Biological molecules(section 1) part 2 (Proteins (Structure of an amino…
Biological molecules(section 1) part 2
reducing sugars (benedicts test)
add 2cm^3 of food sample to test tube. if sample not liquid, grind up in water.
add equal volume of benedicts reagent
heat mixture in gently boiling water bath for 5 min
if reducing sugar present, solution = orange-brown
theory behind the test
all monosaccharides and some disaccharides (maltose) are reducing sugars.
reduction = gain of electrons or hydrogen.
reducing sugar = sugar that can donate electrons to (or reduce) another chemical in this case = benedicts reagent.
Benedict's reagent = alkaline solution of copper2 sulfate.
when reducing sugar heated with benedicts reagent it forms and insoluble red precipitate of copper1 oxide.
theory behind test
Known as the emulsion test.
The cloudy colour is dur to the lipid sample being finely dispersed in the water to form an emulsion.
light passing through this emulsion is refracted as it passes from oil droplets to water droplets making it appear cloudy.
Take completely dry and grease-free test tube.
Add 5cm^3 of ethanol to 2cm^3 of sample being tested
Shake tube thoroughly to dissolve any lipid In sample
Add 5cm^3 of water and shake gently
A cloudy-white colour indicates the presence of a lipid.
As a control, repeat the procedures using water instead of the sample; the final solution should remain clear.
theory behind test
easily detected by its ability to change colour of iodine in potassium iodine solution from yellow to blue-black.
test carried out at room temp
2cm^3 of sample in test tube.
Add 2 drops of iodine solution and shake/stir.
Presence of starch indicated by blue- black coloration.
if sample is not liquid, grind up in water
Add 2cm^3 of benedict's reagent in test tube and filter
Place test tube in gently boiling water bath for 5 mins, if solution does not change colour reducing sugar is not present
Add another 2cm^3of dilute HCL in a test tube and place it in a gently water bath for 5 mins. the dilute HCL will hydrolyse any disaccharide present into monosaccharides.
Slowly add sodium hydrogencarbonate to test tube to neutralise the HCL. test with PH paper to check solution is alkaline
Re-test solution by heating with 2cm^3 of benedict's reagent in gently boiling water bath for 5 mins
if non-reducing sugar was present in original sample Benedict's reagent will now turn orange-brown. due to reducing sugars that were produced in hydrolysis of non-reducing sugar.
Test for proteins(Biuret test- detects peptide bonds)
Place sample of solution to be tested in test tube. Add equal vol of sodium hydroxide at room temp.
Add few drops of very dilute (0.05%) copper2 sulfate solution and mix.
Purple coloration indicates presence of peptide bonds = protein
no protein = solution remains blue.
Structure of an amino acid
Amino acids = basic monomer units which combine to make up a polymer = polypeptide. Polypeptides can be combined to form proteins. About 100 amino acids have been identified, of which 20 occur naturally in proteins. indirect evidence for evolution.
Every amino acid has a central carbon atom to which are attached four different chemical groups:
Amino group (-NH2) = basic group
Carboxyl group (-COOH) = an acidic group, gives amino acid the acid part of its name
Hydrogen atom (-H)
R (side) group = a variety of diff chemical groups. each amino acid has a diff R group. these 20 naturally occurring amino acids only differ in their R (side) group.
Formation of peptide bond
Amino acid monomers can combine to form a dipeptide.
Process = removal of a water molecule in condensation reaction.
Water made from OH from carboxyl group and H from amino group of another amino acid.
The two amino acids become linked by new peptide bond between carbon atom of one amino acid and nitrogen atom of the other.
Peptide bond can be broken down by Hydrolysis
Primary structure of proteins-Polypeptides
Through a series of condensation reactions, many amino acids can be joined together in process called polymerisation.
Resulting in a chain of many hundreds of amino acids called polypeptide.
Sequence of amino acids in polypeptide chain forms primary structure of any protein
Polypeptides have usually hundreds of the 20 naturally occurring amino acids joined together in different sequences. there are almost a limitless number of possible combinations and therefore types of primary protein structure.
Secondary structure of Proteins
The linked amino acids that make up a polypeptide possess both-NH and -C=O groups on either side of every peptide bond.
The H of the -NH group has an overall positive charge, the O has an overall negative charge.
These 2 groups form readily weak bonds = Hydrogen bonds.
Causes the long polypeptide chain to be twisted into a 3D shape such as the coil known as an alpha-helix
Tertiary structure of proteins:
The alpha-helixes of the secondary protein structure can be twisted and folded more to give the complex 3D structure of each protein.
Where bonds occur depends on primary structure of protein.
