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Topic Exploration Pack

Oxygen Dissociation Curves

Activity 1 Teacher Answers....................................................................................................6

Activity 2 Teacher Answers...............................................................................................9

Activity 3 – Teacher Guide..............................................................................................11

Activity 4 – Teacher Answers..........................................................................................12

Activity 5 – Teacher Sheet - Respiratory Gases Hide’n’Seek.........................................15

Learner Activity 1............................................................................................................18

Learner Activity 2 The Graph of Mystery Project............................................................24

Learner Activity 3 - Oxygen Dissociation Curve Groundhog..........................................31

Learner Activity 4 – Haemoglobin Hopscotch.................................................................33

Learner Activity 5 – Respiratory Gases Hide’n’Seek......................................................35

Instructions and answers for teachers

These instructions cover the student activity section which can be found on page 18. This Topic Exploration Pack supports OCR A Level Biology A (H020/H420) and Biology B (Advancing Biology) (H022/H422).

When distributing the activity section to the students either as a printed copy or as a Word file you will need to remove the teacher instructions section.

Version 1 1 Copyright © OCR 2016

Learning outcome This topic exploration pack covers the AS and A Level Biology A Learning Outcomes 3.1.2(i) and (j):

3.1.2(i) the role of haemoglobin in transporting oxygen and carbon dioxide

3.1.2(j) the oxygen dissociation curve for fetal and adult haemoglobin.

This topic exploration pack covers the A Level Biology B Learning Outcomes 4.1.2(g) - (i):

4.1.2(g) the role of haemoglobin in oxygen transport

4.1.2(h) the oxygen dissociation curves for different respiratory pigments

4.1.2(i) the factors which affect oxygen dissociation from respiratory pigments.

IntroductionThe roles of haemoglobin and oxygen dissociation curves form a more challenging end to section 3.1.2 (Biology A) than the foregoing physiology of the cardiovascular system. This is because the topic requires a focus on the molecular and biochemical level. This is harder for some learners to visualise than the gross structure of the heart and blood vessels. Learning outcome 3.1.2(j) also requires strong graph skills and provides an opportunity to reinforce the basics about plotting and using graphs. In Biology B, this topic comes later (4.1.2) and is an A level only topic following cellular respiration and exercise. The activities that follow aim to build up conceptual knowledge of these areas from very simple beginnings.

While prior knowledge of the other learning outcomes in 3.1.2 (Biology A) and section 2.2 (Biology B) is essential, so too is a thorough grounding in levels of protein structure and the structure and function of haemoglobin (2.1.2m - n Biology A, 2.1.3b Biology B). Some revision of these topics could be undertaken in class as an oral or written formative test or as a homework exercise.

The major learner misconceptions that lose marks in examination questions on this topic are as follows:

using inappropriate language to describe the binding of oxygen to haemoglobin

confusing a red blood cell with a haemoglobin molecule – in fact one red blood cell is

said to contain 300 million molecules of haemoglobin

confusing the x and y axis labels on graphs of oxygen dissociation curves

failing to realise that an oxygen dissociation curve shows both association and

dissociation depending on whether the curve is followed from left to right or from right

to left relative to the x axis.

These misconceptions are addressed in the activities that follow.


Suggested activitiesThis topic may be seen as presenting a dense section of theory teaching with no opportunity for practical work. A range of new activities are provided here which allow learners to take a more hands-on approach and to gradually build their understanding of the concepts, rather than being bamboozled by the subtleties of oxygen dissociation curve graphs from the outset. External sources that can be used to inform and broaden teaching include:

https://www.youtube.com/watch?v=9fxm85Fy4sQ&list=PL3EED4C1D684D3ADF&index=27

This is a 12 minute video summary of oxygen acquisition and transport and the transport and excretion of carbon dioxide. It could be used as a starter activity before Activity 1 to summarise the combination of sections 3.1.1 and the rest of 3.1.2, to consolidate knowledge before introducing topic, 3.1.2 (i) and (j) (Biology A).

http://courses.washington.edu/conj/resp/oxygen.htm

This webpage calculates the total volume of oxygen in a litre of oxygenated blood. The total comprises a tiny amount as gaseous oxygen dissolved in plasma, and a much greater proportion bound to haemoglobin. The figures are useful to show the importance of haemoglobin in boosting the total volume of oxygen that can be transported. This could be used to introduce the revision of haemoglobin structure from 2.1.1(n) (Biology A) and 2.1.3(b) (Biology B).

http://www.interactive-biology.com/2631/060-hemoglobin-and-the-oxygen-dissociation-curve/

This video tutorial explains haemoglobin structure and the oxygen dissociation curve very clearly in simple terms. This could follow Activity 2 to reinforce the concepts. Note: the units on the graph shown here for partial pressure of oxygen are mm Hg. Partial pressure of oxygen is more usually reported in kPa.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230298/

This short paper for teachers describes how to talk through a visual demonstration of cooperativity, using the postage stamp analogy to explain the pattern of release of oxygen from oxyhaemoglobin. Each learner or pair of learners requires a block of four 1p postage stamps for the practical activity. The labels on the axes in this resource are oxygen tension (instead of partial pressure of oxygen) with units as mm Hg on the x axis and percentage oxyhaemoglobin (instead of percentage oxygen saturation) on the y axis, but learners can be told that these mean the same as the preferred terms in brackets.


http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230298/

http://www.interactive-biology.com/2631/060-hemoglobin-and-the-oxygen-dissociation-curve/

http://courses.washington.edu/conj/resp/oxygen.htm

https://www.youtube.com/watch?v=9fxm85Fy4sQ&list=PL3EED4C1D684D3ADF&index=27

The five OCR activity sheets produced for this topic exploration pack are summarised here. Each has an accompanying learner worksheet containing instructions for carrying out the activity and some written tasks or follow-up work, plus a separate teacher answer sheet.

