2.4 Proteins
Protein functions acvy
Living organisms synthesize many diferent proteins with du xpm
a wide range o unctions.
A solution oegg albumen
Other groups o carbon compounds have important roles in the cell, but in a test tube can be heated
none can compare with the versatility o proteins. They can be compared in a water bath to nd the
to the worker bees that perorm almost all the tasks in a hive. All o the temperature at which it
unctions listed here are carried out by proteins. denatures. The efects opH
can be investigated by adding
Catalysis there are thousands o dierent enzymes to catalyse acids and alkalis to test tubes
specifc chemical reactions within the cell or outside it. oegg albumen solution.
To quantiy the extent o
Muscle contraction actin and myosin together cause the denaturation, a colorimeter
muscle contractions used in locomotion and transport around can be used as denatured
the body. albumen absorbs more light
than dissolved albumen.
Cytoskeletons tubulin is the subunit o microtubules
that give animals cells their shape and pull on chromosomes acvy
during mitosis.
bx
Tensile strengthening fbrous proteins give tensile strength
needed in skin, tendons, ligaments and blood vessel walls. Botox is a neurotoxin
obtained rom Clostridium
Blood clotting plasma proteins act as clotting actors that cause botulinum bacteria.
blood to turn rom a liquid to a gel in wounds.
1 What are the reasons
Transport of nutrients and gases proteins in blood help or injecting it into
transport oxygen, carbon dioxide, iron and lipids. humans?
Cell adhesion membrane proteins cause adjacent animal cells 2 What is the reason or
to stick to each other within tissues. Clostridium botulinum
producing it?
Membrane transport membrane proteins are used or
acilitated diusion and active transport, and also or electron 3 What are the reasons or
transport during cell respiration and photosynthesis. injecting it rather than
taking it orally?
Hormones some such as insulin, FSH and LH are proteins,
but hormones are chemically very diverse.
Receptors binding sites in membranes and cytoplasm or
hormones, neurotransmitters, tastes and smells, and also
receptors or light in the eye and in plants.
Packing of DNA histones are associated with DNA in eukaryotes
and help chromosomes to condense during mitosis.
Immunity this is the most diverse group o proteins, as cells can
make huge numbers o dierent antibodies.
There are many biotechnological uses or proteins including enzymes
or removing stains, monoclonal antibodies or pregnancy tests or
insulin or treating diabetics. Pharmaceutical companies now produce
many dierent proteins or treating diseases. These tend to be very
expensive, as it is still not easy to synthesize proteins artifcially.
Increasingly, genetically modifed organisms are being used as
microscopic protein actories.
93
2 MOLECULAR BIOLOGY
exampls of protins
Rubisco, insulin, immunoglobulins, rhodopsin, collagen and spider silk as
examples o the range o protein unctions.
Six proteins which illustrate some o the unctions o proteins are described in table 4.
rubo inuln
This name is an abbreviation or ribulose bisphosphate This hormone is produced as a signal to many cells in
carboxylase, which is arguably the most important the body to absorb glucose and help reduce the glucose
enzyme in the world. The shape and chemical properties concentration o the blood. These cells have a receptor
o its active site allow it to catalyse the reaction that xes or insulin in their cell membrane to which the hormone
carbon dioxide rom the atmosphere, which provides binds reversibly. The shape and chemical properties o
the source o carbon rom which all carbon compounds the insulin molecule correspond precisely to the binding
needed by living organisms can be produced. It is site on the receptor, so insulin binds to it, but not other
present at high concentrations in leaves and so is molecules. Insulin is secreted by cells in the pancreas
probably the most abundant o all proteins on Earth. and is transported by the blood.
immunoglobuln rhodopn
These proteins are also known as antibodies. They have Vision depends on pigments that absorb light. One o
sites at the tips o their two arms that bind to antigens these pigments is rhodopsin, a membrane protein o rod
on bacteria or other pathogens. The other parts o the cells o the retina. Rhodopsin consists o a light sensitive
immunoglobulin cause a response, such as acting as a retinal molecule, not made o amino acids, surrounded
marker to phagocytes that can engul the pathogen. The by an opsin polypeptide. When the retinal molecule
binding sites are hypervariable. The body can produce absorbs a single photon o light, it changes shape. This
a huge range o immunoglobulins, each with a diferent causes a change to the opsin, which leads to the rod cell
type o binding site. This is the basis o specic immunity sending a nerve impulse to the brain. Even very low light
to disease. intensities can be detected.
collagen spde lk
There are a number o diferent orms o collagen but all Diferent types o silk with diferent unctions are
are rope-like proteins made o three polypeptides wound produced by spiders. Dragline silk is stronger than steel
together. About a quarter o all protein in the human body and tougher than Kevlar. It is used to make the spokes
is collagen it is more abundant than any other protein. o spiders webs and the lielines on which spiders
It orms a mesh o bres in skin and in blood vessel suspend themselves. When rst made it contains
walls that resists tearing. Bundles o parallel collagen regions where the polypeptide orms parallel arrays.
molecules give ligaments and blood vessel walls their Other regions seem like a disordered tangle, but when
immense strength. It orms part o the structure o teeth the silk is stretched they gradually extend, making the
and bones, helping to prevent cracks and ractures. silk extensible and very resistant to breaking.
Protoms
Every individual has a unique proteome.
A proteome is all o the proteins produced by a cell, a tissue or an
organism. By contrast, the genome is all o the genes o a cell, a tissue or
an organism. To fnd out how many dierent proteins are being produced,
mixtures o proteins are extracted rom a sample and are then separated
94
2.4 Proteins
by gel electrophoresis. To identiy whether or not a particular protein is
present, antibodies to the protein that have been linked to a fuorescent
marker can be used. I the cell fuoresces, the protein is present.
Whereas the genome o an organism is xed, the proteome is variable
because dierent cells in an organism make dierent proteins. Even
in a single cell the proteins that are made vary over time depending
on the cells activities. The proteome thereore reveals what is actually
happening in an organism, not what potentially could happen.
Within a species there are strong similarities in the proteome o all
individuals, but also dierences. The proteome o each individual is
unique, partly because o dierences o activity but also because o small
dierences in the amino acid sequence o proteins. With the possible
exception o identical twins, none o us have identical proteins, so each
o us has a unique proteome. Even the proteome o identical twins can
become dierent with age.
Figure 8 Proteins rom a nematode worm have been separated by gel
electrophoresis. Each spot on the gel is a diferent protein
acvy
acv cc: gm d pm
We might expect the proteome of an organism to be smaller than its genome,
as some genes do not code for polypeptides. In fact the proteome is larger.
How could an organism produce more proteins than the number of genes that
its genome contains?
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2 MOLECULAR BIOLOGY
2.5 enzyms
understnding applictions
Enzymes have an active site to which specic Methods o production o lactose-ree milk and
substrates bind. its advantages.
Enzyme catalysis involves molecular motion
and the collision o substrates with the
active site.
Temperature, pH and substrate concentration
afect the rate o activity o enzymes.
Enzymes can be denatured.
Immobilized enzymes are widely used in
industry.
Ntre of science Skills
Experimental design: accurate quantitative Design o experiments to test the efect o
measurements in enzyme experiments require temperature, pH and substrate concentration
replicates to ensure reliability. on the activity o enzymes.
Experimental investigation o a actor afecting
enzyme activity. (Practical 3)
Figure 1 Computer-generated image of the active sites nd enzymes
enzyme hexokinase, with a molecule of its
substrate glucose bound to the active site. The Enzymes have an active site to which specic
enzyme bonds a second substrate, phosphate, substrates bind.
to the glucose, to make glucose phosphate
Enzymes are globular proteins that work as catalysts they speed up
96 chemical reactions without being altered themselves. Enzymes are oten
called biological catalysts because they are made by living cells and speed
up biochemical reactions. The substances that enzymes convert into
products in these reactions are called substrates. A general equation or
an enzyme-catalysed reaction is:
substrate __en_z_y_m_e_ product
Enzymes are ound in all living cells and are also secreted by some cells
to work outside. Living organisms produce many dierent enzymes
literally thousands o them. Many dierent enzymes are needed, as
enzymes only catalyse one biochemical reaction and thousands o
reactions take place in cells, nearly all o which need to be catalysed.
This property is called enzymesubstrate specifcity. It is a signifcant
dierence between enzymes and non-biological catalysts such as the
metals that are used in catalytic converters o vehicles.
To be able to explain enzymesubstrate specifcity, we must look at the
mechanism by which enzymes speed up reactions. This involves the
2.5 enzyMes
substrate, or substrates binding to a special region on the surace o the
enzyme called the active site (see fgure 1 ) . The shape and chemical
properties o the active site and the substrate match each other. This allows
the substrate to bind, but not other substances. Substrates are converted
into products while they are bound to the active site and the products are
then released, reeing the active site to catalyse another reaction.
data-ba qutio: Biosynthesis of glycogen
The Nobel Prize or Medicine was won in 1 947 by glycogen. Glycogen is a polysaccharide, composed
Gerty Cori and her husband Carl. They isolated o glucose molecules bonded together in two
two enzymes that convert glucose phosphate into ways, called 1 ,4 and 1 ,6 bonds (see fgure 2) .
4 Curve B was obtained using enzymes that
had not been heat-treated.
1 4 bonding 1 4 bonding plus a a) Describe the shape o Curve B. [2]
b) Explain the shape o Curve B. [2]
1 6 bond forming a side-branch % conversion
Figure 2 Bonding in glycogen 80 B
1 Explain why two dierent enzymes are [2] 60
needed or the synthesis o glycogen 40
rom glucose phosphate.
2 The ormation o side-branches increases the 20
rate at which glucose phosphate molecules A
can be linked on to a growing glycogen [2] 10 20 30 40 50
molecule. Explain the reason or this. min
Figure 3 shows the percentage conversion of glucose
3 Curve A was obtained using heat-treated phosphate to glycogen by the two enzymes, over a
enzymes. Explain the shape o curve A. [2] 50-minute period
enzym activity 97
Enzyme catalysis involves molecular motion and the
collision of substrates with the active site.
Enzyme activity is the catalysis o a reaction by an enzyme. There are
three stages:
The substrate binds to the active site o the enzyme. Some enzymes
have two substrates that bind to dierent parts o the active site.
While the substrates are bound to the active site they change into
dierent chemical substances, which are the products o the reaction.
The products separate rom the active site, leaving it vacant or
substrates to bind again.
A substrate molecule can only bind to the active site i it moves very
close to it. The coming together o a substrate molecule and an active
site is known as a collision. This might suggest a high velocity impact
between two vehicles on a road, but that would be a misleading image
and we need to think about molecular motion in liquids to understand
how substrateactive site collisions occur.
With most reactions the substrates are dissolved in water around
the enzyme. Because water is in a liquid state, its molecules and all
2 MOLECULAR BIOLOGY
toK the particles dissolved in it are in contact with each other and are in
continual motion. E ach particle can move separately. The direction of
Why hs he lck nd key mdel movement repeatedly changes and is random, which is the basis of
n been lly superseded by he diffusion in liquids. Both substrates and enzymes with active sites are
induced-f mdel? able to move, though most substrate molecules are smaller than the
The lock and key model and the enzyme so their movement is faster.
induced-t model were both developed So, collisions between substrate molecules and the active site occur
to help to explain enzyme activity. because of random movements of both substrate and enzyme. The
Models like these are simplied substrate may be at any angle to the active site when the collision
descriptions, which can be used to occurs. Successful collisions are ones in which the substrate and active
make predictions. Scientists test these site are correctly aligned to allow binding to take place.
predictions, usually by perorming
experiments. I the results agree water molecules
with the predictions, then the model
is retained; i not then the model is substrates
modied or replaced. The German
scientist Emil Fischer introduced the active site
lock and key model in 1890. Daniel
Koshland suggested the induced-t part of enzyme
model in 1959 in the United States. The
conormational changes predicted by Figure 4 Enzyme-substrate collisions. If random movements bring any of the substrate
Koshland's model were subsequently molecules close to the active site with the correct orientation, the substrate can bind to the
observed using high-resolution X-ray active site
analysis o enzymes and other newly
developed techniques. Although Factors afecting enzyme activity
much experimental evidence has
accumulated conrming predictions Temperature, pH and substrate concentration afect the
based on the induced-t model, it is rate o activity o enzymes.
still just viewed as a model o enzyme
activity. Enzyme activity is afected by temperature in two ways
aciviy In liquids, the particles are in continual random motion. When a liquid is
heated, the particles in it are given more kinetic energy. Both enzyme and
Mking hyphesis substrate molecules therefore move around faster at higher temperatures
Bacillus licheniformis lives and the chance of a substrate molecule colliding with the active site of the
in soil and on decomposing enzyme is increased. Enzyme activity therefore increases.
eathers. What is the reason
or it producing a protease When enzymes are heated, bonds in the enzyme vibrate more and
that works best at alkaline the chance of the bonds breaking is increased. When bonds in the
pH? Make a hypothesis to enzyme break, the structure of the enzyme changes, including the
explain the observations. active site. This change is permanent and is called denaturation.
How could you test your When an enzyme molecule has been denatured, it is no longer able
hypothesis? to catalyse reactions. As more and more enzyme molecules in a
solution become denatured, enzyme activity falls. Eventually it stops
98 altogether, when the enzyme has been completely denatured. So, as
temperature rises there are reasons for both increases and decreases
in enzyme activity. Figure 5 shows the effects of temperature on a
typical enzyme.
2.5 enzyMes
Enzymes are sensitive to pH rate at which reaction decreases owing
to denaturation of enzyme molecules
The pH scale is used to measure the acidity or alkalinity o a solution.
