Biological Molecules

organic molecules

any molecule containing carbon that can be found in living things. The 4 classes are:

• carbohydrates: respiratory substrates that provide energy for cells, used for structure in cell membranes and cell walls

• proteins: main component of many cellular structures, form enzymes and chemical messengers e.g RNA

• lipids: can be used as respiratory substrates to provide energy for cells, from a bilayer in cell membranes and make up some hormones

• nucleic acids: form polymers(DNA and RNA) to makeup genomes.They code for the sequence of amino acids to makeup all proteins

hydrolysis

breaking down of large molecules into smaller ones by addition of water molecules(like the opposite of condensation)

condensation reaction

chemical process when 2 molecules combine to form a more complex one whilst eliminating a simple substance(usually water)
how many biological polymers are formed

what supports the theory that all organisms share a common ancestor

• all organisms use the same nucleic acid(DNA and RNA) as genetic material

• all build proteins using the same 20 amino acids

• all use lipids and carbohydrates as energy stores and to make up their cell membranes and walls

monomer, covalent bond type, polymer made and polymer example for: carbohydrates, proteins and nucleic acids

protein functions

• main component of many cellular structures

• form enzymes and chemical messengers

• amino acid→polypeptide(peptide bond)

nucleic acids functions

• form polymers(DNA and RNA) that make up genomes of organisms and code for the sequence of amino acids to make proteins

• neucleotides→polyneucleotides (phosphodiester bond)

lipids functions

• used as respiratory substrates providing energy for cells

• form a bilayer in cell membranes and makeup some hormones

• not polymers because made of different smaller units, not one repeating smaller unit

• glycerol + fatty acids→lipids (ester bond)

carbohydrates functions

• respiratory substrate providing energy for cells

• can be used for structure in cell membranes & cell walls in plants

• monosaccharides→polysaccharides (glycosidic bond)

bonds(in order of strength)

hydrogen: weakest

• electrons are unevenly distributed in some molecules so some regions are - and others are _

• this is polarised/a polar molecule

• there are weak electrostatic forces of attraction between the + and - regions of polar molecules, especially in water

ionic:

• ions with oppositite charges(+ metal and - non metal) attract eachother.

• the electrostatic force of attraction is called an ionic bond

covalent:

• atoms share electrons in the outershell to make a more stable compound with a full outershell, called a molecule

• the bond is the electrostatic force of attraction between + nuclei and - shared electrons

valency of: carbon, oxygen, hydrogen and nitrogen

valency- number of bonds it can form

carbon-4
nitrogen-3
oxygen-2
hydrogen-1

alpha glucose

• an isomer of glucose that can bond together to form starch or glycogen

• forms a helical chain→compact→more can be stored

beta glucose

• an isomer of glucose that can bond together to form cellulose

• forms long straight unbranched chains bc each B-glucose molecule is inverted so all 1-4 glycosidic bonds

isomer

compounds with the same formula but different structure

monosaccharides

• general formula is (CH₂O)_n

• monomers that make up disaccharides and polymers

• e.g. fructose(in sweets, sweetest, most soluble so more water can enter), glucose, galactose(least soluble)

• hexose sugar- 6 carbons e.g glucose, pentose sugar- 5 carbons

disaccharides

• 2 monosaccharides joined by a condensation reaction forming a glycosidic bond between two -OH groups

• reducing sugars can lose/donate electrons to other compounds

• maltose(reducing)= glucose + glucose

• lactose(reducing) = glucose + galactose

• sucrose(non-reducing) = glucose + fructose

• enzymes catalyse hydrolysis to break di→mono e.g lactase for lactose→glucose +galactose

polysaccharides

• more than 2 monosacharides joined by condensation

• polymers of glucose are joined by either a 1-4 or 1-6 glycosidic bond(1-6 means carbon 1 of first glucose joins to carbon 6 of the second glucose) causing different shapes & properties

• amylose and cellulose- unbranched glucose chains w 1-4 glycosidic bonds

• amylopectin and glycogen- branched w 1-6 glycosidic bonds

starch: structure, properties, uses, and diagram

• monosaccharide- alpha glucose

structure:

• mixture of polysaccharides: amylose and amylopectin

• amylose-long unbranched, 1-4 glycosidic bonds, helical so coiled due to H⁺ bonding

• amylopectin- long, branched chains bc of 1-6 glycosidic bonds

properties: 