In activity 1 the learners play a game where they use chopsticks to catch Maltesers® hidden in a bowl of popcorn. They repeat this at five different ‘concentrations’ of Maltesers® to model haemoglobin binding oxygen molecules at different concentrations (partial pressures). The purpose of this activity is to make the concept of haemoglobin binding oxygen real and fun, and to predict a directly proportional relationship. The practical activity should take 50-60 minutes and the Questions and Evaluation section could be completed for homework. See Activity 1 Teacher Answers for more information to support the learner worksheet.

Activity 2 is presented in the context of a mystery. Learners are told that the shape of the oxygen dissociation curve caused consternation for 45 years before the reason for the strange shape was fully understood. Learners then plot the oxygen dissociation curve themselves, analyse the graph and explore why the curve is sigmoidal and not directly proportional as predicted in Activity 1. The worksheet explains how the sigmoidal shape relates to the function of haemoglobin in binding, transporting and releasing oxygen, and to the molecular structure of haemoglobin. This worksheet could occupy 40-50 minutes.

Activity 3 is a team game that reinforces understanding of the oxygen dissociation curve of haemoglobin and improves learner descriptions and explanations using biological terms appropriate to A level. The game could proceed for about ten minutes, perhaps as a recap starter activity in the lesson following activity 2, before moving on to the written exercise.

Activity 4 takes place outside and involves learners jumping to the right or left of a chalked oxygen dissociation curve in response to teacher instructions about changes in conditions that affect formation and dissociation of oxyhaemoglobin. This could also follow activity 2 and could be allowed 15-20 minutes before proceeding to the written task.

Activity 5 is a game that can be played in pairs or small groups. It familiarises learners with the different forms adopted by oxygen and carbon dioxide and their different possible locations when they are transported from the alveoli to the tissues or vice versa. This activity requires knowledge of the reactions in red blood cells that lead to the formation of hydrogen carbonate ions and carbaminohaemoglobin. The game could be played for 10-15 minutes before moving to the written task.


Additional teacher preparationLearners may want explanations of the following Physics concepts:

1. the reason that oxygen concentration is described and measured as partial pressure of oxygen. A good explanation, suitable for projecting for the class, can be found here: http://www.diatronic.co.uk/nds/webpub/partial_pressures.htm

This webpage also gives data for partial pressures of oxygen and carbon dioxide in the air, in venous and arterial blood, and in alveolar air.

2. the difference between the torr units mm Hg (millimetres of mercury) and kPa (kilopascals). If learners have never seen a mercury barometer (an EU directive ended the production of new mercury barometers in Europe in 2007) a diagram could be shown, e.g. http://www.schoolphysics.co.uk/age11-14/Mechanics/Statics/text/Barometer_mercury/index.html

3. Definitions of the two units are:

The millimetre of mercury (mm Hg) is the non-SI unit of pressure. 1 mm Hg equals the atmospheric pressure that supports a column of mercury 1 millimetre high. This unit dates from 1643.

The pascal (symbol Pa) is the SI unit of pressure. It is equivalent to one newton per square metre. 1 kilopascal = 103 pascals.

The relationship between them is that 1 mm Hg = 0.133322 kPa.

Learners may also want explanations of the terms loading tension and unloading tension. The loading tension is the oxygen partial pressure in kPa when the haemoglobin is 95% saturated with oxygen. The unloading tension is the oxygen partial pressure in kPa when the haemoglobin is 50% saturated with oxygen.


http://www.schoolphysics.co.uk/age11-14/Mechanics/Statics/text/Barometer_mercury/index.html

http://www.schoolphysics.co.uk/age11-14/Mechanics/Statics/text/Barometer_mercury/index.html

http://www.diatronic.co.uk/nds/webpub/partial_pressures.htm

Activity 1 ‘Huma-globin’ and the Malteser® Game – an experiment to model the effect of oxygen concentration on the ability of haemoglobin to bind oxygen molecules

Teacher AnswersRunning the activity: The materials needed are listed on the learner worksheet. Large bags of sweet popcorn can be bought in supermarkets, e.g. a 175 g size toffee popcorn is suitable for mixing in a large bowl with the 40 Malteser® sweets. Cheap disposable chopsticks can be sourced online or at some supermarkets.

Correct learner answers to questions on the main text of the worksheet are shown below.