The lower the pH, the more acid or the less alkaline a solution is. Acidity rate of reaction rate at which optimum
is due to the presence o hydrogen ions, so the lower the pH, the higher reaction increases temperature
the hydrogen ion concentration. The pH scale is logarithmic. This means owing to increased
that reducing the pH by one unit makes a solution ten times more acidic. kinetic energy of actual
A solution at pH 7 is neutral. A solution at pH 6 is slightly acidic; pH 5 is substrate and rate of
ten times more acidic than pH 6, pH 4 is one hundred times more acidic enzyme reaction
than pH 6, and so on. molecules
Most enzymes have an optimum pH at which their activity is 0 10 20 30 40 50 60
highest. I the pH is increased or decreased rom the optimum, t e m p e ra t u re / C
enzyme activity decreases and eventually stops altogether. When
the hydrogen ion concentration is higher or lower than the level at Figure 5 Temperature and enzyme activity
which the enzyme naturally works, the structure o the enzyme is
altered, including the active site. Beyond a certain pH the structure Key
o the enzyme is irreversibly altered. This is another example o 1 stomach
denaturation.
acidic hot springs
Enzymes do not all have the same pH optimum in act, there is 2 decaying plant matter
a wide range. This relects the wide range o pH environments in
which enzymes work. For example, the protease secreted by Bacillus large intestine
lichen iform is has a p H o p timum b e twe e n 9 and 1 0 . This b acte rium 3 small intestine
is cultured to produce its alkaline-tolerant protease or use in 4 alkaline lakes
biological laundry detergents, which are alkaline. Figure 6 shows
the pH range o some o the places where enzymes work. Figure 7 5
shows the eects o pH on an enzyme that is adapted to work at
neutral pH.
Enzyme activity is afected by substrate concentration 6
7
Enzymes cannot catalyse reactions until the substrate binds to the active 8
site. This happens because o the random movements o molecules in 9
liquids that result in collisions between substrates and active sites. I the 10
concentration o substrates is increased, substrateactive site collisions
will take place more requently and the rate at which the enzyme
catalyses its reaction increases.
However, there is another trend that needs to be enzyme activity Figure 6
considered. Ater the binding o a substrate to
an active site, the active site is occupied and Optimum pH at which enzyme
unavailable to other substrate molecules until activity is fastest (pH 7 is
products have been ormed and released rom the optimum for most enzymes) .
active site. As the substrate concentration rises,
more and more o the active sites are occupied at As pH increases or decreases from the
any moment. A greater and greater proportion o optimum, enzyme activity is reduced.
substrateactive site collisions are thereore blocked. This is because the shape of the active
For this reason, the increases in the rate at which site is altered so the substrate does not
enzymes catalyse reactions get smaller and smaller t so well. Most enzymes are denatured
as substrate concentration rises. by very high or low pH, so the enzyme
no longer catalyses the reaction.
I the relationship between substrate concentration
and enzyme activity is plotted on a graph, a pH
distinctive curve is seen (fgure 8) , rising less and
less steeply, but never quite reaching a maximum. Figure 7 pH and enzyme activity
99
2 MOLECULAR BIOLOGY
enzyme activity Denaturation
substrate concentration Enzymes can be denatured.
Figure 8 The efect o substrate Enzymes are proteins, and like other proteins their structure can be
concentration on enzyme activity irreversibly altered by certain conditions. This process is denaturation
and both high temperatures and either high or low pH can cause it.
When an enzyme has been denatured, the active site is altered so the
substrate can no longer bind, or i its binds, the reaction that the enzyme
normally catalyses does not occur. In many cases denaturation causes
enzymes that were dissolved in water to become insoluble and orm a
precipitate.
Quantitative experiments
Experimental design: accurate quantitative measurements in enzyme
experiments require replicates to ensure reliability.
Our understanding o enzyme activity is based measurements should be accurate, which in
on evidence rom experiments. To obtain strong science means close to the true value; and
evidence these experiments must be careully
designed and ollow some basic principles: the experiment should be repeated, so that
the replicate results can be compared to assess
the results o the experiment should be how reliable they are.
quantitative, not just descriptive;
data-base questions: Digesting jello cubes a) describing whether the solution around the
cubes is colourless or a shade o pink or red
Figure 9 shows apparatus that can be used to
investigate protein digestion.
tube tight-tting lid b) taking a sample o the solution and
measuring its absorbance in a colorimeter
c) nding the mass o the cubes using an
electronic balance. [3]
2 I method (c) was chosen, discuss whether it
protease in a solution gelatine cubes would be better
with known pH
to nd the mass o all o the cubes o jello
Figure 9 Tube used to investigate the rate o digestion o gelatine together, or nd
the mass o each one separately. [2]
I the cubes are made rom sugar-ree j ello ( j elly) , 3 I the jello cubes have a mass o 0.5 grams,
the colouring that they contain will gradually be state whether it is accurate enough to
released as the protein is digested by the protease. measure their mass to:
The questions below assume that strawberry-
favoured jello with red colouring has been used! a) the nearest gram (g)
1 Explain whether these methods o assessing b) the nearest milligram (mg)
the rate o protein digestion are acceptable:
c) the nearest microgram (g) . [3]
100
2.5 enzyMes
4 To obtain accurate mass measurements o 7 Draw a graph o the results in the table. [5]
the jello cubes, it is necessary to remove 8 Describe the relationship between pH and
them rom the tube and dry their surace papain activity. [3]
to ensure that there are no drips o solution 9 Discuss the conclusions that can be drawn
rom the tube adhering. Explain the reason [2] rom this data about the precise optimum
or drying the surace o the blocks. pH o papain. [2]
Table 1 gives the results that were obtained using
sugar-ree jello cubes and a protease called papain, ph Ma dcra (mg)
extracted rom the fesh o resh pineapples. 2 80 87 77
5 Discuss whether the results in table 1 are
reliable. [2] 3 122 127 131
6 Most o the results were obtained using an 4 163 166 164
extract o protease rom one pineapple, but 5 171 182 177
ater this ran out, a second pineapple was 6 215 210 213
used to obtain more protease or use in the 7 167 163 84
experiment. 8 157 157 77
9 142 146 73
a) Deduce which results were obtained [1 ]
using the second extract. Table 1
b) Suggest how the use o a second
extract could have aected the results. [2]
Designing enzyme experiments
Design o experiments to test the efect o temperature, pH and substrate
concentration on the activity o enzymes.
1 The actor that you are going to investigate is the clock could be used to measure the time
independent variable. You need to decide: taken or a colour change;
how you are going to vary it, or example what units should be used or measuring
with substrate concentration you would the dependent variable, or example
obtain a solution with the highest seconds rather than minutes or hours
concentration and dilute it to get lower would be used or measuring a rapid
concentrations; colour change;
what units should be used or measuring how many repeats you need to get reliable
the independent variable, or example enough results.
temperature is measured in degrees C elsius; 3 Other actors that could aect the dependent are
what range you need or the independent control variables. You need to decide:
variable, including the highest and lowest what all the control variables are;
levels and the number o intermediate levels.
how each o them can be kept constant;
2 The variable that you measure to nd out how
ast the enzyme is catalysing the reaction is the what level they should be kept at, or
dependent variable. You need to decide: example temperature should be kept at
the optimum or the enzyme i pH is being
how you are going to measure it, including investigated, but actors that might inhibit
the choice o meter or other measuring enzymes should be kept at a minimum level.
device, or example an electronic stop
101
2 MOLECULAR BIOLOGY
enzym xprimnts
Experimental investigation o a actor afecting enzyme activity.
There are many worthwhile enzyme experiments. constant i investigating the eect o [2]
The method that ollows can be used to substrate concentration.
investigate the eect o substrate concentration on
the activity o catalase. 4 Predict whether the enzyme activity will
Catalase is one o the most widespread enzymes. change more i substrate concentration is
It catalyses the conversion o hydrogen peroxide,
a toxic by-product o metabolism, into water and increased by 0.2 mol dm-3 or i it is decreased
oxygen. The apparatus shown in fgure 1 0 can be
used to investigate the activity o catalase in yeast. by the same amount. [2]
The experiment could be repeated using the same 5 Explain why tissues such as liver must be
concentration o yeast, but dierent hydrogen
peroxide concentrations. Another possible macerated beore investigating catalase
investigation would be to assess the catalase
concentrations in other cell types, such as liver, activity in them. [2]
kidney or germinating seeds. These tissues would
have to be macerated and then mixed with water Safety goggles must be worn if this experiment
at the same concentration as the yeast. is performed. Care should be taken not to get
hydrogen peroxide on the skin.
oxygen
yeast measuring cylinder
1 Describe how the activity o the enzyme three-way tap water
catalase could be measured using the
apparatus shown in fgure 1 0. [2]
2 Explain why a yeast suspension must always
be thoroughly stirred beore a sample o it is 0.8 mol dm23 water
hydrogen peroxide
taken or use in an experiment. [2]
3 State two actors, apart rom enzyme
concentration, that should be kept Figure 10 Apparatus for measuring catalase activity
Figure 11 Enzyme experiment
102
2.5 enzyMes
data-ba qutio: Designing an experiment to fnd the eect o temperature on lipase.
Lipase converts fats into fatty acids and glycerol. It 2 a) Explain how you would measure the
therefore causes a decrease in pH. This pH change dependent variable accurately. [2]
[1 ]
can be used to measure the activity of lipase. b) State the units for measuring the
Figure 1 2 shows suitable apparatus. dependent variable.
tube contents mixed when both c) Explain the need for at least three [2]
have reached target temperature replicate results for each temperature [3]
in this experiment.
thermometer 3
a) List the control factors that must be
kept constant in this experiment.
b) Explain how these control factors can
be kept constant. [2]
c) Suggest a suitable level for each [3]
control factor.
4 Suggest reasons for:
a) milk being used to provide a source of
thermostatically lipase milk mixed with lipids in this experiment rather than
controlled sodium carbonate (an alkali)
water bath and phenolphthalein vegetable oil. [1 ]
(a pH indicator)
b) the thermometer being placed in the
Figure 12 Apparatus for investigating the activity of lipase tube containing the larger, rather than
Phenolphthalein is pink in alkaline conditions, the smaller, volume of liquid [1 ]
but becomes colourless when the pH drops to c) the substrate being added to the
7. The time taken for this colour change can be enzyme, rather than the enzyme to
used to measure the activity of lipase at different the substrate. [1 ]
temperatures. Alternatively, pH changes could 5 Sketch the shape of graph that you would
be followed using a pH probe and data-logging expect from this experiment, with a
software. temperature range from 0 C to 80 C on
1 a) State the independent variable in this the x-axis and time taken for the indicator
experiment and how you would vary it. [2] to change colour on the y-axis. [2]
b) State the units for measuring the 6 Explain whether lipase from human pancreas
independent variable.
[1 ] or from germinating castor oil seeds would
c) State an appropriate range for the
independent variable. be expected to have the higher optimum
[2] temperature. [2]
Immobilized enzymes
Immobilized enzymes are widely used in industry.
In 1 897 the Buchner brothers, Hans and Eduard, showed that an
extract of yeast, containing no yeast cells, would convert sucrose into
alcohol. The door was opened to the use of enzymes to catalyse chemical
processes outside living cells.
Louis Pasteur had claimed that fermentation of sugars to alcohol could
only occur if living cells were present. This was part of the theory of
103
2 MOLECULAR BIOLOGY
toK vitalism, which stated that substances in animals and plants can only
be made under the infuence o a vital spirit or vital orce. The
Wha is he diference beween articial synthesis o urea, described in sub-topic 2.1 , had provided
dgma and hery? evidence against vitalism, but the Buchners research provided a clearer
alsication o the theory.
Ater the discovery in the 19th century
o the conversion o sugar into alcohol More than 500 enzymes now have commercial uses. Figure 1 3 shows a
by yeast, a dispute developed between classication o commercially useul enzymes. Some enzymes are used in
two scientists, Justus von Liebig and more than one type o industry.
Louis Pasteur. In 1860 Pasteur argued
that this process, called ermentation, miscellaneous 4% other industries 5%
could not occur unless live yeast cells agriculture 11%
were present. Liebig claimed that
the process was chemical and that medical 21% biosensor 16%
living cells were not needed. Pasteurs food & nutrition 23%
view refected the vitalistic dogma
that the substances in animals and biotechnology 46%
plants could only be made under the
infuence o a vital spirit or vital environment 13%
orce. These contrasting views were
as much infuenced by political and energy 3%
religious actors as by scientic
evidence. The dispute was only Figure 13
resolved ater the death o both men.
In 1897 the Buchner brothers, Hans The enzymes used in industry are usually immobilized. This is
and Eduard, showed that an extract o attachment o the enzymes to another material or into aggregations,
yeast, containing no yeast cells, did so that movement o the enzyme is restricted. There are many ways o
indeed convert sucrose into alcohol. doing this, including attaching the enzymes to a glass surace, trapping
The vitalistic dogma was overthrown them in an alginate gel, or bonding them together to orm enzyme
and the door was opened to the use aggregates o up to 0. 1 mm diameter.
o enzymes to catalyse chemical
processes outside living cells. Enzyme immobilization has several advantages.
The enzyme can easily be separated rom the products o the
reaction, stopping the reaction at the ideal time and preventing
contamination o the products.
Ater being retrieved rom the reaction mixture the enzyme may be
recycled, giving useul cost savings, especially as many enzymes are
very expensive.
Immobilization increases the stability o enzymes to changes in
temperature and pH, reducing the rate at which they are degraded
and have to be replaced.
Substrates can be exposed to higher enzyme concentrations than
with dissolved enzymes, speeding up reaction rates.
104
2.6 structure of dna and rna
lctose-free mik
Methods o production o lactose-ree milk and its advantages.
