Biological molecules

Testing for biological molecules: the four standard tests

Every one of these tests works the same way: a reagent is added to a sample and a colour change shows that a particular chemical group is present. The Benedict's test detects reducing sugars. Add an equal volume of Benedict's solution (blue, because it contains copper(II) sulphate in alkaline solution) to the sample and heat it in a water bath at about 80 °C for several minutes. The blue colour passes through green, yellow and orange to a brick-red precipitate as the amount of reducing sugar rises; the precipitate is copper(I) oxide, formed because the sugar reduces the copper(II) ions.

The iodine test detects starch. Add two or three drops of iodine in potassium iodide solution to the sample at room temperature and a blue-black colour appears. The colour is not the colour of iodine itself: iodine molecules slip inside the coiled helix formed by amylose and the trapped iodine changes the way light is absorbed. No other carbohydrate gives this result with iodine.

The emulsion test detects lipids. Shake the sample with about 2 cm3 of ethanol so that any lipid dissolves in it, then pour the ethanol into water. A lipid is soluble in ethanol but not in water, so it comes out of solution as billions of tiny droplets, and a cloudy white emulsion is seen. The cloudiness can be graded from faint to milky to judge roughly how much lipid is present.

The biuret test detects proteins and depends on the peptide bonds that join amino acids together. Add dilute sodium hydroxide to make the solution alkaline, then add a few drops of dilute copper(II) sulphate solution. A purple (lilac) colour appears because the copper(II) ions form a coloured complex with the nitrogen atoms of the peptide bonds. A single amino acid solution also gives a faint colour, but only peptides and proteins give the strong purple.

The semi-quantitative Benedict's test and testing for non-reducing sugars

A plain Benedict's test only tells you whether a reducing sugar is there. To make it semi-quantitative, standardise the method: keep the volume of sample, the volume of Benedict's solution, the temperature of the water bath and the length of the heating time all the same, and change only the concentration of the reducing sugar. There are two ways to use the result. The first is the time to first colour change: the more reducing sugar there is, the more quickly the blue colour disappears, because the copper(II) ions are reduced faster. The second is to make a set of colour standards from glucose solutions of known concentration and match the unknown sample to the closest standard.

The method is only as good as its controls, and the colour judgement is subjective. Two people looking at the same tube can disagree about whether the colour is yellow or orange, so the ranking of a set of tubes is more reliable than a single reading. A colorimeter makes the test quantitative: the wavelength is set to the red part of the spectrum because a reduced solution has lost its blue colour, and the higher the concentration of reducing sugar, the lower the absorbance (the higher the percentage transmission).

Sucrose gives a negative Benedict's result, so if a sample tests negative you must carry out a second test to find out whether a non-reducing sugar is present. Add dilute hydrochloric acid and heat the solution in a water bath: the acid hydrolyses the glycosidic bond, releasing glucose and fructose. Then neutralise the acid by adding sodium hydrogencarbonate until the solution stops fizzing and pH paper shows it is neutral. Finally add Benedict's solution and heat again. A brick-red precipitate in this second test shows that reducing sugar has been released, so the original solution contained a non-reducing sugar.

The neutralising step is the one candidates leave out. Benedict's solution works because its copper(II) ions are reduced in alkaline conditions, so if the acid is still in the tube the second test stays stubbornly blue and you would wrongly conclude that the sample contained no sugar at all. Never add both reagents at once either: the acid must do its work and then be neutralised, because the two steps do opposite things.

• Standardise the test: same volume of sample, same volume of Benedict's solution, same water bath temperature, same heating time.
• Time to first colour change: shorter time means more reducing sugar.
• Colour standards: match the sample to a series of glucose solutions of known concentration.
• Non-reducing sugar test: acid hydrolysis, then neutralise with sodium hydrogencarbonate, then Benedict's test.
• Bench technique: label the tubes, use a separate sample for each test, and judge all the tubes in one sitting so the comparison is fair.

Monosaccharides, disaccharides and the glycosidic bond

A monomer is a small molecule that can be joined to many other similar molecules. A polymer is a large molecule built from many monomers joined together, and a very large polymer is called a macromolecule. The monomers of the carbohydrates are the monosaccharides, single sugar units such as glucose and fructose with the formula C6H12O6. Two monosaccharides joined together form a disaccharide, and many joined together form a polysaccharide.