Method Learners are asked to fill in two gaps:

1. The other chemical components of the blood plasma are:

2. As the concentration of Maltesers® increases, the number of Maltesers® caught should:

ResultsThe independent variable is:

The dependent variable is:

It is preferable if class results can be pooled to calculate a mean for plotting the graph.

Graphs should have on the x axis and

on the y axis. The expectation is that the results

follow a straight directly proportional line.


water

other dissolved gases such as nitrogen and carbon dioxide

dissolved solutes such as glucose, urea and ions (from dissociated salts such as

sodium chloride)

small soluble proteins such as clotting factors, fibrinogen, hormones,

immunoglobulins (antibodies) and albumin.

increase.

number of Maltesers® in bowl

total number of Maltesers® caught

number of Maltesers® in bowl

total number of Maltesers® caught

If learners count every piece of popcorn in the bowl they can calculate an ‘oxygen’ or Malteser® concentration as a percentage figure and plot this on the x axis.

Questions and Evaluation – Expected Answers1. Define the terms independent variable and dependent variable.

2. State where the columns of values for the independent and dependent variables should appear in a results table.

3. State where the independent and dependent variables should be plotted on a graph.

4. Sketch graphs to show the meaning of the terms ‘directly proportional’ and ‘inversely proportional’.


The independent variable is the one that the experimenter chooses to vary and decides on the range of values. The dependent variable is the one that is measured. Its values depend on the value chosen for the independent variable.

Independent goes to the left, dependent to the right of this. In our experiment we had to calculate the concentration, which is the true independent variable, from two separate columns specifying the relative numbers of the two types of sweet.

Independent goes on the x axis, dependent on the y axis.

Directly proportional

x – independent variable

y – dependent variable

5. Why did each group consist of four learners?

6. What variables were controlled in the Malteser® experiment?

7. Explain why plotting the mean class results is an improvement on just plotting your own results.

8. Explain why working in the order given in the instructions could be considered a limitation on the validity of the experiment.


Inversely proportional

The learners are modelling the haemoglobin molecule which has four sub-units. Each sub-unit can bind one molecule of oxygen.

Controlled variables were the time allowed for catching the Maltesers®, the size of the bowl, the distance of the bowl to the cup, the size and shape and material of the chopsticks.

One set of results may contain errors. The class data provides many replicates of the experiment. If there is little variation between all these replicates the data is considered precise. Results which do not fit the trend of the majority of replicates should be considered. Calculating the mean provides a better estimate of how Malteser® concentration affects the number of Maltesers® caught.

Learners will at first be learning the best way of picking up a Malteser® with chopsticks. With practice they will get quicker and this will affect the results. Starting with the lowest concentration biases the results in favour of showing a trend of catching more Maltesers® when there is a greater concentration of them.

x – independent variable

y – dependent variable

Activity 2 The Graph of Mystery Project

Teacher AnswersThe full text of Dr. Adair’s paper can be found here: http://www.jbc.org/content/63/2/529.full.pdf

The abstract of Max Perutz’ paper describing the allosteric basis of cooperativity of oxygen binding in haemoglobin can be found here: http://www.nature.com/nature/journal/v228/n5273/abs/228726a0.html

Note that the term ‘salt link’ would now at A level be termed an ionic bond.

The most reasonable prediction to make about the binding of oxygen and haemoglobin is that as oxygen availability increases, more is bound to haemoglobin in a directly proportional relationship.

1. What units are used to measure the concentration or availability of oxygen in the environment of the haemoglobin?

2. At the range of partial pressures that haemoglobin experiences in the body, is haemoglobin ever fully (100%) saturated with oxygen?

3. Describe the shape of the curve.

4. State the percentage saturation of haemoglobin in (a) the alveolar capillaries and (b) in venous blood.

5. Calculate how much oxygen (as a percentage) is released from haemoglobin when red blood cells move from capillaries in the alveoli to veins leading away from capillaries in tissues.


kilopascals, kPa.

No, it reaches 96.5% saturated.

S-shaped or sigmoid.

(a) 96.5% (b) 24.0%.

72.5% is released from haemoglobin.

http://www.nature.com/nature/journal/v228/n5273/abs/228726a0.html

http://www.jbc.org/content/63/2/529.full.pdf

6. This graph is called the oxygen dissociation curve of haemoglobin. Could it also be called the oxygen association curve?

Solving the Mystery WhyLearner explanation should focus on the ability of haemoglobin to unload oxygen fast when needed (at tissues) and to remain bound to oxygen to transport it when needed (at the alveoli and en route to the tissues).

Solving the Mystery HowHaemoglobin is a conjugated protein. This means it consists of a protein part and a

that is non-protein. It has a quaternary structure because it

consists of polypeptide chains called . These are of two different

sorts, two and two Each polypeptide has its own non-protein group

attached, which contains an atom where one molecule of may bind.

The number of molecules of oxygen that may bind to one molecule of haemoglobin is

Stretch and Challenge Question:If one haemoglobin molecule can bind four oxygen molecules, how can the graph show haemoglobin as 66% saturated?


Yes. Reading from left to right along the x axis we visualise the association of more and more oxygen molecules with haemoglobin. To see dissociation we need to read from right to left, ‘backwards’ along the curve.

prosthetic / haem group

four globins

alpha beta.

iron oxygen

four.