Lactose is the sugar that is naturally present in milk. Lactose tends to crystallize during the
It can be converted into glucose and galactose by the production o ice cream, giving a gritty
enzyme lactase: lactose glucose + galactose. texture. Because glucose and galactose
Lactase is obtained rom Kluveromyces lactis, are more soluble than lactose they remain
a type o yeast that grows naturally in milk. dissolved, giving a smoother texture.
Biotechnology companies culture the yeast, Bacteria erment glucose and galactose more
extract the lactase rom the yeast and puriy quickly than lactose, so the production o
it or sale to ood manuacturing companies. yoghurt and cottage cheese is aster.
There are several reasons or using lactase in Th a i l a n d
ood processing: South India
Some people are lactose-intolerant and cannot Crete
drink more than about 2 5 0 ml o milk per day, Fra n ce
unless it is lactose-reduced (see fgure 1 4) . Fi n l a n d
Galactose and glucose are sweeter than Sweden
lactose, so less sugar needs to be added to 0% 50% 100%
sweet oods containing milk, such as milk lactose intolerance
shakes or ruit yoghurt. Figure 14 Rates of lactose intolerance
2.6 s dna rna
understnding appictions
The nucleic acids DNA and RNA are polymers o Crick and Watsons elucidation o the structure
nucleotides. o DNA using model-making.
DNA difers rom RNA in the number o strands Skis
normally present, the base composition and
the type o pentose. Drawing simple diagrams o the structure o
single nucleotides and o DNA and RNA, using
DNA is a double helix made o two antiparallel circles, pentagons and rectangles to represent
strands o nucleotides linked by hydrogen phosphates, pentoses and bases.
bonding between complementary base pairs.
Ntre of science
Using models as representation o the real
world: Crick and Watson used model-making to
discover the structure o DNA.
105
2 MOLECULAR BIOLOGY
phosphate sugar base Nucleic cids nd nucleotides
O N
5CH2 1 The nucleic acids DNA and RNA are polymers o
O P O O nucleotides.
O C
C Nucleic acids were frst discovered in material extracted rom the nuclei
o cells, hence their name. There are two types o nucleic acid: DNA
4 and RNA. Nucleic acids are very large molecules that are constructed by
linking together nucleotides to orm a polymer.
CC
Nucleotides consist o three parts:
32
a sugar, which has fve carbon atoms, so is a pentose sugar;
OH OH
a phosphate group, which is the acidic, negatively-charged part o
Figure 1 The parts of a nucleotide nucleic acids; and
Figure 2 A simpler representation of a a base that contains nitrogen and has either one or two rings o
nucleotide atoms in its structure.
HOH2C O OH Figure 1 shows these parts and how they are linked together. The base
HH and the phosphate are both linked by covalent bonds to the pentose
sugar. Figure 2 shows a nucleotide in symbolic orm.
H H
OH H To link nucleotides together into a chain or polymer, covalent bonds are
ormed between the phosphate o one nucleotide and the pentose sugar
HOH2C O OH o the next nucleotide. This creates a strong backbone or the molecule o
alternating sugar and phosphate groups, with a base linked to each sugar.
HH
There are our dierent bases in both DNA and RNA, so there are our
HH dierent nucleotides. The our dierent nucleotides can be linked
together in any sequence, because the phosphate and sugar used to link
OH OH them are the same in every nucleotide. Any base sequence is thereore
possible along a DNA or RNA molecule. This is the key to nucleic acids
Figure 3 The sugar within DNA is acting as a store o genetic inormation the base sequence is the store
deoxyribose (top) and the sugar in o inormation and the sugar phosphate backbone ensures that the store
RNA is ribose (bottom) is stable and secure.
Difeences between DNa nd rNa
DNA difers rom RNA in the number o strands normally
present, the base composition and the type o pentose.
There are three important dierences between the two types o nucleic
acid:
1 The sugar within DNA is deoxyribose and the sugar in RNA is ribose.
Figure 3 shows that deoxyribose has one ewer oxygen atom than
ribose. The ull names o DNA and RNA are based on the type o
sugar in them deoxyribonucleic acid and ribonucleic acid.
2 There are usually two polymers o nucleotides in DNA but only one
in RNA. The polymers are oten reerred to as strands, so DNA is
double-stranded and RNA is single-stranded.
3 The our bases in DNA are adenine, cytosine, guanine and
thymine. The our bases in RNA are adenine, cytosine, guanine
and uracil, so the dierence is that uracil is present instead o
thymine in RNA.
106
2.6 structure of dna and rna
d-b qi: Chargafs data
DNA samples from a range of species were 3 Evaluate the claim that in the DNA of
analysed in terms of their nucleotide composition eukaryotes and prokaryotes the amount
by Edwin Chargaff, an Austrian biochemist, and of adenine and thymine are equal and
by others. The data is presented in table 1 . the amounts of guanine and cytosine
are equal.
1 Compare the base composition of [2]
Mycobacterium tuberculosis ( a prokaryote) 4 Explain the ratios between the amounts
with the base composition of the eukaryotes of bases in eukaryotes and prokaryotes in
shown in the table. [2] terms of the structure of DNA. [2]
2 Calculate the base ratio A+ G/T + C, for 5 Suggest reasons for the difference in the
humans and for Mycobacterium tuberculosis. base composition of bacteriophage T2 and
Show your working. [2] the polio virus. [2]
s dna Gp ai Gi cyi thymi
31.0 19.1 18.4 31.5
Human Mammal 28.7 22.2 22.0 27.2
29.7 20.8 20.4 29.1
Cattle Mammal 32.8 17.7 17.4 32.1
27.3 22.7 22.8 27.1
Salmon Fish 31.3 18.7 17.1 32.9
15.1 34.9 35.4 14.6
Sea urchin I n ve rte b ra te 32.6 18.2 16.6 32.6
30.4 25.4 19.5 0.0
Wheat Plant
Yeast Fungus
Mycobacterium tuberculosis Bacterium
Bacteriophage T2 Virus
Polio virus Virus
Table 1
Dwing DNa nd rNa molecules
Drawing simple diagrams of the structure of single
nucleotides and of DNA and RNA, using circles,
pentagons and rectangles to represent phosphates,
pentoses and bases.
The structure of DNA and RNA molecules can be shown in diagrams
using simple symbols for the subunits:
circles for phosphates;
pentagons for pentose sugar;
rectangles for bases.
Figure 2 shows the structure of a nucleotide, using these symbols. The
base and the phosphate are linked to the pentose sugar. The base is
linked to C1 the carbon atom on the right hand side of the pentose
sugar. The phosphate is linked to C 5 the carbon atom on the side
Figure 4 Simplifed diagram o RNA
107
2 MOLECULAR BIOLOGY
covalent bond P chain on the upper let side o the pentose sugar. The positions o
P these carbon atoms are shown in fgure 1 .
S AT S To show the structure o RNA, draw a polymer o nucleotides, with a
line to show the covalent bond linking the phosphate group o each
PP nucleotide to the pentose in the next nucleotide. The phosphate is
linked to C3 o the pentose the carbon atom that is on the lower let.
S CG S
I you have drawn the structure o RNA correctly, the two ends o
PP the polymer will be dierent. They are reerred to as the 3 and the 5
terminals.
S S
TA The phosphate o another nucleotide could be linked to the C3
atom o the 3 terminal.
PP
The pentose o another nucleotide could be linked to the
S GC S phosphate o the 5 terminal.
PP To show the structure o DNA, draw a strand o nucleotides, as with
Hydrogen bonds are formed RNA, then a second strand alongside the frst. The second strand
between two bases should be run in the opposite direction, so that at each end o the DNA
molecule, one strand has a C3 terminal and the other a C5 terminal. The
Key: S sugar P phosphate two strands are linked by hydrogen bonds between the bases. Add letters
or names to indicate the bases. Adenine (A) only pairs with thymine (T)
A C nitrogenous bases and cytosine (C) only pairs with guanine (G) .
TG
Figure 5 Simplifed diagram o DNA
5 end Structure of DNa
3 end DNA is a double helix made of two antiparallel strands
of nucleotides linked by hydrogen bonding between
S complementary complementary base pairs.
PP base pairs
Drawings o the structure o DNA on paper cannot show all eatures o
ST A S the three-dimensional structure o the molecule. Figure 6 represents
some o these eatures.
SG P hydrogen
C S bonds Each strand consists o a chain o nucleotides linked by covalent bonds.
P
S P The two strands are parallel but run in opposite directions so they are
CG S said to be antiparallel. One strand is oriented in the direction 5 to 3
and the other is oriented in the direction 3 to 5 .
P
S T AS The two strands are wound together to orm a double helix.
PP The strands are held together by hydrogen bonds between the
nitrogenous bases. Adenine (A) is always paired with thymine
S (T) and guanine (G) with cytosine (C) . This is reerred to as
PP complementary base pairing, meaning that A and T complement
SC GS each other by orming base pairs and similarly G and C complement
each other by orming base pairs.
P AS
ST
PP
S GCS
P
S T AS
PP sugarphosphate
S backbone
PP
SC G S
P
SGC S 3 end
P
5 end
Figure 6 The double helix
108
2.6 structure of dna and rna
d-b qi: The bases in DNA
Look at the molecular models in fgure 7 and 3 Identiy three similarities between adenine
answer the ollowing questions.
and guanine. [3]
1 State one dierence between adenine and 4 Compare the structure o cytosine and
thymine.
the other bases. [1 ] [4]
2 Each o the bases in DNA has a nitrogen 5 Although the bases have some shared
atom bonded to a hydrogen atom in a eatures, each one has a distinctive chemical
similar position, which appears in the lower structure and shape. Remembering the
let in each case in fgure 7. Deduce how unction o DNA, explain the importance or
this nitrogen is used when a nucleotide is the bases each to be distinctive. [5]
being assembled rom its subunits. [2]
Guanine Adenine Cytosine Thymine
Figure 7 109
Molecular models
Using models as representation of the real world:
Crick and Watson used model-making to discover the
structure of DNA.
The word model in English is derived rom the Latin word modus,
meaning manner or method. Models were originally architects
plans, showing how a new building might be constructed. Three-
dimensional models were then developed to give a more realistic
impression o what a proposed building would be like.
Molecular models also show a possible structure in three dimensions,
but whereas architects models are used to decide whether a building
should become reality in the uture, molecular models help us to
discover what the structure o a molecule actually is.
Models in science are not always three-dimensional and do not
always propose structures. They can be theoretical concepts and
they can represent systems or processes. The common eature o
models is that they are proposals, which are made to be tested. As
with architecture, models in science are oten rejected and replaced.
Model-making played a critical part in C rick and Watsons discovery
o the structure o DNA, but it took two attempts beore they were
successul.
2 MOLECULAR BIOLOGY
toK crik nd Wtsons models of DNa struture
Wha is he relaive rle Crick and Watsons discovery o the structure o DNA
cmpeiin and cperain in using model-making.
scienifc research?
C rick and Watsons success in discovering the structure o D NA was
Three prominent research groups based on using the evidence to develop possible structures or DNA
openly competed to elucidate the and testing them by model-building. Their rst model consisted o a
structure o DNA: Watson and Crick triple helix, with bases on the outside o the molecule and magnesium
were working at Cambridge; Maurice holding the two strands together with ionic bonds to the phosphate
Wilkins and Rosalind Franklin were groups on each strand. The helical structure and the spacing between
working at Kings College o the subunits in the helix tted the X-ray diraction pattern obtained by
University o London; and Linus Rosalind Franklin.
Pauling's research group was operating
out o Caltech in the United States. It was dicult to get all parts o this model to t together satisactorily
and it was rejected when Franklin pointed out that there would not
A stereotype o scientists is that they be enough magnesium available to orm the cross links between the
take a dispassionate approach to strands. Another deciency o this rst model was that is that it did
investigation. The truth is that science is not take account o Chargas nding that the amount o adenine
a social endeavour involving a number equals the thymine and the amount o cytosine equals the amount
o emotion-infuenced interactions o guanine.
between science. In addition to the
joy o discovery, scientists seek the To investigate the relationship between the bases in D NA pieces o
esteem o their community. Within cardboard were cut out to represent their shapes. These showed that
research groups, collaboration is A-T and C-G base pairs could be ormed, with hydrogen bonds linking
important, but outside o their research the bases. The base pairs were equal in length so would t between
group competition oten restricts open two outer sugar-phosphate backbones.
communication that might accelerate
the pace o scientic discovery. On the Another fash o insight was needed to make the parts o the
other hand, competition may motivate molecule t together: the two strands in the helix had to run in
ambitious scientists to work tirelessly. opposite directions they must be antiparallel. C rick and Watson
were then able to build their second model o the structure o
DNA. They used metal rods and sheeting cut to shape and held
together with small clamps. Bond lengths were all to scale and bond
angles correct. Figure 8 shows C rick and Watson with the newly
constructed model.
The model convinced all those who saw it. A typical comment was It
just looked right. The structure immediately suggested a mechanism
or copying DNA. It also led quickly to the realization that the genetic
code must consist o triplets o bases. In many ways the discovery o
DNA structure started the great molecular biology revolution, with
eects that are still reverberating in science and in society.
Figure 8 Crick and Watson and their DNA model
110
2.7 dn a rePlication , transcriPtion and translation
2.7 dna p, p
understnding applictions
The replication o DNA is semi-conservative and Use o Taq DNA polymerase to produce multiple
depends on complementary base pairing. copies o DNA rapidly by the polymerase chain
reaction (PCR) .
Helicase unwinds the double helix and
separates the two strands by breaking Production o human insulin in bacteria as an
hydrogen bonds. example o the universality o the genetic code
allowing gene transer between species.
DNA polymerase links nucleotides together to
orm a new strand, using the pre-existing strand Skills
as a template.