Tertiary structure is maintained by a number of diff bonds:
Disulfide bridges: Fairly strong and therefore not easily broken
Ionic bonds: formed between any carboxyl and amino groups that are not involved in forming peptide bonds. weaker than disulfide bonds, easily broken by changes of pH.
Hydrogen bonds: numerous but easily broken.
Quaternary Structure of proteins
Large proteins often form complex molecules containing a number of individual polypeptide chains that are linked in various ways.
There may also be non-protein (prosthetic) groups associated with the molecules. E.g. the iron-containing haem group in haemoglobin.
it is the sequence of amino acids that determines the 3-D shape in the first place.
Enzymes = globular proteins, act as catalysts
Catalysts alter rate of reaction without undergoing permanent change themselves.
Enzymes as catalysts lowering activation energy:
Typical chemical reaction:
sucrose + water --> glucose + fructose
For reactions like this:
sucrose and water molecules must collide with sufficient energy to alter arrangement of their atoms to form glucose and fructose.
The free energy of products (glucose and fructose) must be less than that of the substrates (Sucrose and Fructose)
Many reactions require an initial amount of energy to start. minimum amount of energy needed to activate reaction = activation energy.
Enzymes work by lowering activation energy level. enzymes allow reactions to take place at a lower temp than normal
Active site made up of a relatively small number of amino acids. The active site forms a small depression within the much larger enzyme molecule.
The molecule on which the enzyme acts = substrate.
Fits neatly into this depression and forms an enzyme-substrate complex.
Substrate molecule held within active site by bonds that temporarily form between amino acids of active site and groups on the substrate molecule
Induced fit model of enzyme action:
Suggests that the enzyme = flexible and can mould itself around the substrate to fit the shape of the substrate.
As enzyme changes shape it puts a strain on the substrate molecule. Strain distorts a particular bond/ bonds in substrate --> lowers activation energy needed to break bond.
Factors affecting enzyme action
Effect of temperature on enzyme action
Rise in temp increases kinetic energy of molecules.
molecules move around rapidly and collide more often.
In enzyme-catalysed reaction, enzyme and substrate come together more often.
Usually at 60 degrees enzyme is disrupted and stops working= denatured.
Denature = permanent change, enzyme does not function again.
Effect of pH on enzyme action
PH of solution = conc of H+ ions
Hydrogen ion conc = 1 x 10^-9. pH = 9
Increase/ decrease in pH reduces rate of enzyme action. If change in pH = more extreme pH becomes denatured.
Change in pH alters charges on amino acid that make up active site of enzyme. substrate can no longer become attached to active site so enzyme substrate complex cannot be formed.
Depending on how significant the change in pH is, may cause bonds maintaining enzyme's tertiary structure to break. Active site changes shape.
pH fluctuations inside organisms = usually small, means far more likely to reduce an enzymes activity than denature it.
Effect of enzyme concentration on rate of reaction
Enzymes = not used up in reaction and therefore work efficiently at very low concentrations.
As long as there is an excess of substrate, an increase in the amount of enzyme leads to a proportionate increase in the rate of reaction.
If you increase the enzyme conc some of the excess substrate can now also be acted upon and the rate of reaction will increase.
If substrate = limiting, any increase in substrate conc will have no effect on rate of reaction. rate of reaction will stabilise at a constant level --> graph will level off.
Effect of substrate concentration on rate of enzyme action
If conc of enzyme = fixed and substrate conc is slowly increased, rate of reaction increases in proportion to conc of substrate.
Because at a low substrate conc, the enzyme molecules have only a limited number of substrate molecules to collide with, therefore the active sites of enzymes are not working to full capacity.
As more substrate added active sites gradually become filled, until point where all of them working as fast as they can. Rate of reaction at maximum.
After that addition of more substrate will have no effect on RoR --> there is an excess of substrate, RoR levels off.
Substances that directly/ indirectly interfere with functioning of active site of an enzyme and so reduced its activity.
have a molecular shape similar to that of the substrate.
Allows them to occupy the active site of an enzyme.
Therefore they compete with substrate for available active sites.
Its the diff between conc of the inhibitor and the conc of the substrate that determines effect it has on enzyme activity.
If substrate conc = increased, effect of inhibitor = reduced.
Inhibitor not permanently bound to active site.
Example of inhibitor occurs with an important respiratory enzyme that acts on succinate
Malonate --> can inhibit enzyme because it has a very similar molecular shape to succinate.
Combines with enzyme and block succinate from combining with enzyme's active site.
Attach themselves to enzyme at a binding site --> not active site.
Upon attaching to enzyme, inhibitor alters shape of enzyme therefore active site so that a substrate molecule can no longer occupy it so enzyme cannot function.
As substrate and inhibitor are not competing for same site, increase in substrate conc does not decrease effect of inhibitor