Glucose exists as a ring in solution, and it comes in two forms that you must be able to draw. In the ring, carbon 1 is joined to the oxygen that was on carbon 5, leaving an -OH group sticking out from carbon 1. In alpha-glucose that -OH group on carbon 1 points down; in beta-glucose it points up. Nothing else is different: the same six carbons, the same formula, the same everything except the position of one group. That single difference decides whether the polymer built from it coils into a helix or stays straight, and therefore whether the polymer is a storage molecule or a structural one.

Monomers are joined by condensation, in which a hydroxyl group of one sugar and a hydrogen of the other are removed together as a molecule of water, and a glycosidic bond forms between the two carbons. Glucose joined to glucose by an alpha-1,4 glycosidic bond gives maltose; glucose joined to fructose gives sucrose; glucose joined to galactose gives lactose. The bond is broken by hydrolysis, the addition of water, which is exactly what happens when amylase digests starch and when acid hydrolysis is used in the non-reducing sugar test.

The reducing sugars are the ones with a free aldehyde or ketone group that can reduce copper(II) ions. Glucose, fructose and maltose are all reducing sugars, which is why they turn Benedict's solution brick-red. Sucrose is a non-reducing sugar: the glycosidic bond joins carbon 1 of the glucose ring to carbon 2 of the fructose ring, so neither ring has a free reducing group left, and the copper(II) ions are never reduced.

Starch, glycogen and cellulose: polymers of glucose with different jobs

Starch is the storage polysaccharide of plants and is a mixture of two polymers. Amylose is a long, unbranched chain of alpha-glucose joined by alpha-1,4 glycosidic bonds that coils into a helix. Amylopectin is also made of alpha-glucose, but along its chain there are alpha-1,6 glycosidic bonds that create branches. Starch is stored as grains in potato tubers, in the chloroplasts of leaves during the day, and in cereal grains such as rice, wheat and maize.

Every feature of starch suits it to storage. It is insoluble, so it does not dissolve in the cytoplasm, does not change the water potential of the cell and therefore does not draw water in by osmosis. It is a large molecule, so it cannot diffuse out of the cell and is not lost. It is compact, because the helical amylose packs a great deal of glucose into a small volume, so a plant can store a lot of energy without carrying a lot of bulk. The branches of amylopectin give many free ends, and because amylase and other hydrolytic enzymes work inwards from an end, the branching allows glucose to be released rapidly when the plant needs it for respiration.

Glycogen is the storage polysaccharide of animals and is the animal version of amylopectin, but with many more branches and shorter chains. The extra branching gives even more free ends, so glucose can be released even faster, which matters because animals have a much higher metabolic rate than plants and their muscles may need glucose within seconds. Glycogen is stored as granules in liver and muscle cells, and, like starch, it is insoluble and compact so it can be laid down without affecting the water potential of the cytoplasm.

Cellulose is the structural polysaccharide of plant cell walls and is built from beta-glucose. In the chain, every other beta-glucose molecule is turned through 180 degrees so that the -OH groups line up, and this makes the chain straight and unbranched instead of coiled. The chains are held side by side by hydrogen bonds between the -OH groups of neighbouring chains, and bundles of a few dozen chains form microfibrils. Long, straight, hydrogen-bonded microfibrils have a very high tensile strength, so the cell wall can resist the turgor pressure of the cell inside it and stop the cell bursting when it takes up water, and the criss-cross arrangement of the microfibrils keeps the cell's shape and supports the whole plant. The microfibrils are also spaced far enough apart for water and dissolved solutes to pass through the wall, so the wall is freely permeable.

Triglycerides and phospholipids: the non-polar molecules of life

A triglyceride is made of one molecule of glycerol joined to three fatty acids. Glycerol is an alcohol with three hydroxyl groups, and each fatty acid has a carboxyl group (-COOH) at one end and a long hydrocarbon tail at the other. Each fatty acid is joined to the glycerol by an ester bond, formed by a condensation reaction, so three molecules of water are released for every triglyceride made.

Fatty acids come in two kinds. A saturated fatty acid has no carbon to carbon double bonds in its hydrocarbon tail, so the tail is a straight chain and the molecules can pack closely together; fats built from them, such as butter, lard and the fat around a kidney, are solid at room temperature. An unsaturated fatty acid has one double bond (monounsaturated) or several (polyunsaturated), and each double bond puts a kink in the tail. The kinked molecules cannot pack closely, so the interactions between them are weaker and unsaturated oils, such as olive oil and sunflower oil, are liquid at room temperature.