Use a table o the genetic code to deduce which
Transcription is the synthesis o mRNA codon(s) corresponds to which amino acid.
copied rom the DNA base sequences by RNA
polymerase. Analysis o Meselson and Stahls results
to obtain support or the theory o semi-
Translation is synthesis o polypeptides on conservative replication o DNA.
ribosomes.
Use a table o mRNA codons and their
The amino acid sequence o polypeptides is corresponding amino acids to deduce the
determined by mRNA according to the genetic sequence o amino acids coded by a short
code. mRNA strand o known base sequence.
Codons o three bases on mRNA correspond to Deducing the DNA base sequence or the
one amino acid in a polypeptide. mRNA strand.
Translation depends on complementary Ntre of science
base pairing between codons on mRNA and
anticodons on tRNA. Obtaining evidence or scientifc theories:
Meselson and Stahl obtained evidence or the
semi-conservative replication o DNA.
Semi-conservtive repliction of DNa 111
The replication o DNA is semi-conservative and depends
on complementary base pairing.
When a cell prepares to divide, the two strands o the double helix
separate (see fgure 2) . Each o these original strands serves as a guide,
or template, or the creation o a new strand. The new strands are
ormed by adding nucleotides, one by one, and linking them together.
The result is two DNA molecules, both composed o an original strand
and a newly synthesized strand. For this reason, DNA replication is
reerred to as being semi-conservative.
2 MOLECULAR BIOLOGY
adenine thymine The base sequence on the template strand determines the base sequence on
cytosine guanine the new strand. Only a nucleotide carrying a base that is complementary to
cytosine the next base on the template strand can successully be added to the new
guanine strand (fgure 1 ) .
This is because complementary bases orm hydrogen bonds with each
other, stabilizing the structure. I a nucleotide with the wrong base started
to be inserted, hydrogen bonding between bases would not occur and the
nucleotide would not be added to the chain. The rule that one base always
pairs with another is called complementary base pairing. It ensures
that the two DNA molecules that result rom DNA replication are identical
in their base sequences to the parent molecule that was replicated.
thymine adenine obtaining evidence fr the thery f semi-
Figure 1 cnservative replicatin
Parental DNA Obtaining evidence or scientifc theories: Meselson
and Stahl obtained evidence or the semi-conservative
GC replication o DNA.
CG
CG Semi-conservative replication is an example o a scientifc theory that
AT seemed intuitively right, but nonetheless needed to be backed up
with evidence. Laboratories around the world attempted to confrm
GC experimentally that replication o DNA is semi-conservative and soon
TA convincing evidence had been obtained.
TA
CG In 1 958 Matthew Meselson and Franklin Stahl published the results
o exceedingly elegant experiments that provided very strong
Replication fork evidence or semi-conservative replication. They used 15N, a rare
isotope o nitrogen that has one more neutron than the normal
AT 14N isotope, so is denser. In the 1 9 3 0s Harold Urey had developed
methods o puriying stable isotopes that could be used as tracers in
G C biochemical pathways. 15N was one o these.
T
A C Meselson and Stahl devised a new method o separating DNA
TA containing 15N in its bases rom DNA with 14N. The technique is
GC called caesium chloride density gradient centriugation. A solution
TA TA o caesium chloride is spun in an ultracentriuge at nearly 45,000
TA CG revolutions per minute or 20 hours. The dense caesium ions tend
C to move towards the bottom o the tube but do not sediment ully
C because o diusion. A gradient is established, with the greatest
caesium concentration, and thereore density, at the bottom and
G CG the lowest at the top o the tube. Any substance centriuged with
A TA the caesium chloride solution becomes concentrated at a level
AT AT corresponding with its density.
AT AT
Meselson and S tahl cultured the bacterium E. coli or ourteen
GC GC generations in a medium where the only nitrogen source was 15N.
AT AT Almost all nitrogen atoms in the bases o the DNA in the bacteria
TA TA were thereore 15N. They then transerred the bacteria abruptly to a
G GC medium in which all the nitrogen was 14N. At the temperature used
to culture them, the generation time was 50 minutes the bacteria
Parental New New Parental divided and thereore replicated their DNA once every 50 minutes.
strand strand strand strand
Figure 2 Semi-conservative replication
112
2.7 dn a rePlication , transcriPtion and translation
Meselson and Stahl collected samples o DNA rom the bacterial avy
culture or several hours rom the time when it was transerred to
the 14N medium. They extracted the D NA and measured its density nw xpm hqu
by caesium chloride density gradient centriugation. The DNA
could be detected because it absorbs ultraviolet light, and so Meselson and Stahl used three
created a dark band when the tubes were illuminated with techniques in their experiments
ultraviolet. Figure 3 shows the results. In the next part o this that that were relatively new.
sub-topic there is guidance in how to analyse the changes in Identiy a technique used by
position o the dark bands. them that was developed:
a) by Urey in the 1930s
b) by Pickels in the 1940s
c) by Meselson and Stahl
themselves in the 1950s.
0 0.3 0.7 1.0 1.5 2.0 2.5 3.0 4.0 avy
generations Mg h vy
Figure 3 To model helicase activity you
could use some two-stranded rope
Meselson nd Sthls DNa repliction or string and a split key ring. The
experiments strands in the rope are helical and
represent the two strands in DNA.
Analysis o Meselson and Stahls results to obtain support Open the key ring and put one
or the theory o semi-conservative replication o DNA. strand othe rope inside it. Close
the ring so that the other strand
The data-based question below will guide you through the analysis o is outside. Slide the ring along the
Meselson and Stahls results and help to build your skills in this aspect string to separate the strands.
o science. What problems are revealed by this
model othe activity ohelicase?
Use the internet to fnd the solution
used by living organisms.
d-b qu: The Meselson and Stahl experiment
In order or cell division to occur, DNA must be to a 14N medium. Samples o the bacteria were
duplicated to ensure that progeny cells have the taken over a period o time and separated by
same genetic inormation as the parent cells. The density gradient centriugation, a method in
process o duplicating DNA is termed replication. which heavier molecules settle urther down
The MeselsonStahl experiment sought to in acentriuge tube than lighter ones.
understand the mechanism o replication. Did it
occur in a conservative ashion, a semi-conservative 1 The single band o D NA at the start
ashion or in a dispersive ashion (see fgure 4) ? (0 generations) had a density o 1 .724 g cm-3.
The main band o DNA ater our generations
Meselson and Stahl grew E. coli in a medium had a density o 1 .71 0 g cm-3. Explain how
containing heavy nitrogen (15N) or a number DNA with a lower density had been produced
o generations. They then transerred the bacteria by the bacteria. [2]
113
2 MOLECULAR BIOLOGY
2 a) Estimate the density o the DNA ater one 6 Predict the results o centriuging a
mixture o DNA rom 0 generations and
generation. [2] 2 generations.
b) Explain whether the density o DNA ater [2]
one generation alsifes any o the three
possible mechanisms or DNA replication
shown in fgure 4. [3]
3 a) Describe the results ater two generations,
including the density o the DNA. [3]
b) Explain whether the results ater [3]
two generations alsiy any o the
three possible mechanisms or DNA
replication.
4 Explain the results ater three and our [2]
generations.
5 Figure 4 shows D NA rom E. coli at the start
(0 generations) and ater one generation,
with strands o DNA containing 15N shown
red and strands containing 14N shown green.
Redraw either (a) , (b) or (c) , choosing the
mechanism that is supported by Meselson
and Stahls experiment. Each DNA molecule Dispersive Conservative Semi-conservative
can be shown as two parallel lines rather Newly synthesized strand
Original template strand
than a helix and the colours do not have to
Figure 4 Three possible mechanisms for
be red and green. Draw the DNA or two DNA replication
more generations o replication in a medium
containing 14N. [3]
Helicase
Helicase unwinds the double helix and separates the two
strands by breaking hydrogen bonds.
B eore D NA replication can occur, the two strands o the molecule
must separate so that they can each act as a template or the ormation
o a new strand. The separation is carried out by helicases, a group o
enzymes that use energy rom ATP. The energy is required or breaking
hydrogen bonds between complementary bases.
One well-studied helicase consists o six globular polypeptides arranged
in a donut shape. The polypeptides assemble with one strand o the DNA
molecule passing through the centre o the donut and the other outside
it. Energy rom ATP is used to move the helicase along the DNA molecule,
breaking the hydrogen bonds between bases and parting the two stands.
Double-stranded DNA cannot be split into two strands while it is still
helical. Helicase thereore causes the unwinding o the helix at the same
time as it separates the strands.
114
2.7 dn a rePlication , transcriPtion and translation
DNa polymese
DNA polymerase links nucleotides together to form a new
strand, using the pre-existing strand as a template.
Once helicase has unwound the double helix and split the DNA into two
strands, replication can begin. Each o the two strands acts as a template
or the ormation o a new strand. The assembly o the new strands is
carried out by the enzyme DNA polymerase.
DNA polymerase always moves along the template strand in the same
direction, adding one nucleotide at a time. Free nucleotides with each
o the our possible bases are available in the area where DNA is being
replicated. Each time a nucleotide is added to the new strand, only
one o the our types o nucleotide has the base that can pair with the
base at the position reached on the template strand. DNA polymerase
brings nucleotides into the position where hydrogen bonds could orm,
but unless this happens and a complementary base pair is ormed, the
nucleotide breaks away again.
Once a nucleotide with the correct base has been brought into position
and hydrogen bonds have been ormed between the two bases, DNA
polymerase links it to the end o the new strand. This is done by
making a covalent bond between the phosphate group o the ree
nucleotide and the sugar o the nucleotide at the existing end o the
new strand. The pentose sugar is the 3 terminal and the phosphate
group is the 5 terminal, so D NA polymerase adds on the 5 terminal o
the ree nucleotide to the 3 terminal o the existing strand.
DNA polymerase gradually moves along the template strand, assembling
the new strand with a base sequence complementary to the template
strand. It does this with a very high degree o fdelity very ew mistakes
are made during DNA replication.
Pcr the polymese hin etion
Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the
polymerase chain reaction (PCR) .
The polymerase chain reaction (PCR) is a o them so they hold the two strands together
technique used to make many copies o a selected successully at the temperatures normally
DNA sequence. Only a very small quantity o the encountered by most cells. I DNA is heated to a
DNA is needed at the start. The DNA is loaded high temperature, the hydrogen bonds eventually
into a PCR machine in which a cycle o steps break and the two strands separate. I the DNA
repeatedly doubles the quantity o the selected is then cooled hydrogen bonds can orm, so the
DNA. This involves double-stranded DNA being strands pair up again. This is called re-annealing.
separated into two single strands at one stage o
the cycle and single strands combining to orm The PCR machine separates DNA strands by heating
double-stranded DNA at another stage. them to 95 C or fteen seconds. It then cools
the DNA quickly to 54 C. This would allow re-
The two strands in DNA are held together by annealing o parent strands to orm double-stranded
hydrogen bonds. These are weak interactions, DNA. However, a large excess o short sections o
but in a DNA molecule there are large numbers single-stranded DNA called primers is present. The
115
2 MOLECULAR BIOLOGY
primers bind rapidly to target sequences and as a strands. It would work at the lower temperature
large excess o primers is present, they prevent the o 54 C that is used to attach the primers, but
re-annealing o the parent strands. Copying o the its optimum temperature is 72 C. The reaction
single parent strands then starts rom the primers. mixture is thereore heated to this temperature or
the period when Taq DNA polymerase is working.
The next stage in PCR is synthesis o double- At this temperature it adds about 1 ,000 nucleotides
stranded DNA, using the single strands with per minute, a very rapid rate o DNA replication.
primers as templates. The enzyme Taq DNA
polymerase is used to do this. It was obtained rom When enough time has elapsed or replication
a bacterium, Thermus aquaticus, ound in hot springs, o the selected base sequence to be complete,
including those o Yellowstone National Park. The the next cycle is started by heating to 95 C. A
temperatures o these springs range rom 50 C to cycle o PCR can be completed in less than two
80 C. Enzymes in most organisms would rapidly minutes. Thirty cycles, which ampliy the DNA
denature at such high temperatures, but those o by a actor o a billion, take less than an hour.
Thermus aquaticus, including its DNA polymerase, are With the help o Taq D NA polymerase, PC R allows
adapted to be very heat-stable to resist denaturation. the production o huge numbers o copies o a
selected base sequence in a very short time.
Taq DNA polymerase is used because it can resist
the brie period at 95 C used to separate the DNA
Select the DNA
sequence to be copied
Twice as many DNA Raise temperature
molecules can be copied
15 seconds to 95C to separate
in the next cycle the two strands
80 seconds Lower temperature
25 seconds abruptly to 54C to
Raise temperature to 72C to
allow rapid DNA replication by allow binding of
primers to DNA
Taq DNA polymerase
Figure 5
Figure 6
116
Transcription
Transcription is the synthesis of mRNA copied from the
DNA base sequences by RNA polymerase.
This sequence o bases in a gene does not, in itsel, give any observable
characteristic in an organism. The unction o most genes is to speciy the
sequence o amino acids in a particular polypeptide. It is proteins that
oten directly or indirectly determine the observable characteristics o an
individual. Two processes are needed to produce a specifc polypeptide,
using the base sequence o a gene. The frst o these is transcription.
Transcription is the synthesis o RNA, using D NA as a template. B ecause
RNA is single-stranded, transcription only occurs along one o the two
strands o DNA. What ollows is an outline o transcription:
The enzyme RNA polymerase binds to a site on the DNA at the start
o a gene.
2.7 dn a rePlication , transcriPtion and translation
RNA polymerase moves along the gene separating DNA into single
strands and pairing up RNA nucleotides with complementary bases
on one strand o the DNA. There is no thymine in RNA, so uracil
pairs in a complementary ashion with adenine.