One haemoglobin molecule can only bind up to four oxygen molecules, so could be 25%, 50%, 75% or 100% saturated depending on whether one, two, three or four haem groups had bound oxygen. The graph however concerns a large number of haemoglobin molecules. At 66% saturation we can assume that all have at least two oxygen molecules bound and that around half have bound three oxygen molecules.

Activity 3 Oxygen Dissociation Curve Groundhog

Teacher Guide

Groundhog is inspired by the radio 4 panel game ‘Just a Minute’. The purpose of this game is to familiarise learners with the inappropriate ways of describing haemoglobin binding to oxygen which can lose learners marks in assessments. The list of ‘banned’ words provided is taken from the legacy biology past paper mark scheme for F211 June 2015.

This could be played with the whole class divided into two teams and the speakers coming up to the whiteboard to draw their graph and attempt to describe and explain it without making any errors. Any member of the opposing team and the teacher can shout ‘Groundhog!’ in this case, and the teacher can oversee the accuracy and clarity of learner answers. In the version of the game with several sets of smaller opposing teams more learners have an active role to play and the teacher can move between the groups listening and contributing if necessary.

Follow-Up Task: Learners are asked to write a final polished answer to the challenge to describe and explain the shape of the oxygen dissociation curve for haemoglobin, using only the terms considered acceptable at A level. A sample answer is given below.


Description: As the partial pressure of oxygen increases from 0 to 14 kPa, the percentage saturation of haemoglobin with oxygen increases in a sigmoid curve. At the oxygen partial pressure typical of the tissues (2 kPa) haemoglobin is 24% saturated with oxygen. At the oxygen partial pressure typical of the alveoli (12 kPa) haemoglobin reaches 96.5% saturation with oxygen.

Explanation: At 0 kPa of oxygen partial pressure haemoglobin molecules have no oxygen bound to them. Binding of oxygen to haemoglobin is cooperative. There are four ferrous iron atoms (Fe2+) in four haem prosthetic groups in each haemoglobin molecule. It is difficult for the first oxygen molecule (O2) to bind so the slope of the graph is initially shallow. Once the first oxygen molecule has bound, it causes an allosteric shape change in the haemoglobin molecule, making it easier for a second oxygen molecule to bind. The slope of the graph therefore become steeper, as a small increase in oxygen partial pressure results in a large increase in percentage saturation. The binding of the third and fourth oxygen molecules to each haemoglobin molecule is easier again, so the shape of the curve is a steep increase followed by a plateau at the maximum saturation of around 96%. The sigmoidal shape of the binding curve means that oxygen is bound at the alveoli (high oxygen partial pressures) forming oxyhaemoglobin which then dissociates efficiently to unload the oxygen transported at the tissues (low oxygen partial pressures).

Activity 4 Haemoglobin Hopscotch

Teacher Answers

Haemoglobin Hopscotch is an outdoor activity, something which always rouses enthusiasm from an A level class provided the weather is clement. Learners chalk large graphs on the playground and hop to the right or left of the curve depending on instructions given by you, the teacher, or a chosen learner. Chalk on concrete, asphalt, etc, washes off after rainfall, so the learners’ work will only be a temporary addition to the school decor. An unobtrusive spot away from the Head’s study, visitors’ car park and marked out tennis and netball courts may be prudent.

A list of instructions and the correct moves for the ‘tissues’ learner and the ‘alveoli’ learner are provided here. The order of the instructions can be varied, some can be omitted or alternatively the same instruction can be repeated until learners have improved their understanding.

Where learners go wrong there needs to be time allowed for them to sketch in new curves on their chalk graphs. A learner could be used to shout the instructions, allowing the teacher the freedom to observe more closely which way learners jump in order to identify and correct errors.

This list includes specialised animals adapted to low oxygen environments. This goes beyond the specification and these calls can be omitted if desired. Similarly ‘surprise’ situations for learners to consider are carbon monoxide poisoning and change of body temperature.

If time is short the teacher or lab technician can chalk a giant oxygen dissociation graph on the ground and a group of up to ten learners can stand along the line, process each instruction and decide to jump right or left. All learners have to engage in deciding where to position themselves so as the teacher you can easily see how confident learners are with the concepts.


Instruction Direction of movement of tissues learner at 2 kPa

Direction of movement of alveoli learner at 12 kPa

increase in partial pressure of carbon dioxide

RIGHT RIGHT

llama living high in the Andes

LEFT LEFT

fetal haemoglobin LEFT LEFT

lugworm buried in anaerobic mud

LEFT LEFT

increase in acidity RIGHT RIGHT

decrease in pH RIGHT RIGHT

physiological curve RIGHT NO MOVEMENT

hill breed of sheep LEFT LEFT

lowland breed of sheep RIGHT RIGHT

6 month old baby NO MOVEMENT NO MOVEMENT

decreased affinity for oxygen

RIGHT RIGHT

increased affinity for oxygen LEFT LEFT

trainer mountaineer acclimatised to high

altitude*

NO MOVEMENT NO MOVEMENT

decrease in partial pressure of carbon dioxide

LEFT LEFT

increase in alkalinity LEFT LEFT

increase in pH LEFT LEFT

strenuous exercise RIGHT RIGHT

carbon monoxide poisoning LEFT LEFT

hyperthermia (increase in body temperature)

RIGHT RIGHT


*Acclimatisation involves production of more haemoglobin but the genetically determined shape of the curve does not alter.