• amlose-coiled so its compact to store more in a smaller space

• amylopectin- branches increase surface area→faster hydrolysis→more glucose released for respiration

uses: 

• plants- store excess glucose as starch in grains & granules bc starch is too large to leave the cell and is insoluble so doesnt affect water potential(amount of water in/out of cell) and is hydrolysed by amylase and maltase for glucose for respiration

glycogen: structure, properties, uses, and diagram

• monosaccharide- alpha glucose

structure:

• long branched chain w 1-6 glycosidic bonds and lots of long side branches(more than amylopectin)

properties: 

• lots of branches increase surface area for enzymes to hydrolyse glycosidic bonds for quick release of glucose. also compact→good for storage

• less dense & more soluble than starch→broken down quicker

uses:

• animals store excess glucose as glycogen in the liver and muscles in granules that can quickly be hydrolysed to release glucose for respiration when needed e.g during exercise

cellulose: structure, properties, uses, and diagram

• monomer- beta glucose

structure:

• long unbranched, straight chains, 1-4 glycosidic bonds

• the cellulose chains are then linked by hydrogen bonds between the glucose molecules in each chain to form thicker fibres called microfibrils

• each B-glucose is inverted to the ones either side of it so the chains are straight

• hydrogen bonds between each parallel chain

properties: 

• hydrogen bonds between cellulose chains make the microbrils very strong but still flexible, allowing them to provide support

uses: 

• plant cell walls use cellulose as a structural component as it provides support and allows cell walls to become turgid

• large gaps between microfibrils so substances can move to cell membranes for selective absorption

testing for reducing sugars

• all monosaccharides and some disaccharides e.g maltose, lactose

method:

1. add excess benedict’s reagent to sample

2. heat in a water bath at boiling/above 80c

result:

blue precipitate(none/orignal colour) →green(trace amounts)→yellow(low)→orange(moderate)→brick red(high)

quantitative measurements:

filter solution and weigh precipitate or remove precipitate and use a colourimeter to measure absorbance of remaining benedict’s solution

how to test for non reducing sugars

• some polymers and disaccharides e.g sucrose

method: 

1. add dilute HCl and heat in a water bath to break bonds and produce monosaccharides

2. add an alkali to neutralize it e.g NaOH

3. follow method for reducing sugars

hazards and precautions for testing for sugars

• benedicts reagent is an irritant→wear goggles, wash hands if contact w skin

• hot water can cause burns→use caution when carrying or pouring

• (non reducing only) HCl and alkalis e.g NaOH can be corrosive→ wear goggles, wash hands if contact w skin

testing for starch

• add iodine

• if starch is present it turns from brown to blue-black

• this is qualitative only

uses of lipids

• electrical insulation for nerves and thermal insulation in adipose(fat) cells

• steroid hormones e.g testosterone

• forms the plasma membrane

• protect delicate organs

• waterproofing and buoyancy→lipids are less dense than water so float

triglycerides

1 glycerol + 3 fatty acids

properties:

• non polar and hydrophobic(repels water) →insoluble in water but soluble in organic(carbon containing) substances e.g ethanol

• in water triglycerides bundle together as insoluble droplets bc the tails face inwards and the glycerol heads shield them from water

• ester bonds form between each glycerol’s OH group and each fatty acids’s OH group through a condensation reaction also called esterification

• triglycerides are stored in adipose(fat) tissue and are used as an energy store bc lots of energy is released when fatty acid chains break down

mono and diglycerides

monoglycerides- 1 fatty acid+ 1 glycerol
diglycerides- 2 fatty acids+ 1 glycerol

fats vs oils, saturated vs unsaturated fatty acids

fat-saturated, solid at room temp, higher density
oil-(poly)unsaturated, liquid at room temperature, less dense

saturated-no double bonds between carbon atoms in the hydrocarbon chain
unsaturated-has double bonds between carbons in the hydrocarbon chain so fewer hydrogen atoms. Double bonds cause the chain to kink/bend so its less dense, can be one-monounsaturated or many carbon double bonds-polyunsaturated

fatty acids

• the -OH reacts w glycerol

• the hydrocarbon chain is non-polar and hydrophobic

• the variable region can vary in length of hydrocarbon chain, or if it is saturated or unsaturated

phospholipids

• glycerol + phosphate + 2 fatty acids

• phospholipids are polar- 2 ends/poles that behave differently. Because phosphate is hydrophyllic, the head is attracted to water but the tail is hydrophobic so repels water

• hormones are phospholipids

phospholipid bilayers

• When water is in(intracellular) and out(extracellular)of cells, the hydrophobic tails move inwards and the hydrophyllic heads move outwards forming a double layer called a bilayer, making cell membranes.