RNA polymerase orms covalent bonds between the RNA nucleotides.
The RNA separates rom the DNA and the double helix reorms.
Transcription stops at the end o the gene and the completed RNA
molecule is released.
The product o transcription is a molecule o RNA with a base sequence
that is complementary to the template strand o DNA. This RNA has a
base sequence that is identical to the other strand, with one exception
there is uracil in place o thymine. So, to make an RNA copy o the
base sequence o one strand o a DNA molecule, the other strand is
transcribed. The DNA strand with the same base sequence as the RNA
is called the sense strand. The other strand that acts as the template
and has a complementary base sequence to both the RNA and the sense
strand is called the antisense strand.
direction of RNA polymerase
transcription free RNA nucleotides
antisense strand of DNA
5 3
3 5
RNA molecule
sense strand of DNA
Figure 7
Translation DNA TRAN S CRI PTI O N
Translation is synthesis of polypeptides on ribosomes. RNA TRAN S LATI O N
The second o the two processes needed to produce a specifc PO LYPEPTI D E
polypeptide is translation. Translation is the synthesis o a polypeptide,
with an amino acid sequence determined by the base sequence o a Figure 8
molecule o RNA. The production o RNA by transcription and how its
base sequence is determined by a gene was described in the previous
part o this sub-topic.
Translation takes place on cell structures in the cytoplasm known as
ribosomes. Ribosomes are complex structures that consist o a small and
a large subunit, with binding sites or each o the molecules that take part
in the translation. Figure 9 shows the two subunits o a ribosome. Each is
composed o RNA molecules (pink and yellow) and proteins (purple) . Part
o the large subunit (green) is the site that makes peptide bonds between
amino acids, to link them together into a polypeptide.
117
2 MOLECULAR BIOLOGY
Figure 9 Large and small subunits of the ribosome with proteins shown in purple, ribosomal
RNA in pink and yellow and the site that catalyses the formation of peptide bonds green
Messenge rNa nd the genetic code
The amino acid sequence of polypeptides is determined
by mRNA according to the genetic code.
RNA that carries the inormation needed to synthesize a polypeptide
is called messenger RNA, usually abbreviated to mRNA. The length o
mRNA molecules varies depending on the number o amino acids in the
polypeptide but an average length or mammals is about 2,000 nucleotides.
In the genome there are many dierent genes that carry the inormation
needed to make a polypeptide with a specifc amino acid sequence. At
any time a cell will only need to make some o these polypeptides. Only
certain genes are thereore transcribed and only certain types o mRNA
will be available or translation in the cytoplasm. Cells that need or
secrete large amounts o a particular polypeptide make many copies o
the mRNA or that polypeptide. For example, insulin-secreting cells in
the pancreas make many copies o the mRNA needed to make insulin.
Although most RNA is mRNA, there are other types; or example,
transer RNA is involved in decoding the base sequence o mRNA into an
amino acid sequence during translation and ribosomal RNA is part o the
structure o the ribosome. They are usually reerred to as tRNA and rRNA.
data-base questions: Interpreting electron micrographs
The electron micrographs in fgure 1 0 show show up more clearly. Identiy each o these
transcription, translation and DNA replication. structures:
1 Deduce, with reasons, which process is [5] a) the red structure in the central micrograph
occurring in each electron micrograph.
b) the thin blue molecule near the lower
2 The colour in the electron micrographs has edge o the right-hand micrograph
been added to make the dierent structures
118
2.7 dn a rePlication , transcriPtion and translation
c) the blue molecules o variable length e) the green molecules in the let-hand
attached to this thin blue molecule
micrograph. [5]
d) the red molecule in the let-hand micrograph
Figure 10
codons
Codons of three bases on mRNA correspond
to one amino acid in a polypeptide.
The translation dictionary that enables the f s p th
cellular machinery to convert the base sequence on p p
the mRNA into an amino acid sequence is called (5 ) u ca G (3 )
the genetic code. There are our dierent bases and U Phe U
twenty amino acids, so one base cannot code or Phe Ser Tyr Cys C
one amino acid. There are sixteen combinations o C Leu A
two bases, which is still too ew to code or all o Leu Ser Tyr Cys G
the twenty amino acids. Living organisms thereore Leu U
use a triplet code, with groups o three bases coding Leu Ser Stop Stop C
or an amino acid. Leu A
Leu Ser Stop Trp G
IIe U
IIe Pro His Arg C
IIe A
A sequence o three bases on the mRNA is called Met Pro His Arg G
a codon. Each codon codes or a specifc amino Val U
acid to be added to the polypeptide. Table 1 lists Val Pro Gln Arg C
all o the 64 possible codons. The three bases o an Val A
mRNA codon are designated in the table as frst, Val Pro Gln Arg G
second and third positions.
A Thr Asn Ser
Thr Asn Ser
Note that dierent codons can code or the same Thr Lys Arg
amino acid. For example the codons GUU and
GUC both code or the amino acid valine. For this Thr Lys Arg
reason, the code is said to be degenerate. Note
also that three codons are stop codons that code G Ala Asp Gly
or the end o translation.
Ala Asp Gly
Ala Glu Gly
Ala Glu Gly
Amino acids are carried on another kind o RNA, Table 1
called tRNA. Each amino acid is carried by a
specifc tRNA, which has a three-base anticodon
complementary to the mRNA codon or that
particular amino acid.
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2 MOLECULAR BIOLOGY
Deoding base sequenes
Use of a table of the genetic code to deduce which codon(s) corresponds to which
amino acid; use of a table of mRNA codons and their corresponding amino acids to
deduce the sequence of amino acids coded by a short mRNA strand of known base
sequence; deducing the DNA base sequence for the mRNA strand.
There is no need to try to memorize the genetic base sequence complementary to the mRNA. For
code, but i a table showing it is available, you example, the codon AUG in mRNA is transcribed
should be able to make various deductions. rom the base sequence TAC on the antisense
strand o the DNA. A longer example is that
1 Which codons correspond to an amino acid? the base sequence GUACGUACG is transcribed
rom C ATGC ATGC . Note that adenine pairs with
Three letters are used to indicate each amino acid in thymine in DNA but with uracil in RNA.
the table o the genetic code. Each o the 20 amino
acids has between one and six codons. Read o the Questions
three letters o each codon or the amino acid. For
example, the amino acid methionine, shown as 1 Deduce the codons or
Met on the table, has one codon which is AUG.
a) Tryptophan ( Trp)
2 What amino acid sequence would be b) Tyrosine ( Tyr) [3]
translated from a sequence of codons in a c) Arginine (Arg)
strand of mRNA?
The frst three bases in the mRNA sequence are the 2 Deduce the amino acid sequences that [3]
codon or the frst amino acid, the next three bases correspond to these mRNA sequences:
are the codon or the second base and so on. Look a) ACG b) CACGGG c) CGCGCGAGG [3]
down the let hand side o the table to fnd the frst 3 I mRNA contains the base sequence
base o a codon, across the top o the table to fnd the C UC AUC GAAUAAC C C
second base and down the right hand side to fnd the
third base. For example, GCA codes or the amino a) deduce the amino acid sequence o
acid alanine, which is abbreviated to Ala in the table. the polypeptide translated rom the
3 What base sequence in DNA would be mRNA [2]
transcribed to give the base sequence of a b) deduce the base sequence o the
strand of mRNA? antisense strand transcribed to produce
A strand o mRNA is produced by transcribing the the mRNA. [2]
anti-sense strand o the DNA. This thereore has a
codons and antiodons
Translation depends on complementary base pairing
between codons on mRNA and anticodons on tRNA.
Three components work together to synthesize polypeptides by translation:
mRNA has a sequence o codons that specifes the amino acid
sequence o the polypeptide;
tRNA molecules have an anticodon o three bases that binds to a
complementary codon on mRNA and they carry the amino acid
corresponding to that codon;
ribosomes act as the binding site or mRNA and tRNAs and also
catalyse the assembly o the polypeptide.
120
2.7 dn a rePlication , transcriPtion and translation
A summary o the main events o translation ollows:
1 An mRNA binds to the small subunit o the ribosome.
2 A molecule o tRNA with an anticodon complementary to the frst
codon to be translated on the mRNA binds to the ribosome.
3 A second tRNA with an anticodon complementary to the second
codon on the mRNA then binds. A maximum o two tRNAs can be
bound at the same time.
4 The ribosome transers the amino acid carried by the frst tRNA to the
amino acid on the second tRNA, by making a new peptide bond. The
second tRNA is then carrying a chain o two amino acids a dipeptide.
5 The ribosome moves along the mRNA so the frst tRNA is released,
the second becomes the frst.
6 Another tRNA binds with an anticodon complementary to the next
codon on the mRNA.
7 The ribosome transers the chain o amino acids carried by the frst
tRNA to the amino acid on the second tRNA, by making a new
peptide bond.
Stages 4, 5 and 6 are repeated again and again, with one amino acid
added to the chain each time the cycle is repeated. The process continues
along the mRNA until a stop codon is reached, when the completed
polypeptide is released.
The accuracy o translation depends on complementary base pairing
between the anticodon on each tRNA and the codon on mRNA.
Mistakes are very rare, so polypeptides with a sequence o hundreds o
amino acids are regularly made with every amino acid correct.
growing polypeptide chain amino acid
large sub unit of ribosome
tRNA
tRNA
mRNA
anticodon
Figure 11
Production of human insulin in bacteria
Production of human insulin in bacteria as an example of the universality of the
genetic code allowing gene transfer between species.
Diabetes in some individuals is due to destruction been widely used. Porcine insulin has only one
o cells in the pancreas that secrete the hormone dierence in amino acid sequence rom human
insulin. It can be treated by injecting insulin into insulin and bovine insulin has three dierences.
the blood. Porcine and bovine insulin, extracted Shark insulin, which has been used or treating
rom the pancreases o pigs and cattle, have both diabetics in Japan, has seventeen dierences.
121
2 MOLECULAR BIOLOGY
Despite the dierences in the amino acid sequence This may seem
between animal and human insulin, they all bind
to the human insulin receptor and cause lowering obvious, but it
o blood glucose concentration. However, some
diabetics develop an allergy to animal insulins, depends on each
so it is preerable to use human insulin. In 1 982
human insulin became commercially available or tRNA with a
the rst time. It was produced using genetically
modied E. coli bacteria. S ince then methods o particular anticodon
production have been developed using yeast cells
and more recently safower plants. having the same
Each o these species has been genetically amino acid attached
modied by transerring the gene or making
human insulin to it. This is done in such a way to it as in humans. In
that the gene is transcribed to produce mRNA and
the mRNA is translated to produce harvestable other words, E. coli,
quantities o insulin. The insulin produced has
exactly the same amino acid sequence as i the yeast and safower
gene was being transcribed and translated in
human cells. (a prokaryote, a
ungus and a plant)
all use the same
genetic code as
humans (an animal) .
It is ortunate or Figure 12
genetic engineers
that all organisms, with very ew exceptions, use
the same genetic code as it makes gene transer
possible between widely diering species.
2.8 cell respiration
undFiegurrse 1t2Tenxtdtoibnegadded. applictions
Cell respiration is the controlled release o Use o anaerobic cell respiration in yeasts to
energy rom organic compounds to produce produce ethanol and carbon dioxide in baking.
ATP.
Lactate production in humans when anaerobic
ATP rom cell respiration is immediately respiration is used to maximize the power o
available as a source o energy in the cell. muscle contractions.
Anaerobic cell respiration gives a small yield o
ATP rom glucose.
Aerobic cell respiration requires oxygen and
gives a large yield o ATP rom glucose.
Ntre of science Skills
Assessing the ethics o scientifc research: Analysis o results rom experiments involving
the use o invertebrates in respirometer measurement o respiration rates in germinating
experiments has ethical implications. seeds or invertebrates using a respirometer.
122
2.8 cell resPiration
relese of enegy by cell espition Figure 1 Breaking down 8 grams of glucose
in cell respiration provides enough energy to
Cell respiration is the controlled release of energy from sprint 100 metres
organic compounds to produce ATP.
Cell respiration is one o the unctions o lie that all living cells perorm.
O rganic compounds are broken down to release energy, which can then
be used in the cell. For example, energy is released in muscle fbres by
breaking down glucose into carbon dioxide and water. The energy can
then be used or muscle contraction.
In humans the source o the organic compounds broken down in cell
respiration is the ood that we eat. Carbohydrates and lipids are oten
used, but amino acids rom proteins may be used i we eat more protein
than needed. Plants use carbohydrates or lipids previously made by
photosynthesis.
Cell respiration is carried out using enzymes in a careul and controlled
way, so that as much as possible o the energy released is retained
in a usable orm. This orm is a chemical substance called adenosine
triphosphate, almost always abbreviated to ATP. To make ATP, a
phosphate group is linked to adenosine diphosphate, or AD P. E nergy
is required to carry out this reaction. The energy comes rom the
breakdown o organic compounds.
ATP is not transerred rom cell to cell and all cells require a continuous
supply. This is the reason or cell respiration being an essential unction
o lie in all cells.
aTP is souce of enegy cell respiration
ATP from cell respiration is immediately available as a ADP 1 ATP
source of energy in the cell. phosphate
C ells require energy or three main types o activity. active cell processes
Synthesizing large molecules like DNA, RNA and proteins. Figure 2
Pumping molecules or ions across membranes by active transport. Figure 3 Infra red photo of toucan
showing that it is warmer than its
Moving things around inside the cell, such as chromosomes, surroundings due to heat generated
vesicles, or in muscle cells the protein fbres that cause muscle by respiration. Excess heat is
contraction. dissipated by sending warm blood
to the beak
The energy or all o these processes is supplied by ATP. The
advantage o ATP as an energy supply is that the energy is
immediately available. It is released simply by splitting ATP into AD P
and phosphate. The ADP and phosphate can then be reconverted to
ATP by cell respiration.