Follow-Up Task: Learners could be asked to summarise in a two column table the situations that cause a shift in the curve to the right and to the left. A sample table based on the teacher instructions above is provided here:

Situations that shift curve to the left Situations that shift curve to the right

decrease in partial pressure of carbon dioxide

increase in partial pressure of carbon dioxide

increase in alkalinity increase in acidity

increase in pH decrease in pH

hill breed of sheep (genetic adaptation) lowland breed of sheep (genetic adaptation)

increased affinity for oxygen decreased affinity for oxygen

fetal haemoglobin strenuous exercise

living at high altitude (species adaptation) hyperthermia (increase in body temperature)

living in anaerobic mud (species adaptation)

carbon monoxide poisoning


Activity 5 Respiratory Gases Hide’n’Seek

Teacher Sheet

This game is a version of an imagination game called ‘Fireside Hide’n’Seek’ where a player pretends to hide anywhere in the universe and the other player has to narrow down their location in twenty questions. Here it is restricted to locations in the body important in understanding the transport of oxygen and carbon dioxide. The object of playing the game is to improve learners’ knowledge of the geography of where and in what form the respiratory gases exist in the body of a mammal.

The learner worksheet explains the rules. For maximum participation it can be played in pairs with the two learners taking it in turns to ‘hide’. To explain the rules and get learners started it can be played as a whole class activity.

Learners will need to refer to a diagram of the reaction in red blood cells catalysed by carbonic anhydrase, the subsequent dissociation of carbonic acid, the buffering effect that occurs when haemoglobin binds hydrogen ions and the chloride shift. The diagram can be in their text books, notes or projected on the whiteboard. A suitable diagram showing all the above plus dissolved carbon dioxide in the plasma and the formation of carbaminohaemoglobin in the red blood cell may be found here:

https://classconnection.s3.amazonaws.com/650/flashcards/1289650/jpg/picture11331078569098.jpg

The game could be extended to include carbon monoxide and carboxyhaemoglobin, or to include other relevant ions involved in the reactions in the red blood cell, such as hydrogen, chloride and potassium ions.

A table showing the range of basic hider identities and locations is given on the following page. The follow-up work for learners asks them to recreate this table from a starting framework.




Identity Location

1 dissolved carbon dioxide / CO2 tissue cell cytoplasm

2 dissolved carbon dioxide / CO2 interstitial fluid

3 dissolved carbon dioxide / CO2 capillary endothelial cell cytoplasm

4 dissolved carbon dioxide / CO2 plasma

5 dissolved carbon dioxide / CO2 red blood cell cytoplasm

6 dissolved carbon dioxide / CO2 alveolar / squamous epithelial, cell cytoplasm

7 dissolved carbon dioxide / CO2 layer of moisture on, alveolar / squamous epithelial, cells

8 gaseous carbon dioxide / CO2 alveolar air sacs

9 carbonic acid / H2CO3 red blood cell cytoplasm

10 carbonic acid / H2CO3 plasma

11 hydrogen carbonate ion / HCO3– red blood cell cytoplasm

12 hydrogen carbonate ion / HCO3– plasma

13 carbaminohaemoglobin red blood cell cytoplasm

14 oxyhaemoglobin red blood cell cytoplasm

15 dissolved oxygen / O2 tissue cell cytoplasm

16 dissolved oxygen / O2 interstitial fluid

17 dissolved oxygen / O2 capillary endothelial cell cytoplasm

18 dissolved oxygen / O2 plasma

19 dissolved oxygen / O2 red blood cell cytoplasm

20 dissolved oxygen / O2 alveolar / squamous epithelial, cell cytoplasm

21 dissolved oxygen / O2 layer of moisture on, alveolar / squamous epithelial, cells

22 oxygen / O2 gas alveolar air sacs


Follow-Up Task: Learners complete the summary on their sheet. The table above contains the correct answers. For example, the seven locations requested on the learner table for dissolved carbon dioxide are here numbered 1-7, the one for gaseous carbon dioxide is number 8, the two for carbonic acid are 9 and 10, etc.


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Topic Exploration Pack

Oxygen Dissociation Curves Learner Activity 1 ‘Huma-globin’ and the Malteser® Game – an experiment to model the effect of oxygen concentration on the ability of haemoglobin to bind oxygen molecules.

In this activity you will join with three classmates to become ‘Huma-globin’ – a cooperative of four humans jointly pretending to be a haemoglobin molecule. Maltesers® take the place of oxygen molecules for you to ‘bind’ (catch) with chopsticks. As a transport molecule, you will then carry each oxygen molecule and release it in a capillary within a respiring tissue.

You will investigate how varying the concentration of Maltesers® in a bowl of popcorn affects how many of them you can catch and transport in thirty seconds.

We will extrapolate from this to predict how the concentration of oxygen should, in theory, affect the saturation of haemoglobin with oxygen. The saturation means the quantity of oxygen that binds to haemoglobin.