• The center is hydrophobic so water soluble molecules can’t easily enter, creating a barrier separating solutions and creating different conditions either side of the membrane

• This structure allows the phospholipid to form glycolipids by combining w carbohydrates within the cell-surface membrane. Glycolipids are important in cell recognition

micelles

• phospholipid structure that forms when water surrounds it, but there is no water inside

• can be used to transport monoglycerides

testing for lipids

emulsion test
1. add ethanol to sample(bc insoluble in water) and shake
2. add water to sample and shake for a minute

positive-lipids form a milky white emulsion(layer at the top)
more lipids present=more milky/opaque emulsion but qualitative only

ethanol is flammable so do not conduct the test near open flames

amino acids

• proteins are made of the monomer amino acids

• there are only 20 possible amino acids but if the amino acids are in a different sequence, the (disulfide bridges/hydrogen/ionic) bonds are in different places so the protein folds in a different way and has different 3D shapes in the tertiary structure so form different proteins

• R group is the variable region(only part that changes), it is usually lots of carbon

dipeptides

• two amino acids joined by a condensation reaction to form a dipeptide w a peptide bond

• the bond is between the amine group and the carboxyl group of another

polypeptides

• more than 2 amino acids joined together

• proteins are folded chains of single or multiple polypeptides

• There are 4 levels to protein structure, based on how much it folds/changes shape. Single chain polypeptides can fold to the tertiary level, but only multichain polypeptides can fold to the quaternary level

• in all proteins, more bonds=more stable

primary structure

the sequence of amino acids in a polypeptide chain

• the number and sequence of amino acids in a polypeptide chain determines its structure and shape

• a protein’s shape is specific to its function so change in amino acids→changes shape→can’t function

secondary structure

coil spiral e.g alpha/beta helice or a beta pleated sheet

• amino acids in the chain have -NH and -C=O groups on either side of the peptide bond

• the H in -NH has a + charge and the O in -C=O is - charged

• both groups form weak hydrogen bonds causing the polypeptide chain to be twisted into a 3D shape

tertiary structure

the secondary structure twists and folds even more, into a 3D globular stucture e.g enzymes

• the 3D shape is what makes each protein distinctive and recognisable to other molecules to interact in specific ways

• their bonds depend on the primary structure. The 3 possible bonds are:
  disulfide bridges- fairly strong and not easily broken
  ionic bonds-form between any carboxyl and amine groups that are involved in peptide bonds, weaker than disulfide bridges and easily broken by changes in pH
  hydrogen bonds-numerous but easily broken

quaternary structure

multiple polypeptide chains linked together to form compelx molecules

• haemoglobin has haem groups that are prosthetic(non protein) and contains ferrous iron that binds to oxygen. Haemoglobin has 2 alpha helices/chains and 2 beta helices/chains

fibrous proteins

e.g collagen

• structural functions such as tendons to join muscles to bones

• long chains that run parallel to eachother. Chains are linked by cross-bridges so they form very stable molecules w a high tensile strength

• collagen has 3 alpha helice chains

biuret’s test

detects the peptide bonds in protein

1. add buiret’s agent to sample

2. if proteins/peptide bonds are present it turns purple. If not, the solution remains blue

how do enzymes work

• enzymes are globular 3D catalysts that act as biological catalysts to speed up the rate of metabolic reactions without being used up

• They decrease activation energy to allow reactions to happen at lower temperatures

• depending on the reaction, they either hold substrates close together, reducing repulsion and allowing them to bond more easily or put more strain on bonds allowing them to break apart more easily

lock and key model

• active site is always complementary to substrate

• acrive site is rigid(disproved bc other molecules can bind to different sites to the active site on enzyme and alter the enzymes shape and activity-so active site must be flexible)

• basic explanation, doesn’t show how activation energy is lowered

induced fit model

• active site isn’t complementary at first, but undergoes a confirmational change to become complementary

• active site is flexible and moulds around the shape, putting strain around bonds to decrease activation enregy