When energy rom ATP is used in cells, it is ultimately all converted
to heat. Although heat energy may be useul to keep an organism
warm, it cannot be reused or cell activities and is eventually lost to the
environment. This is the reason or cells requiring a continual source o
ATP or cell activities.
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2 MOLECULAR BIOLOGY
anerobic respirtion
Anaerobic cell respiration gives a small yield of
ATP from glucose.
Glucose is broken down in anaerobic cell respiration without using any
oxygen. The yield o ATP is relatively small, but the ATP can be produced
quickly. Anaerobic cell respiration is thereore useul in three situations:
when a short but rapid burst o ATP production is needed;
when oxygen supplies run out in respiring cells;
in environments that are decient in oxygen, or example
waterlogged soils.
Figure 4 The mud in mangrove swamps is The products o anaerobic respiration are not the same in all organisms.
defcient in oxygen. Mangrove trees have In humans, glucose is converted to lactic acid, which is usually in a
evolved vertical roots called pneumatophores dissolved orm known as lactate. In yeast and plants glucose is converted
which they use to obtain oxygen rom the air to ethanol and carbon dioxide. Both lactate and ethanol are toxic in
excess, so must be removed rom the cells that produce them, or be
activity produced in strictly limited quantities.
does bioethnol solve or mke more Summary equations
problems?
There has been much debate about glucose lactate
bioethanol production. A renewable
fuel that cuts down on carbon AD P ATP
emissions is obviously desirable.
What are the arguments against This occurs in animals including humans.
bioethanol production?
glucose ethanol + carbon dioxide
AD P ATP
This occurs in yeasts and plants.
Yest nd its uses
Use of anaerobic cell respiration in yeasts to produce
ethanol and carbon dioxide in baking.
Yeast is a unicellular ungus that occurs naturally in habitats where
glucose or other sugars are available, such as the surace o ruits.
It can respire either aerobically or anaerobically. Anaerobic cell
respiration in yeast is the basis or production o oods, drinks and
renewable energy.
B read is made by adding water to four, kneading the mixture to make
dough and then baking it. Usually an ingredient is added to the dough
to create bubbles o gas, so that the baked bread has a lighter texture.
Yeast is oten this ingredient. Ater kneading, the dough is kept
warm to encourage the yeast to respire. Any oxygen in the dough is
soon used up so the yeast carries out anaerobic cell respiration. The
carbon dioxide produced by anaerobic cell respiration cannot escape
rom the dough and orms bubbles. The swelling o the dough due to
Figure 5
124
2.8 cell resPiration
the production o bubbles o carbon dioxide is called rising. Ethanol
is also produced by anaerobic cell respiration, but it evaporates
during baking.
Bioethanol is ethanol produced by living organisms, or use as
a renewable energy source. Although any plant matter can be
utilized as a eed stock and various living organisms can be used
to convert the plant matter into ethanol, most bioethanol is
produced rom sugar cane and corn ( maize) , using yeast. Yeast
converts sugars into ethanol in large ermenters by anaerobic
respiration. Only sugars can be converted, so starch and cellulose
must rst be broken down into sugars. This is done using enzymes.
The ethanol produced by the yeasts is puried by distillation
and various methods are then used to remove water rom it to
improve its combustion. Most bioethanol is used as a uel in
vehicles, sometimes in a pure state and sometimes mixed with
gasoline (petrol) .
Figure 6
d-b qu: Monitoring anaerobic cell respiration in yeast
The apparatus in gure 7 was used to monitor 2 Explain the loss o mass. [3]
mass changes during the brewing o wine. The
fask was placed on an electronic balance, which 3 Suggest two reasons or the increasing rate
was connected to a computer or data-logging. The
results are shown in gure 8. o mass loss rom the start o the experiment
until day 6. [2]
1 Calculate the total loss o mass during the 4 Suggest two reasons or the mass remaining
experiment and the mean daily loss. [3] constant rom day 1 1 onwards. [2]
airlock to electronic 560
prevent balance
entry connected 555
to a data-
of oxygen logging 550
computer
yeast in a mass / g545
solution of 0 1 2 3 4 5 6 7 8 9 10 11 12 13
sugar and time / days
nutrients Figure 8 Monitoring anaerobic cell respiration in yeast
555.00
Figure 7 Yeast data-logging apparatus
anerobic respirtion in humns
Lactate production in humans when anaerobic respiration is used to maximize the
power of muscle contractions.
The lungs and blood system supply oxygen to resort to anaerobic cell respiration in muscles. The
most organs o the body rapidly enough or reason is that anaerobic respiration can supply
aerobic respiration to be used, but sometimes we ATP very rapidly or a short period o time. It is
125
2 MOLECULAR BIOLOGY
thereore used when we need to maximize the Ater vigorous muscle contractions, the lactate
power o muscle contractions. must be broken down. This involves the use o
oxygen. It can take several minutes or enough
In our ancestors maximally powerul muscle oxygen to be absorbed or all lactate to be broken
contractions will have been needed or survival down. The demand or oxygen that builds up
by allowing escape rom a predator or catching o during a period o anaerobic respiration is called
prey during times o ood shortage. These events the oxygen debt.
rarely occur in our lives today. Instead anaerobic
respiration is more likely to be used during
training or sport. These are examples:
weight liters during the lit;
short-distance runners in races up to 400
metres;
long-distance runners, cyclists and rowers
during a sprint fnish.
Anaerobic cell respiration involves the production Figure 9 Short bursts of intense exercise are fuelled
o lactate, so when it is being used to supply ATP, by ATP from anaerobic cell respiration
the concentration o lactate in a muscle increases.
There is a limit to the concentration that the body
can tolerate and this limits how much anaerobic
respiration can be done. This is the reason or the
short timescale over which the power o muscle
contractions can be maximized. We can only sprint
or a short distance not more than 400 metres.
aerobic respirtion
Aerobic cell respiration requires oxygen and gives a large
yield of ATP from glucose.
I oxygen is available to a cell, glucose can be more ully broken down
to release a greater quantity o energy than in anaerobic cell respiration.
Whereas the yield o ATP is only two molecules per glucose with
anaerobic cell respiration, it is more than thirty per glucose with aerobic
cell respiration.
Aerobic cell respiration involves a series o chemical reactions. Carbon
dioxide and water are produced. In most organisms carbon dioxide is a
waste product that has to be excreted, but the water is oten useul. In
humans about hal a litre is produced per day.
glucose + oxygen carbon dioxide + water
AD P to ATP
Figure 10 The desert rat never needs to drink In eukaryotic cells most o the reactions o aerobic cell respiration,
despite only eating dry foods, because aerobic including all o the reactions that produce carbon dioxide, happen inside
cell respiration supplies its water needs the mitochondrion.
126
2.8 cell resPiration
respiometes
Analysis of results from experiments involving measurement of respiration rates in
germinating seeds or invertebrates using a respirometer.
A respirometer is any device that is used to in volume. I possible the temperature inside
measure respiration rate. There are many possible the respirometer should be controlled using a
designs. Most involve these parts: thermostatically controlled water bath.
A sealed glass or plastic container in which the Respirometers can be used to perorm various
organism or tissue is placed. experiments:
An alkali, such as potassium hydroxide, to the respiration rate o dierent organisms
absorb carbon dioxide. could be compared;
A capillary tube containing fuid, connected to the eect o temperature on respiration rate
the container. could be investigated;
One possible design o respirometer is shown respiration rates could be compared in active
in gure 1 1 , but it is possible to design simpler and inactive organisms.
versions that require only a syringe with a
capillary tube attached to it. The table below shows the results o an experiment
in which the eect o temperature on respiration in
I the respirometer is working correctly and germinating pea seeds was investigated.
the organisms inside are carrying out aerobic
cell respiration, the volume o air inside the To analyse these results you should rst check to
respirometer will reduce and the fuid in the see i the repeats at each temperature are close
capillary tube will move towards the container enough or you to decide that the results are reliable.
with the organisms. This is because oxygen is used You should then calculate mean results or each
up and carbon dioxide produced by aerobic cell temperature. The next stage is to plot a graph o the
respiration is absorbed by the alkali. mean results, with temperature on the horizontal
x-axis and the rate o movement o fuid on the
The position o the fuid should be recorded vertical y-axis. Range bars can be added to the
several times. I the rate o movement o the graph by plotting the lowest and highest result at
fuid is relatively even, the results are reliable. each temperature and joining them with a ruled
I the temperature inside the respirometer line. The graph will allow you to conclude what the
fuctuates, the results will not be reliable because relationship is between the temperature and the
an increase in air temperature causes an increase respiration rate o the germinating peas.
tmpu Mvm fud pm
(c) (mm m-1)
graduated 1 cm3 5 1 2d 3d
syringe 10 dg dg dg
15
wire basket containing 20 2.0 1.5 2.0
animal tissue 25
lter paper rolled 30 2.5 2.5 3.0
to form a wick
potassium hydroxide 3.5 4.0 4.0
solution
5.5 5.0 6.0
capillary tube 6.5 8.0 7.5
Figure 11 Diagram of a respirometer 11.5 11.0 9.5
127
2 MOLECULAR BIOLOGY
data-bas qustions: Oxygen consumption in tobacco hornworms
Tobacco hornworms are the larvae o Manduca sexta. b) Suggest reasons or the dierence in the
Adults o this species are moths. Larvae emerge
rom the eggs laid by the adult emale moths. There trends between the periods below and
are a series o larval stages called instars. Each
instar grows and then changes into the next one above the critical weight. [2]
by shedding its exoskeleton and developing a new
larger one. The exoskeleton includes the tracheal The researchers reared some tobacco hornworms
tubes that supply oxygen to the tissues. in air with reduced oxygen content. They ound
that the instar larvae moulted at a lower body mass
than larvae reared in normal air with 20% oxygen.
The graphs below (fgure 1 2) show measurements 3 Suggest a reason or earlier moulting in larvae
made using a simple respirometer o the respiration reared in air with reduced oxygen content. [2]
rate o 3rd, 4th and 5th instar larvae. Details o
the methods are given in the paper published by before critical weight after critical weight
the biologists who carried out the research. The 5th instar
reerence to the research is Callier V and Nijhout 0.16
H F (201 1 ) Control o body size by oxygen supply 0.12 0.14
reveals size-dependent and size-independent 0.10 0.12
mechanisms o molting and metamorphosis. 0.08 0.10
PNAS;1 08:1 46641 4669. This paper is reely 0.06 0.08
available on the internet at http://www.pnas.org/ 0.04
content/1 08/35/1 4664.ull.pd+html. 0.02 7 8 9 10 11 12 13
123456
Each data point on the graphs shows the body mass respiration rate (ml O2/min) 0.025 4th instar 0.032
and respiration rate o one larva. For each instar the 0.020 0.030
results have been divided into younger larvae with 0.028
low to intermediate body mass and older larvae 0.015 0.026
with intermediate to high body mass. The results 0.024
are plotted on separate graphs. The intermediate 0.010 0.022
body mass is reerred to as the critical weight. 0.020
0.005 0.018
0.20.30.40.50.60.70.80.9
1.0 1.1 1.2 1.3 1.4
0.007 3rd instar 0.009
0.006 0.008
1 a) Predict, using the data in the graphs, how 0.007
0.005 0.006
the respiration rate o a larva will change 0.005
0.004
as it grows rom moulting until it reaches 0.004
0.003 0.003
the critical weight. [1 ]
0.002 0.160.18
weight (g)
b) Explain the change in respiration rate that 0.001
you have described. [2] 0.000 000...110208
0.04 0.06 0.14
0000....22222640
2 a) Discuss the trends in respiration rate in weight (g)
larvae above the critical weight. [2] Figure 12 Respiration rates of tobacco hornworms (after
Callier and Nijhout, 2011)
ethics of animal us in rspiromtrs
Assessing the ethics o scientifc research: the use o invertebrates in
respirometer experiments has ethical implications.
It is important or all scientists to assess the we consider the consequences such as benefts
ethics o their research. There has been intense to students who are learning science? Do we
debate about the ethics o using animals in consider intentions? For example, i the animals
experiments. When discussing ethical issues, do are harmed unintentionally does that change
128
2.9 Photosynthesis
whether the experiment was ethical or not? Are 3 Can the risk o accidents that cause pain or
there absolute principles o right and wrong: or suering to the animals be minimized during
example, can we say that animals should never the experiment? In particular, can contact
be subject to conditions that are outside what with the alkali be prevented?
they would encounter in their natural habitat?
4 Is the use o animals in the experiment
Beore carrying out respirometer experiments essential or is there an alternative method that
involving animals these questions should avoids using animals?
be answered to help to decide whether the
experiments are ethically acceptable: It is particularly important to consider the ethics o
animal use in respirometer experiments because the
1 Is it acceptable to remove animals rom their International Baccalaureate Organization has issued
natural habitat or use in an experiment and a directive that laboratory or eld experiments and
can they be saely returned to their habitat? investigations need to be undertaken in an ethical
way. An important aspect o this is that experiments
2 Will the animals suer pain or any other harm should not be undertaken in schools that infict
during the experiment? pain or harm on humans or other living animals.
2.9 P
understnding applictions
Photosynthesis is the production o carbon Changes to the Earths atmosphere, oceans and
compounds in cells using light energy. rock deposition due to photosynthesis.
Visible light has a range o wavelengths with Skills
violet the shortest wavelength and red the
longest. Design o experiments to investigate limiting
actors on photosynthesis.