Materials per group of four learners

40 Malteser® sweets (or similar alternative chocolates)

large bag of sweet popcorn

four pairs of chopsticks

large clean bowl labelled ‘alveolar capillary’

four clean disposable cups labelled ‘tissue capillary’

stopclock


Method

The Maltesers® represent oxygen molecules.

The popcorn represents other chemical components present in the blood plasma within an alveolar capillary. You can list what you think these are here:

You and three classmates are going to combine to play the role of one haemoglobin

molecule. You will each use a pair of chopsticks to ‘bind’ oxygen molecules, that is,

to select and pick up Maltesers® present in a bowl of popcorn. When you have

caught a Malteser® in your chopsticks, transfer it to your cup placed 5 cm away from

the bowl.

The bowl represents a capillary at the alveolus. Your cup represents a tissue

capillary where you will release ‘oxygen’. In a real tissue capillary it would then

diffuse to aerobically respiring cells.

You will run the experiment at five values of the independent variable, which is

oxygen concentration represented by the proportion of Maltesers® in the bowl. Add 8

Maltesers® to a large bag of popcorn for the first run, 16 for the second, then 24, 32

and 40 Maltesers®. If you counted every piece of popcorn in the bowl it would be

possible to calculate your ‘oxygen’ or Malteser® concentration as a percentage but

this is not essential.

In each case set the timer for 30 seconds and see how many Maltesers® your

foursome can bind and transfer to the cups.

Do not eat the caught Maltesers® as you will need to re-use them after each run of

the experiment.

Before you begin, predict what you think should happen here:

As the concentration of Maltesers® in the bowl of popcorn increases, the number of Maltesers® caught should:


Results

Record your results here:

Number of Maltesers® in bowl Total number of Maltesers® caught

8

16

24

32

40

The column that represents the independent variable is:

The column that represents the dependent variable is:


Plot your group’s results or the mean class results on a graph, labelling the x axis with the independent variable and units, and the y axis with the dependent variable.


Conclusion

You may have found that the effect of Malteser® concentration on the number you could pick up was a directly proportional relationship. This is a general rule when two chemical entities are able to bind or react together. If the concentration of one is made greater, then more successful combinations or reactions will occur within a period of time (i.e. the rate of the reaction increases).

The ability of the large conjugated protein haemoglobin to bind to oxygen was investigated in a series of lab experiments, where different concentrations of oxygen were provided. However, the surprise was that the results were NOT in fact directly proportional. This shows that there is something special and interesting going on when haemoglobin binds to oxygen that is not taken account of in the standard model.

The real relationship between oxygen concentration and the ability of haemoglobin to bind to it is shown in a graph called an oxygen dissociation curve graph. You will draw and analyse this type of graph in activity 2.

Questions and Evaluation

The first four questions check your understanding of experimental design and the presentation of results in tables and graphs.

1. Define the terms independent variable and dependent variable.

2. State where the columns of values for the independent and dependent variables should appear in a results table.

3. State where the independent and dependent variables should be plotted on a graph.


4. Sketch graphs to show the meaning of the terms ‘directly proportional’ and ‘inversely proportional’.

The next four questions evaluate the Malteser® experiment.

5. Why did each group consist of four learners?

6. What variables were controlled in the Malteser® experiment?

7. Explain why plotting the mean class results is an improvement on just plotting your own results.

8. Explain why working in the order given in the instructions could be considered a limitation on the validity of the experiment.


Learner Activity 2 The Graph of Mystery Project

Background

In 1925 Dr. G.S.Adair of the Massachusetts General Hospital published a paper on ‘The Oxygen Dissociation Curve of Haemoglobin’. In this he presented data tables and graphs to show the relationship between two variables:

the oxygen pressure (effectively, the concentration or availability of oxygen) on the x

axis and

the percentage of haemoglobin that is oxygenated on the y axis.

Dr. Adair then writes,

‘There has been much speculation over the meaning of these curves. We cannot attempt to do justice to all the points of view now held.’ In 1925, there were at least four theories attempting to explain the mysterious shape of this graph. The mystery was not fully solved until Max Perutz published a landmark paper in Nature in 1970.

So what does this mysterious graph look like and why was it considered so peculiar? Let’s find out.

Plotting the Graph

Table 1 provides the necessary data for you to plot on graph paper. The independent variable is partial pressure of oxygen. This measures how much free molecular oxygen there is in the environment of the haemoglobin. In real life, the haemoglobin is inside red blood cells in the blood vessels. The partial pressure of oxygen is 12 kPa in an alveolar capillary but only 2 kPa in venous blood after it has passed through capillaries of an organ or tissue. To obtain the data in Table 1 however, haemoglobin was subjected to different concentrations (partial pressures) of oxygen in vitro and the saturation of the haemoglobin with oxygen as a percentage of the maximum possible was measured.

Before you plot the data, what would you predict would be the effect of more oxygen available on the quantity of oxygen that haemoglobin molecules can attach to? If you have done Activity 1, you should be able to describe the predicted relationship precisely.