• more complex and more widely accepted explanation

intercellular vs extracellular

anabolic and catabollic

intercellular- in cells e.g hydrogen peroxide→oxygen + water, enzyme: catalase
extracellular- outside cells e.g protein→amino acids enzyme: trypsin

catabollic reaction- breaks down.e.g. hydrolysis
anabolic reaction-builds e.g condensation

graph showing rate of reaction as substrate concentration increases with no inhibitor, a competitive inhibitor and a non-competitive inhibitor

competitive inhibitors

• competitive inhibitors are molecules that have a similar shape to a substrate, so they can occupy the active site of an enzyme

• therefore, they compete w substrates for available active sites

• increasing substrate conc reduces the effects of competitive inhibitors by reducing the chance of inhibitors colliding w active site

• some competitive inhibitors bind irreversibly(permanently) to an active site

e.g. succinate is a substrate for an enzyme in respiration but malonate has a similar structuer and can be a reversible competitive inhibitor
e.g. pencilin is an irreversible competitive inhibitor to transpeptidase- an enzyme for the synthesis of bacterial cell walls, so is used to treat bacterial infections

non-competitive inhibitors

• binds to enzyme’s allosteric site

• this causes the enzyme’s tertiary structure to undergo a confirmational change in shape, so active site changes

• so the active site is no longer complementary to substrate so it can’t bind to form enzyme-substrate complexes

• This reduces rate of reaction and unlike with competitive inhibitors, its effects cannot be overcome by increasing substrate concentration

enzyme cofactors

non protein structures that bind to active site to make it complementary to a substrate

co-enzymes- e.g NAD, FAD, NAPD→gain electrons to accept hydrogen to assist in reactions, organic molecules, often from vitamins e.g vitamin B5 is used to make coenzyme A used in aerobic respiration, temporarily bind to enzymes

inorganic cofactors- don’t contain carbon, usually metal ions

prosthetic groups- tightly bound cofactors that permanently attach to enzymes, e.g zinc ions are a prosthetic group for carbonic anhydrase- an enzyme to regulate pH in blood

how does pH affect enzyme action

• pH is the measure of H⁺ ions in a solution

• the shape/arrangement of an enzyme’s active site is partly determined by the hydrogen and ionic bonds in the enzyme’s tertiary structure

• the change in H⁺ ions affects this bonding so

• outside of optimum pH the hydrogen and ionic bonds holding the enzyme’s tertiary structure together break so it unfolds and the enzyme denatures

• so the active site is no longer complementary to the substrate, so enzyme-substrate complexes can’t form, reducing the rate of reaction

how does temperature affect enzyme action

• at first, increasing temperature increases rate of reaction

• bc particles have more kinetic energy so move faster

• so more frequent successful collisions, so more enzyme-substrate complexes formed

but past optimum temperature:

• increasing temp decreases rate of reaction bc there is too much kinetic energy so

• hydrogen bonds in tertiary structure of active site break and the enzyme unfolds

• the active site changes shape and is no longer complementary to substrates since the enzyme is denatured

• so fewer enzyme-substrate complexes are made

how does concentration of enzyme/substrate affect enzyme action

1:

• increasing substrate concentration increases the rate of reaction bc there are more frequent successful collisions and the substrate is the limiting reactant

• can replace substrate for enzyme

2: the rate of reaction is limited by:

• number of enzymes available(if increasing substrate conc) so all active sites are saturated by substrates so it plateaus

• number of substrates available(if increasing enzyme conc) so not enough substrates to supply enzyme’s active sites so it plateaus

how to measure rate of reaction

• draw a tangent with an equal number of squares either side

• change in y/change in x

end point inhibition/feedback inhibition

water properties overview

• is a major compontent of cells

• is a metabolite(necessary for metabollism)

• is a solvent

• has a large latent heat of vaporisation

• has a high specific heat capacity

• has strong cohesion between molecules

water structure

• 2 hydrogen atoms covalently bonded to 1 oxygen

• polar molecule- electrons aren’t equally shared(hydrogen is slighly +, O is slighly -)

• dipolar

hydrogen bonds in water:

• the slightly negative charge on the oxygen atom attracts it to the slightly positive hydrogen atom of another water molecule

• many of the properties of water are due to its ability to form hydrogen bonds

• the numerous hydrogen bonds in water make it a very stable structure

water’s specific heat capacity

the energy to raise the temperature of 1kg of water by 1 degree

• water has a high specific heat capacity because water molecules stick together bc of the hydrogen bonds so more energy is needed to seperate them