Chlorophyll absorbs red and blue light most
eectively and refects green light more than Separation o photosynthetic pigments by
other colours. chromatography.
Oxygen is produced in photosynthesis rom Drawing an absorption spectrum or chlorophyll
photolysis o water. and an action spectrum or photosynthesis.
Energy is needed to produce carbohydrates and Ntre of science
other carbon compounds rom carbon dioxide.
Experimental design: controlling relevant
Temperature, light intensity and carbon dioxide variables in photosynthesis experiments is
concentration are possible limiting actors on essential.
the rate o photosynthesis.
129
2 MOLECULAR BIOLOGY
What is photosynthesis?
Photosynthesis is the production of carbon compounds in
cells using light energy.
Living organisms require complex carbon compounds to build the
structure of their cells and to carry out life processes. Some organisms
are able to make all the carbon compounds that they need using only
light energy and simple inorganic substances such as carbon dioxide and
water. The process that does this is called photosynthesis.
Photosynthesis is an example of energy conversion, as light energy
is converted into chemical energy in carbon compounds. The carbon
compounds produced include carbohydrates, proteins and lipids.
Figure 2 The trees in one hectare of redwood
forest in California can have a biomass of more
than 4,000 tonnes, mostly carbon compounds
produced by photosynthesis
Figure 1 Leaves absorb carbon dioxide and light and use them in photosynthesis
Separating photosynthetic pigments by chromatography
Separation of photosynthetic pigments by chromatography. (Practical 4)
Chloroplasts contain several types of chlorophyll
and other pigments called accessory pigments.
Because these pigments absorb different ranges of
wavelength of light, they look a different colour to
us. Pigments can be separated by chromatography.
You may be familiar with paper chromatography
but thin layer chromatography gives better results.
This is done with a plastic strip that has been
coated with a thin layer of a porous material.
A spot containing pigments extracted from leaf
tissue is placed near one end of the strip. A
solvent is allowed to run up the strip, to separate
the different types of pigment.
1 Tear up a leaf into small pieces and put them Figure 3 Thin layer chromatography
in a mortar.
2 Add a small amount of sand for grinding.
130
2.9 Photosynthesis
3 Add a small volume o propanone (acetone) . Pgm clu f r
pgm f
4 Use the pestle to grind the lea tissue and Carotene orange
dissolve out the pigments. Chlorophyll a blue green 0.98
Chlorophyll b yellow green 0.59
5 I the propanone all evaporates, add a little more. Phaeophytin olive green 0.42
Xanthophyll 1 yellow 0.81
6 When the propanone has turned dark green, Xanthophyll 2 yellow 0.28
allow the sand and other solids to settle, then 0.15
pour the propanone o into a watch glass.
12 Mark the outside o the tube just below the
7 Use a hair drier to evaporate o all the level o the spot on the TLC strip.
propanone and water rom the cells cytoplasm.
1 3 Take the strip and cork out o the
8 When you have just a smear o dry pigments tube.
in the watch glass, add 34 drops o propanone
and use a paint brush to dissolve the pigments. 14 Pour running solvent into the specimen tube
up to the level that you marked.
9 Use the paint brush to transer a very small
amount o the pigment solution to the 15 Place the specimen tube on a lab bench
TLC strip. Your aim is to make a very small where it will not be disturbed. Careully
spot o pigment in the middle o the strip, lower the TLC strip and cork into the
1 0 millimetres rom one end. It should be very tube, so that the tube is sealed and the
dark. This is achieved by repeatedly putting a TLC strip is just dipping into the running
small drop onto the strip and then allowing it solvent. The solvent must NOT touch the
to dry beore adding another amount. You can pigment spot.
speed up drying by blowing on the spot or by
using the hair drier. 16 Leave the tube completely alone or about
fve minutes, to allow the solvent to run
10 When the spot is dark enough, slide the other up through the TLC strip. You can watch
end o the strip into the slot in a cork or bung the pigments separate, but DO NOT TOUCH
that fts into a tube that is wider than the TLC THE TUBE.
strip. The slot should hold the strip frmly.
11 Insert the cork and strip into a specimen tube.
The TLC strip should extend nearly to the
bottom o the tube, but not quite touch.
sp clu da r nam f 17 When the solvent has nearly reached the
f top o the strip, remove it rom the tube and
umb mv
pgm separate it rom the cork.
(mm)
1 18 Rule two pencil lines across the strip, one at
the level reached by the solvent and one at the
2 level o the initial pigment spot.
3 19 Draw a circle around each o the separated
4 pigment spots and a cross in the centre o
5 the circle.
6
7
8 Figure 4 Chromatogram of leaf pigments
Table o standard R values
131
2 MOLECULAR BIOLOGY
20 Using a ruler with millimetre markings, 21 Calculate the R or each pigment, where R is
measure the distance moved by the running the distance run by the pigment divided by the
solvent (the distance between the two lines) distance run by the solvent.
and the distance moved by each pigment (the
distance between the lower line and the cross 22 Show all your results in the table above, starting
in the centre o the circle) . with the pigment that had moved least ar.
Figure 5 In a rainbow the wavelengths of solar radiation reaching theWaveengths of ight
visible light are separated Earths surface/W m2 2
Visible light has a range o wavelengths with violet the
shortest wavelength and red the longest.
Sunlight or simply light is made up o all the wavelengths o electromagnetic
radiation that our eyes can detect. It is thereore visible to us and other
wavelengths are invisible. There is a spectrum o electromagnetic radiation
rom very short to very long wavelengths. Shorter wavelengths such as
X-rays and ultraviolet radiation have high energy; longer wavelengths such
as inrared radiation and radio waves have lower energy. Visible light has
wavelengths longer than ultraviolet and shorter than inrared. The range o
wavelengths o visible light is 400 to 700 nanometres.
When droplets o water in the sky split sunlight up and a rainbow is
ormed, dierent colours o light are visible. This is because sunlight is
a mixture o dierent wavelengths, which we see as dierent colours,
including violet, blue, green and red. Violet and blue are the shorter
wavelengths and red is the longest wavelength.
The wavelengths o light that are detected by the eye are also those used
by plants in photosynthesis. A reason or this is that they are emitted by
the sun and penetrate the Earths atmosphere in larger quantities than
other wavelengths, so are particularly abundant.
1.5 blue 5 4502 500 nm
green 5 5252 575 nm
red 5 6502 700 nm
1.0
0.5
0
500 1000 1500 2000 2500 3000
wavelength /nm
Figure 6 The spectrum of electromagnetic radiation reaching the Earths surface
light absorption by chorophy
Chlorophyll absorbs red and blue light most eectively
and refects green light more than other colours.
The frst stage in photosynthesis is the absorption o sunlight. This
involves chemical substances called pigments. A white or transparent
substance does not absorb visible light. Pigments are substances that do
132
2.9 Photosynthesis
absorb light and thereore appear coloured to us. Pigments that absorb Figure 7 Gentian fowers contain the
all o the colours appear black, because they emit no light. pigment delphinidin, which refects blue
light and absorbs all other wavelengths.
There are pigments that absorb some wavelengths o visible light but
not others. For example, the pigment in a gentian fower absorbs all
colours except blue. It appears blue to us, because this part o the
sunlight is refected and can pass into our eye, to be detected by cells in
the retina.
Photosynthesizing organisms use a range o pigments, but the main
photosynthetic pigment is chlorophyll. There are various orms o
chlorophyll but they all appear green to us. This is because they absorb
red and blue light very eectively, but the intermediate green light
much less eectively. Wavelengths o green light thereore are refected.
This is the reason or the main colour in ecosystems dominated by plants
being green.
absorption nd ction spectr
Drawing an absorption spectrum for chlorophyll and an action spectrum
for photosynthesis.
An action spectrum is a graph showing the rate It is not dicult to explain why action and absorption
o photosynthesis at each wavelength o light. spectra are very similar: photosynthesis can only
An absorption spectrum is a graph showing the occur in wavelengths o light that chlorophyll or the
percentage o light absorbed at each wavelength other photosynthetic pigments can absorb.
by a pigment or a group o pigments.
100 chlorophyll a
When drawing both action and absorption chlorophyll b
spectra, the horizontal x-axis should have the carotenoids
legend wavelength, with nanometres shown % absorption
as the units. The scale should extend rom 400
to 700 nanometres.
On an action spectrum the y-axis should be 400 500 600 700
used or a measure o the relative amount wavelength (nm)
o photosynthesis. This is oten given as a
percentage o the maximum rate, with a scale Figure 8 Absorption spectra o plant pigments
rom 0 to 1 00%.
100
On an absorption spectrum the y-axis should
have the legend % absorption, with a scale photosynthesis
rom 0 to 1 00%. (% of max rate)
Ideally data points or specic wavelengths 400 500 600 700
should be plotted and then a smooth wavelength (nm)
curve be drawn through them. I this is
not possible, the curve rom a published Figure 9 Action spectrum o a plant pigment
spectrum could be copied.
133
2 MOLECULAR BIOLOGY
data-bas qustions: Growth of tomato seedlings in red, green and blue light
Tomato seeds were germinated and grown 1 Plot a graph to show the relationship between
or 30 days in light produced by red, orange,
green and blue light emitting diodes. Four wavelength, lea area and height. Hint: i
dierent colours o LED were tested and two
combinations o colours. In every treatment you need two dierent scales on the y-axis
the tomato plants received the same intensity
o photons o light. The peak wavelength o you can put one on the let hand side o
light emitted by each wavelength is shown in
the table below, together with the mean lea the graph and the other on the right hand
area and height o the seedlings. Plants oten
grow tall, with weak stems and small leaves side. Do not attempt to plot the results or
when they are receiving insufcient light or
photosynthesis. combinations o LEDs. [6]
2 Using your graph, deduce the relationship
between the lea area o the seedlings and
their height. [1 ]
3 Evaluate the data in the table or a grower
o tomato crops in greenhouses who is
considering using LEDs to provide light. [3]
coours o leds Pak wavngt o igt mitt la ara o sings higt o sings
by led (nm) (m2) (mm)
Red 630 5.26 192
Orange 600 4.87 172
Green 510 5.13 161
Blue 450 7.26 128
Red and Blue 5.62 99
Red, Green and Blue 5.92 85
Source: Xiaoying, Shirong, Taotao, Zhigang and Tezuka (2012) . Regulation o the growth and photosynthesis o cherry tomato
seedlings by diferent light irradiations o light emitting diodes (LED) . African Journal ofBiotechnology Vol. 11(22) , pp. 6169-6177
Figure 10 Photosynthesizing organisms seem oxygen prductin in phtsynthesis
insignicant in relation to the size o the Earth
but over billions o years they have changed it Oxygen is produced in photosynthesis from photolysis
signicantly of water.
One o the essential steps in photosynthesis is the splitting o molecules
o water to release electrons needed in other stages.
H2O 4e + 4H+ + O2
This reaction is called photolysis because it only happens in the light
and the word lysis means disintegration. All o the oxygen generated
in photosynthesis comes rom photolysis o water. O xygen is a waste
product and diuses away.
efts o potosyntsis on t eart
Changes to the Earths atmosphere, oceans and rock
deposition due to photosynthesis.
Prokaryotes were the frst organisms to perorm photosynthesis, starting
about 3,500 million years ago. They were joined millions o years later by
algae and plants, which have been carrying out photosynthesis ever since.
134
2.9 Photosynthesis
One consequence o photosynthesis is the rise in the oxygen concentration av
o the atmosphere. This began about 2,400 million years ago (mya) , rising to
2% by volume by 2,200 mya. This is known as the Great Oxidation Event. dfr mpr
At the same time the Earth experienced its frst glaciation, presumably Pl cmp
due to a reduction in the greenhouse eect. This could have been due mpr (%)
to the rise in oxygenation causing a decrease in the concentration o
methane in the atmosphere and photosynthesis causing a decrease in CO N Ar O H O
carbon dioxide concentration. Both methane and carbon dioxide are 22 22
potent greenhouse gases.
Venus 98 1 1 0 0
The increase in oxygen concentrations in the oceans between 2,400 and
2 , 2 00 mya caused the oxidation o dissolved iron in the water, causing Earth 0.04 78 1 21 0.1
it to precipitate onto the sea bed. A distinctive rock ormation was
produced called the banded iron ormation, with layers o iron oxide Mars 96 2.5 1.5 2.5 0.1
alternating with other minerals. The reasons or the banding are not yet
ully understood. The banded iron ormations are the most important What are the main diferences
iron ores, so it is thanks to photosynthesis in bacteria billions o years between the composition o the
ago that we have abundant supplies o steel today. Earth's atmospheres and the
atmosphere o the other planets.
The oxygen concentration o the atmosphere remained at about 2% rom What is the cause o these
2,200 mya until about 750-635 mya. There was then a signifcant rise to diferences?
20% or more. This corresponds with the period when many groups o
multicellular organisms were evolving.
50
oxygen/% of atmosphere40
CO2 uptake/mol h21
30 av
20 lg
1500
10
1000
0 3.0 2.0 1.0 0 500
4.0 Millions of years ago ( 1,000)
Figure 11 0
200 75 150 225 300
light intensity /J dm22s21
Production of carbohydrates
Figure 12 The graph shows the results
Energy is needed to produce carbohydrates and other of an experiment in which the rate
carbon compounds rom carbon dioxide. of photosynthesis was found by
measuring the uptake of carbon dioxide
Plants convert carbon dioxide and water into carbohydrates by
photosynthesis. The simple equation below summarizes the process: 1 What is the reason or a CO
2
carbon dioxide + water carbohydrate + oxygen uptake rate o 200 in
To carry out this process, energy is required. A chemical reaction that darkness?
involves putting in energy is described as endothermic. Reactions
involving the production o oxygen are usually endothermic in living 2 What can you predict about cell
systems. Reactions involving combining smaller molecules to make respiration and photosynthesis
larger ones are also oten endothermic and molecules o carbohydrate at the point where the net rate o
such as glucose are much larger than carbon dioxide or water. CO uptake is zero?