Table 1

Partial pressure of oxygen / kPa Saturation of haemoglobin with oxygen / %

1 8.5

2 24.0

3 43.0

4 57.5

5 71.5

6 80.0

7 85.5

8 88.0

9 92.0

10 94.0

11 95.5

12 96.5

13 97.5

14 98.0


Now plot the graph and join the points with a smooth free-hand curve.


Understanding the Graph

1. What units are used to measure the concentration or availability of oxygen in the environment of the haemoglobin?

2. At the range of partial pressures that haemoglobin experiences in the body, is haemoglobin ever fully (100%) saturated with oxygen?

3. Describe the shape of the curve.

4. State the percentage saturation of haemoglobin in (a) the alveolar capillaries and (b) in venous blood.

5. Calculate how much oxygen (as a percentage) is released from haemoglobin when red blood cells move from capillaries in the alveoli to veins leading away from capillaries in tissues.

6. This graph is called the oxygen dissociation curve of haemoglobin. Could it also be called the oxygen association curve?


Explaining the Graph and Solving the Mystery

The sigmoidal shape of the graph can be explained at two levels – why it needs to be this shape as an adaptive measure to maximise the efficiency of haemoglobin as an oxygen transporter, and how this effect is achieved in terms of the molecular structure of haemoglobin. Perutz’s discovery answered the second of these questions.

Solving the mystery WHY

You can probably see the answer to the first for yourself. Here is a summary of it. Point to the relevant region of the graph as you read through it.

The sigmoidal shape means that at high oxygen partial pressures, such as those at the alveoli and at the slightly lower oxygen partial pressures in the arteries leading to tissues and organs, haemoglobin remains highly saturated (as oxyhaemoglobin). It stubbornly holds on to the oxygen it has bound.

However a small drop in partial pressure as the haemoglobin enters the capillaries of respiring tissues causes a large quantity of oxygen to ‘unbind’ or dissociate from the haemoglobin molecules in the red blood cells. Suddenly, although this new environment is only slightly oxygen poorer than before, instead of binding to oxygen, the oxyhaemoglobin now releases it.

Essentially, the top of the ‘S’ shape of the graph is a long flat section, so that haemoglobin holds oxygen in these oxygen partial pressures while it moves from alveoli towards the tissues, but the stem of the ‘S’ is a steep gradient, so that a small decrease in oxygen availability causes a big release of oxygen to the tissues that need it.

Explain why this situation is better than if the graph took a directly proportional shape, as we predicted earlier.


Solving the mystery HOW

Think back to what you learnt about the structure of haemoglobin earlier in the course and fill in the gaps:

Haemoglobin is a conjugated protein. This means it consists of a protein part and

a .......................... ............................... that is non-protein. It has a quaternary structure

because it consists of .. ........................ polypeptide chains called ......................... .

These are of two different sorts, two ................... and two ....................... . Each

polypeptide has its own non-protein group attached, which contains an ..........................

atom where one molecule of ............................ may bind. The number of molecules of

oxygen that may bind to one molecule of haemoglobin is ......................... .

Perutz discovered that the binding of oxygen to haemoglobin is cooperative. It is difficult for the first oxygen molecule to bind, but once one oxygen has bound this alters the structure of the rest of the haemoglobin molecule making it easier for the next oxygen to bind, and so on. In other words, haemoglobin shows allosteric shape changes when one, then two and then three oxygen molecules bind.

Following the graph from left to right we see the curve get steeper. It levels off at high oxygen partial pressures (lungs and arteries) at just below 100% saturation.

If we follow the graph from right to left however, we see how the oxyhaemoglobin remains intact until oxygen is needed at tissues and then there is rapid unloading of the transported oxygen.

You may think that some oxygen appears to be wasted. At 2 kPa in the veins, according to your graph, haemoglobin is still 25% saturated. This means that every haemoglobin molecule (and there are 300 million in every red blood cell) still holds on to one of its four possible oxygen molecules. In the physiological conditions within the body however, there is another factor at work that solves this problem. This is explored in Activity 3. The Bohr Shift occurs in the tissues giving rise to what is known as the physiological curve.


Stretch and Challenge Question: If one haemoglobin molecule can bind four oxygen molecules, how can the graph show haemoglobin as 66% saturated?


Learner Activity 3 Oxygen Dissociation Curve GroundhogThe Groundhog game tests your understanding of the basic shape of the oxygen dissociation curve graph for haemoglobin. It encourages you to articulate your knowledge using appropriate A level terminology that will help to access marks in assessments.

The class divides into an even number of teams, so that each team has an opposing

team. Three or four learners per team is perfect.

The first person on the starting team has one minute to try to describe and explain

the shape of the oxygen dissociation curve for haemoglobin. They can use pen and

paper or the whiteboard to illustrate their speech.

The opposing team starts the stopclock and listens closely to the explanation.

A member of the opposing team shouts ‘Groundhog!’ when the speaker makes a

mistake. See below for a list of classic errors to listen out for.

The opposing team can also shout ‘Groundhog!’ if the speaker hesitates, says ‘er’,

‘um’ or some other form of hesitation, or pauses without speaking for three seconds.