• this means water can act as a buffer against sudden temperature variations

• this is useful bc organism’s bodies are mostly made of water. The water in and around our cells absorbs a lot of heat energy without its temperature increasing much to ‘bufffer’ heat changes

• water’s high SHC is also an advantage to aquatic organisms bc large bodies of water(seas and lakes) don’t change temperature as quickly as terrestrial(land) environment

latent heat of vaporisation

energy required to evaporate 1g of water

• water has a high latent heat of vaporization bc there are hydrogen bonds between water molecules which requires lots of energy to break

• in a body of water some molecules are moving at faster speeds so some have enough enrgy to escape the water and move into air-evapouration

• evaporation causes energy loss, decreasing the kinetic energy of water so it cools

importance to organisms:

• animals that sweat can keep cool bc the water in sweat evaporates of the surface of the animal

• plants are also cooled when water evaporates from their leave

water potential

• water moves from high to low concentration- osmosis

• water also tends to move from areas of high hydrostatic pressure to low

• water movement is also affected by gravity and electrostatic forces-such as those that cause surface tension

• the tendency for water to move due to any of these effects is water potential

water’s cohesion

cohesion- an attractive force between particles of the same kind e.g. water and water
adhesion- an attractive force between unalike substances

• water sticks together due to the cohesive forces of hydrogen bonding

• this allows it to be pulled up a tube(like drinking through a straw). This happens in xylem vessels

water and surface tension

• acts as a force pulling droplets of water back to a body of eater

• the water surface acts a skin strong enough to support small organisms e.g pond skaters

cohesion-tension theory

• water is a polar molecule, so its positive and negative charges aren’t evenly distributed

• the O is slighly - and the H is slighly +

• so in the xylem, water molecules spontaneously arrange so the + and - poles lie next to eachother

• this causes the molecules to cohere(stick together) so as some leave a plant by transpiration, others are pulled up behind them

water density

• when cooled all substances move closer together and become more dense

• water is most dense at 4 degrees

• below 4c it becomes less dense and forms a crystalline structure. This structure is unique, hence why all snowflakes are different

importance to organisms: 

• ice is less dense than water so it floats. This insulates lakes and oceans to prevent them from freezing solid, allowing organisms to survive winter. Ice caps are a habitat for polar bears

water as a solvent

• a solvent is a liquid that solutes can dissolve in

• dissovled solutes are free to move

• positive and negative charges of water attract other molecules causing the solute to separate and dissolve

importance:

• the metabolic reactions in all organisms only happens when the reactants are dissolved in water

• substances being dissolved in water allows them to be transported around the bodies of organisms e.g glucose, CO₂ and urea in blood plasma, and ions and sugar dissolved in water are transported by plants phloem and xylem

metabolites

nucleic acids

• DNA is a nucleic acid. Nucleic acid are macromolecules containing O, H, C, N, and P

• nucleic acids are made of nucleotides

• the 2 nucleic acids are ribonucleic acid(RNA) and deoxiribonucleic acid(DNA)

nucleotides

• phosphodiester bonds hold nucleotides together

hydrogen bonding in DNA

DNA structure

• discovered by Rosalin Franklin

• neucleotide chains add stability

• long→ store lots of info/entire genome

• helix→compact

• 3.2bn base pairs in DNA→near infinite order→code for different ordeer of proteins→variety for genetic diversity, and also accurate template for replication so identical copies each time

• sugar-phosphate backbone→helix structure protects reactive bases

• hydrogen bonding→weak so little energy to break apart to act as a template for replication

• complementary base pairs→accurate template for replication

RNA

• contains cytosine, adenine, guanine and uracil(instead of thymine)

• it is a single helix/one strand and is short lived(quickly broken down)

• found in the cytoplasm

• there is tRNA, mRNA, and rRNA

transfer RNA- transfer genetic info from DNA to ribosomes

• one strand folded to form a clover structure with H⁺ bonds between complementary base pairs

• each tRNA can bring one specific amino acid to a ribosome

• anticodons are bases that are complementary to codons on an RNA strand

• codon’s are complementary to DNA triplets

• see image

ribosomal RNA- two strands
messenger RNA-

• small enough to leave nucleus

• makes a copy of bases complementary to one strand of DNA

similarities and differences between DNA and RNA

similarities and differences between rRNA, mRNA, and tRNA

replication theories

DNA has to replicate every time a cell divides so both cells have identical copies of the entire genome. The method of replication is called semi conservative replication