2
135
2 MOLECULAR BIOLOGY
ativityincrease in biomass of grass The energy for the conversion of carbon dioxide into carbohydrate is
/kg ha-1 h-1 obtained by absorbing light. This is the reason for photosynthesis only
co nentrtin occurring in the light. The energy absorbed from light does not
2 disappear it is converted to chemical energy in the carbohydrates.
40 limiting fators
30 Temperature, light intensity and carbon dioxide
concentration are possible limiting factors on the
20 rate of photosynthesis.
10 The rate of photosynthesis in a plant can be affected by three
external factors:
0 100 200 300 400
210 CO2/cm3m-3 air temperature;
Figure 13 In this graph the rate of light intensity;
photosynthesis was measured
indirectly by measuring the change in carbon dioxide concentration.
plant biomass.
Each of these factors can limit the rate if they are below the optimal
1 The maximum carbon level. These three factors are therefore called limiting factors.
dioxide concentration of the According to the concept of limiting factors, under any combination
atmosphere is 380 cm3 m3air. of light intensity, temperature and carbon dioxide concentration, only
Why is the concentration often one of the factors is actually limiting the rate of photosynthesis. This
lower near leaves? is the factor that is furthest from its optimum. If the factor is changed
to make it closer to the optimum, the rate of photosynthesis increases,
2 In what weather conditions is but changing the other factors will have no effect, as they are not the
carbon dioxide concentration limiting factor.
likely to be the limiting factor
for photosynthesis? Of course, as the limiting factor is moved closer to its optimum, while
keeping the other factors constant, a point will be reached where
this factor is no longer the one that is furthest from its optimum and
another factor becomes the limiting factor. For example, at night, light
intensity is presumably the limiting factor for photosynthesis. When
the sun rises and light intensity increases, temperature will usually
take over as the limiting factor. As the temperature increases during
the morning, carbon dioxide concentration might well become the
limiting factor.
controed variabes in imiting fator
experiments
Experimental design: controlling relevant variables in
photosynthesis experiments is essential.
In any experiment, it is important to control all variables other than
the independent and dependent variable that you are investigating.
The independent variable is the one that you deliberately vary in the
experiment with a range of levels that you choose. The dependent
variable is what you measure during the experiment, to see if it is
affected by the independent variable.
136
2.9 Photosynthesis
It is essential during this type o experiment to be sure that the
independent variable is the only actor that could be aecting
the dependent variable. All other variables that might aect the
independent variable must thereore be controlled.
These are questions that you need to answer when you are designing
an experiment to investigate a limiting actor on photosynthesis:
Which limiting actor will you investigate? This will be your
independent variable.
How will you measure the rate o photosynthesis? This will be
your dependent variable.
How will you keep the other limiting actors at a constant and
optimal level? These will be your controlled variables.
Investigating limiting factors % of maximum rateacv
Design of experiments to investigate limiting factors tmprur
on photosynthesis.
100
There are many possible experimental designs. A method that can be
used to investigate the eect o carbon dioxide concentration is given 50
below. You could either modiy this to investigate a dierent limiting
actor or you could develop an entirely dierent design. 0
0 10 20 30 40 50
Investigating the efect o carbon dioxide on photosynthesis tem pera tu re /C
I a stem o pondweed such as Elodea, Cabomba or Myriophyllum is Figure 14 In this graph the
placed upside-down in water and the end o the stem is cut, bubbles rate of photosynthesis was
o gas may be seen to escape. I these are collected and tested, they measured indirectly by
are ound to be mostly oxygen, produced by photosynthesis. The measuring the change in
rate o oxygen production can be measured by counting the bubbles. plant biomass
Factors that might aect the rate o photosynthesis can be varied to
fnd out what eect this has. In the method below carbon dioxide 1 What was the optimum
concentration is varied. temperature for
photosynthesis in this
1 Enough water to fll a large beaker is boiled and allowed to cool. plant?
This removes carbon dioxide and other dissolved gases.
2 What was the maximum
2 The water is poured repeatedly rom one beaker to another, to temperature for
oxygenate the water. Very little carbon dioxide will dissolve. photosynthesis?
3 A stem o pondweed is placed upside-down in the water and the
end o its stem is cut. No bubbles are expected to emerge, as the
water contains almost no carbon dioxide. The temperature o the
water should be about 25 C and the water should be very brightly
illuminated. Suitable apparatus is shown in fgure 1 6.
4 Enough sodium hydrogen carbonate is added to the beaker to raise
the carbon dioxide concentration by 0.01 mol dm-3. I bubbles
emerge, they are counted or 30 seconds, repeating the counts
until two or three consistent results are obtained.
137
2 MOLECULAR BIOLOGY
sodium 5 Enough sodium hydrogen carbonate is added to raise the
hydrogen concentration by another 0.01 mol dm3. B ubble counts are done
carbonate in the same way.
po n d we e d 6 The procedure above is repeated again and again until further
increases in carbon dioxide do not affect the rate of bubble
water at 25 C production.
light source Questions
Figure 15 Apparatus or measuring 1 Why are the following procedures necessary?
photosynthesis rates in diferent
concentrations o carbon dioxide a) Boiling and then cooling the water before the experiment.
b) Keeping the water at 25 C and brightly illuminating it.
c) Repeating bubble counts until several consistent counts have
been obtained.
2 What other factor could be investigated using bubble counts with
pondweed and how would you design the experiment?
3 How could you make the measurement of the rate of oxygen
production more accurate?
138
Questions
Questions
1 Lipase is a digestive enzyme that accelerates a) (i) State the volume units that are shown
the breakdown o triglycerides in the small in the equation. [1 ]
intestine. In the laboratory, the rate o activity (ii) State the mass units that are shown in
o lipase can be detected by a decline in pH. the equation. [2]
Explain what causes the pH to decline. [4] b) ( i) C alculate the mass o ATP produced per
dm3 o oxygen. [2]
2 Papain is a protease that can be extracted rom ( ii) C alculate the mass o ATP produced per
pineapple ruits. Figure 1 7 shows the eect
o temperature on the activity o papain. The race in table 1 . [4]
experiment was perormed using papain dissolved
in water and then repeated with the same c) Explain how it is possible to synthesize such
quantity o papain that had been immobilized by
attaching it to a solid surace. The results show large masses o ATP during races. [3]
the percentage o the protein in the reaction
mixture that was digested in a fxed time. d) D uring a 1 00 m race, 80 g o ATP is needed
100 immobilized but only 0.5 dm3 o oxygen is consumed.
papain
D educe how ATP is being produced. [3]
80
lgh f Vm f xyg cmd c
60 dissolved rac/m rpra drg h rac/dm3
papain
1500 36
40
% of protien digested 10,000 150
rate of CO2 absorption /arbitrary units
42,300 700
20
Table 1
0 4 Figure 1 8 shows the eects o varying light
20 30 40 50 60 70 80
temperature /C
Figure 17 intensity on the carbon dioxide absorption
by leaves, at dierent, fxed carbon dioxide
a) (i) Outline the eects o temperature on concentrations and temperatures.
the activity o dissolved papain. [2] a) Deduce the limiting actor or
photosynthesis at:
(ii) Explain the eects o temperature on
the activity o dissolved papain. [2] (i) W (ii) X (iii) Y (iv) Z. [4]
b) (i) Compare the eect o temperature on b) Explain why curves I and II are the same
between 1 and 7 units o light intensity. [3 ]
the activity o immobilized papain with
the eect on dissolved papain. [2] c) Explain the negative values or carbon
(ii) Suggest a reason or the dierence that dioxide absorption when the leaves were in
you have described. [2] low light intensities. [3]
(iii) In some parts o the human body,
enzymes are immobilized in membranes. Z IV 0.4%CO at 30C
13 2
Suggest one enzyme and a part o the
12
body where it would be useul or it to 11
be immobilized in a membrane. [2] 10
9 III 0.4%CO at 20C
82
7
6 XY
3 The equation below summarizes the results o 5 II 0.13%CO at 30C
metabolic pathways used to produce ATP, using 2
energy rom the oxidation o glucose. 4
glucose + oxygen + (ADP + Pi) 3 I 0.13%CO at 20C
1 80 g 1 34.4 dm3 1 8.25 kg 22
1W
0
-1 1 2 3 4 5 6 7
light intensity /arbitrary units
carbon dioxide + water + ATP Figure 18
1 34.4 dm3 1 08 g 1 8.25 kg 139
2 MOLECULAR BIOLOGY
5 Figure 1 9 shows the results o an experiment a) Describe the relationship between
in which Chlorella cells were given light o wavelength o light and oxygen yield,
wavelengths rom 660 nm (red) up to 700 nm when there was no supplementary light. [2]
(ar red) . The rate o oxygen production by
photosynthesis was measured and the yield b) Describe the eect o the supplementary
o oxygen per photon o light was calculated.
This gives a measure o the efciency light. [2]
o photosynthesis at each wavelength.
The experiment was then repeated with c) Explain how the error bars help in drawing
supplementary light with a wavelength o
650 nm at the same time as each o the conclusions rom this experiment. [2]
wavelengths rom 660 to 700 nm, but with the
same overall intensity o light as in the frst d) The probable maximum yield o oxygen
experiment.
was 0.1 25 molecules per photon o light.
with supplementary light
without supplementary light Calculate how many photons are needed
0.15 to produce one oxygen molecule in
0.10 photosynthesis. [2]
e) Oxygen production by photolysis involves
this reaction:
4H2O O2 + 2H2O + 4H+ + 4e-
yeild of oxygen molecules per photon of light Each photon o light is used to excite an
electron (raise it to a higher energy level) .
Calculate how many times each electron
produced by photolysis must be excited
during the reactions o photosynthesis. [2]
0.05
0 700
660 680
wavelength (nm)
Figure 19 Photon yield o photosynthesis in diferent light
intensities
140
3 GEnEtICs allowing new combinations to be ormed by the
usion o gametes. Biologists have developed
CIEroLduLcioB I O L O G Y techniques or artifcial manipulation o DNA,
cells and organisms.
Every living organism inherits a blueprint or
lie rom its parents. The inheritance o genes
ollows patterns. Chromosomes carry genes in
a linear sequence that is shared by members
o a species. Alleles segregate during meiosis
3.1 Genes
Uderadig Applicaio
A gene is a heritable actor that consists o The causes o sickle cell anemia, including a
a length o DNA and inuences a specic base substitution mutation, a change to the
characteristic. base sequence o mRNA transcribed rom it and
a change to the sequence o a polypeptide in
A gene occupies a specic position on one type hemoglobin.
o chromosome.
Comparison o the number o genes in humans
The various specic orms o a gene are alleles. with other species.
Alleles difer rom each other by one or a ew skill
bases only.
Use o a database to determine diferences in
New alleles are ormed by mutation. the base sequence o a gene in two species.
The genome is the whole o the genetic naure of ciece
inormation o an organism.
Developments in scientic research ollow
The entire base sequence o human genes was improvements in technology: gene sequencers,
sequenced in the Human Genome Project. essentially lasers and optical detectors, are
used or the sequencing o genes.
141
3 Genetics
What is a gene?
A gene is a heritable actor that consists o a length o DNA
and infuences a specic characteristic.
Genetics is the branch o biology concerned with the storage o
inormation in living organisms and how this inormation can be passed
rom parents to progeny. The word genetics was used by biologists long
beore the method o inormation storage was understood. It came
rom the word genesis, meaning origins. Biologists were interested in
the origins o eatures such as baldness, blue eyes and much more.
Something must be the cause o these eatures and be passed on to
ospring where the eatures would again develop.
Experiments in the 1 9th century showed that there were indeed actors
in living organisms that infuenced specic characteristics and that these
actors were heritable. They could be passed on to ospring by pea
plants, ruit fies and all other organisms. There was intense research
into genetics rom the early 20th century onwards and the word gene
was invented or the heritable actors.
One obvious question was the chemical composition o genes. By the
middle o the 20th century there was strong evidence that genes were
made o DNA. There are relatively ew DNA molecules in a cell just 46
in a typical human cell or example yet there are thousands o genes. We
can thereore deduce that each gene consists o a much shorter length o
DNA than a chromosome and that each chromosome carries many genes.
Comparing numbers of genes
Comparison o the number o genes in humans with other species.
How many genes does it take to make a have more genes. The table shows whether this is
bacterium, a banana plant or a bat, and how true. It gives a range o predicted gene numbers.
many are needed to make a human? We They are based on evidence rom the DNA o
see ourselves as more complex in structure, these species but are not precise counts o gene
physiology and behaviour so we might expect to numbers as these are not yet known.
Group Name of species Brief description Numbers of genes
Prokaryotes Haemophilus infuenzae Pathogenic bacterium 1,700
Escherichia coli Gut bacterium 3,200
Protoctista Trichomonas vaginalis Unicellular parasite 60,000
Fungi Saccharomyces cerevisiae (Yeast) Unicellular ungus 6,000
Plants Oryza sativa (Rice) Crop grown or ood 41,000
Arabidopsis thaliana (Thale cress) Small annual weed 26,000
Animals Populus trichocarpa (Black cottonwood) Large tree 46,000
Drosophila melanogaster (Fruit fy) Larvae consume ripe ruit 14,000
Caenorhabditis elegans Small soil roundworm 19,000
Homo sapiens (Humans) Large omnivorous biped 23,000
Daphnia pulex (Water fea) Small pond crustacean 31,000
142
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