The stopclock is re-started and we go back to the start after a ‘Groundhog!’

interruption. The challenger on the opposite team who just shouted ‘Groundhog!’ now

tries to describe and explain the shape of the oxygen dissociation curve for

haemoglobin in one minute. If that person makes an error and the opposing team

shouts ‘Groundhog!’ play starts again with a new speaker from the first team.

Listeners need to try to stop the other team from clocking up a minute of correct

description and explanation.

Speakers need to concentrate on answering the question clearly and precisely using

only terms suitable for an A level Biology answer.

The first team member who answers the question for one minute without any

‘Groundhog!’ interruption wins the game for their team.

Banned terms that are not allowed to describe the haemoglobin molecule binding

molecular oxygen (O2) include: picks up / takes up / gains / absorbs / attracts /

attaches / captures / catches / saturates / reacts with or any other similar terms. The

only correct terms allowed are that haemoglobin binds, combines with, associates

with or loads oxygen.

Oxygen dissociates from oxyhaemoglobin, but oxygen alone cannot dissociate and

haemoglobin alone cannot dissociate. The only other term allowed for the

dissociation of the two is that (oxy)haemoglobin can unload oxygen.


Follow-Up Task: Write a final polished answer to the challenge to describe and explain the shape of the oxygen dissociation curve for haemoglobin, using only the terms considered acceptable at A level.


Learner Activity 4 Haemoglobin HopscotchThis outdoor game allows you to test and extend your understanding of how the basic shape of the oxygen dissociation curve of haemoglobin is distorted in a variety of situations.

You will need:

coloured chalks

an area of playground which can be chalked on

a partner

a graph of the oxygen dissociation curve of haemoglobin to copy, or the data from

Table 1 in Activity 2.

Each pair draws the axes and curve of the oxygen dissociation curve on the ground. The scale should be roughly as follows: x axis – 2 kPa = 0.5 m, y axis – 10% saturation = 0.5 m.

One partner should stand on the curve at the point corresponding to the partial pressure of oxygen at the tissues, while the other stands at the point corresponding to the partial pressure of oxygen at the alveoli.

The teacher or a chosen learner will shout instructions for you to follow. Each partner needs to decide whether to jump right or left or to stay where they are to reflect the distortion to the curve caused by the conditions in the instruction.

These situations alter the position, and maybe the shape, of the basic curve and should be studied or researched before playing the game:

raised partial pressure of carbon dioxide and low pH

mammalian fetal haemoglobin has a different affinity for oxygen than adult

haemoglobin

the physiological curve takes account of the fact that there is higher carbon dioxide

partial pressure at the tissues than at the alveoli

species and breeds of animals show adaptation to low oxygen partial pressure

environments like waterlogged mud or high altitude.


If you jump the wrong way at any point your penalty is to draw in the whole curve in its new position in a different chalk colour. Label the reason why this new curve is different to the original. The winning team where both partners jump the correct way each time will have only one curve drawn on their axes at the end of the game.

Follow-Up Task: Use your experience of Haemoglobin Hopscotch to summarise in a two column table the situations that cause a shift in the curve either to the right or to the left.


Learner Activity 5 Respiratory Gases Hide’n’SeekFor this activity you will need knowledge of how oxygen is transported from the alveoli to the tissues and how carbon dioxide is transported from the tissues to the alveoli. You will need to refer to a diagram in a textbook or one displayed on the whiteboard by your teacher.

Respiratory Gases Hide’n’Seek can be played in a pair or a small group.

One person, the ‘hider’ picks a stage in the transport of respiratory gases and pretends to be that particular molecule in that particular place. For example the hider could pretend to be:

dissolved oxygen molecules in the blood plasma

a hydrogen carbonate ion in the cytoplasm of a red blood cell

carbaminohaemoglobin in the cytoplasm of a red blood cell.

The other player or players now have five questions to try to pin down where and what the hider is. Questions can only be answered by the hider with YES or NO. Suitable questions could be:

Are you in a cell? (YES would cover tissue cells, capillary endothelial cells, red blood

cells and the squamous epithelial cells of alveoli.)

Are you dissolved in a solution? (Yes could include interstitial fluid, blood plasma and

the layer of moisture on the surface of alveolar squamous cells.)

Are you partly protein? (Yes indicates the hider is oxyhaemoglobin or

carbaminohaemoglobin.)

If the hider’s identity and location are not discovered in five questions, the hider wins. The questioner or questioners win if they do pinpoint the hider’s identity and location in five questions or less. It is the hider’s job to keep count of the number of questions that have been asked.

Follow-up Work: Complete the table to summarise the possible locations of each form of respiratory gas. For dissolved carbon dioxide, for example, there are seven possible locations on the journey from the tissue cell cytoplasm as far as the layer of moisture on the surface of alveolar cells.

Respiratory gas identity and form Location


dissolved carbon dioxide / CO2

1

2

3

4

5

6

7

gaseous carbon dioxide / CO2 1

carbonic acid / H2CO3 1

2

hydrogen carbonate ion / HCO3 – 1

2

carbaminohaemoglobin 1

oxyhaemoglobin 1

dissolved oxygen / O2 1

2

3

4

5

6

7

oxygen / O2 gas 1


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