DNA helicase- enzyme that breask down H⁺ bondes between bases(give example of base in ExamQs)
DNA polymerase- join adjacent neucleotides forming phosphodiester bonds to synthesise the sugar phosphate backbone

replication steps

1. the enzyme DNA helicase ‘unwinds’ the double helix by breaking H⁺ bonds between the bases of 2 DNA strands

2. each original strand acts as a template determining the order of bases to build a new strand. Free DNA nucleotides are attracted to the exposed bases on the templates and they attach to the correct base through complementary base pairing(A w T, and C w G)

3. the free nucleotides are joined to the new strand by condensation reactions catalysed by the enzyme DNA polymerase

4. each new DNA molecule contains one strand from original DNA and one new strand

how does DNA polymerase work

• each DNA strand has a directional structure due to the ends of the strand being either a sugar attached to the 5th carbon, 5’(5 prime end), or a hydroxyl group attached to the 3’ end

• DNA polymerase is only complementary to the 3’ end of the template strand, so it can only move along the template strand and add nucleotides in the 3’ to 5’ direction so the new strand is built 5’ to 3’ bc the strands are antiparallel

• 1 strand continously built, whilst the other is built in sections in the opposite direction as DNA is unwound

• the DNA polymerase on the opposite template has to detach and reattach so it often moves more slowly

experimental evidence for semi-conservative replication

conservative replication was still a possibility until Melelson and Stahl proved DNA replication was semi conservative

• nucleotides contain Nitrogen. They used one light N and one heavy N isotope. DNA samples containing different isotopes can be separated by weight in a centrifuge as heavy DNA sinks and light DNA settles out higher up in the solution

• to get samples of light and heavy DNA, they grew 2 cultures of bacteria in nutrient broth containing either light of heavy N so it would be in their nucleotides

• then the heavy N bacteria were put in a broth w only light N, so when they replicated the parent strand would contain heavy nucleotides but only be able to use light nucleotides to make the new strand

• if replication was semi conservative→Dna is a mixture of heavy and light/ one strand of each so DNA would settle in the middle in the centrifuge solution. This is what happened

• The second generation showed that semi conservative replication continued bc a light band was formed. This is because the DNA molecule splits 2 strands would have been formed with the light DNA strand as the template and two would be a mix as they used the original heavy DNA as a template.

ATP

• energy is stored in the bonds between phosphates and released when they are hydrolysed

• phosphates that have been separated from ATP can be added to other molecules to make them more reactive(phosphorylating) Dephosphorylation is the opposite

• ATP is hydrolysed w ATP hydrolase, and synthesised from ADP by ATP synthisase

endergonic and exergonic reactions

endergonic- absorbs free energy from surroundings

exergonic- spontaneously releases energy for other molecuels

phosphorylation reaction

inorganic ions: hydrogen, iron, sodium, phosphate

inorganic ion- ion that doesn’t contain carbon. They occur in solutions in the cytoplasm and body fluids of organism, in various concentrations

hydrogen ions(H⁺): determine pH of substances such as blood, the higher the concentration of H⁺ ions, the lower the pH

Iron ions(Fe²⁺/Fe³⁺): a component of haemoglobin-an oxygen carrying molecule in red blood cells

sodium ions(Na⁺): involved in co-transport of glucose and amino acids

phosphate ions(PO₄^3-): a component in DNA and ATP

method for measuring effect of temperature on rate of enzyme-controlled reactions

1. make 2 controlled variables:

• add 5cm³ milk suspension+5cm³ distilled water to a test tube(no enzyme activity)

• add 5cm³ milk suspension + 5cm³ HCl to a test tube(completely hydrolysed sample)

2. In another 3 test tubes add 5cm³ milk to each and place in a water bath at 10c for 5 minutes to equilibirate 

3. add 5cm³ trypsin to each test tube at the same time and immediately start the timer

4. record how long it takes for the milk samples to completely hydrolyse and turn colourless

5. repeat steps 2-4 at 20c,30c,40c,50c, and 60c

6. find the mean time for milk to hydrolyse at each temperature and work out the rate of reaction rate of reaction= 1/mean time

• milk contains a protein called casein which causes the milk to turn colourless when broken down. Trypsin is a protease enzyme to hydrolyse casein.