Triglycerides are non-polar and hydrophobic. They have no charged groups and no groups carrying a partial charge that could form hydrogen bonds with water, so water molecules are not attracted to them and the two do not mix. This single property explains most of their functions. They are a compact energy store, because the long hydrocarbon tails are rich in carbon to hydrogen bonds and so release roughly twice as much energy per gram as carbohydrate when respired. They are insoluble, so stored fat does not affect the water potential of the cytoplasm and does not drag water into the cell by osmosis. They can also be stored without water, unlike glycogen, which holds water with it, and so save mass - one reason birds and desert animals rely on fat for migration and for survival.

Fat is also used for thermal insulation, as in the blubber of whales and seals and the subcutaneous fat of animals living in cold seas, and for protection, as in the fat that cushions the kidneys and the heart. Oxidising fat releases metabolic water, which is a real advantage for animals living in deserts, such as the camel and the kangaroo rat. A phospholipid is a modified triglyceride: glycerol is joined to two fatty acids and to a phosphate group. The phosphate group is charged, so the head is hydrophilic (polar) and mixes readily with water, while the two fatty acid tails are hydrophobic (non-polar). A molecule that has a water-loving end and a water-hating end arranges itself in water with the tails together and the heads facing the water, and this is the basis of the phospholipid bilayer of every cell membrane.

• Triglyceride = glycerol + 3 fatty acids, joined by 3 ester bonds formed by condensation.
• Saturated fatty acid: no C=C double bonds, straight tail, packs closely, higher melting point.
• Unsaturated fatty acid: one or more C=C double bonds, kinked tail, packs loosely, lower melting point.
• Triglyceride functions: energy store, insulation, protection and a source of metabolic water.
• Phospholipid = glycerol + 2 fatty acids + a phosphate group, with a hydrophilic head and hydrophobic tails.

Amino acids, peptide bonds and the four levels of protein structure

Every amino acid has the same general structure. A central carbon atom is bonded to an amino group (-NH2), a carboxyl group (-COOH), a hydrogen atom and an R group. The R group is the only part that differs from one amino acid to the next, and it is the R group that makes one amino acid acidic, another basic, another polar and another hydrophobic, or gives it a sulphur-containing group. In the conditions inside a cell the amino group carries a positive charge and the carboxyl group a negative charge, so you should draw them as -NH3+ and -COO-.

Two amino acids are joined by a condensation reaction between the carboxyl group of one and the amino group of the next. A molecule of water is released and a peptide bond forms between the carbon of the carboxyl group and the nitrogen of the amino group. Two amino acids joined this way make a dipeptide, and a chain of many makes a polypeptide. Peptide bonds are broken by hydrolysis: proteases hydrolyse them during digestion, and boiling with acid or alkali hydrolyses them too, which is why prolonged boiling destroys a protein permanently while ordinary heat treatment only denatures it.

Primary structure is the sequence and number of amino acids in the polypeptide chain. It is the most important of the four levels, because the R groups along the chain decide where every later interaction can form, so the primary structure determines the secondary, tertiary and quaternary structure. It is held by the strong covalent peptide bonds.

Secondary structure is the local folding of the chain into an alpha-helix or a beta-pleated sheet, held by hydrogen bonds between the partly negative oxygen of one C=O group and the partly positive hydrogen of an N-H group further along the same chain. Tertiary structure is the overall three-dimensional shape of the whole chain, held by four types of interaction between R groups: hydrogen bonds between polar R groups, ionic bonds between R groups carrying opposite charges, hydrophobic interactions where non-polar R groups are pushed together away from water, and covalent disulfide bonds between the sulphur atoms of two cysteine residues. Quaternary structure is the way two or more separate polypeptide chains, and any non-protein groups attached to them, are arranged together in one functional protein - as in haemoglobin, insulin and antibodies.

Haemoglobin and collagen: globular and fibrous proteins

Globular proteins are roughly spherical. The non-polar R groups are tucked away in the interior and the polar and charged R groups face outwards towards the water, so globular proteins are generally soluble. Their chains fold into a precise shape with a specific binding site, so they have physiological (metabolic) roles: enzymes, antibodies, hormones such as insulin and the oxygen carrier haemoglobin. Fibrous proteins are long chains lying parallel to one another and cross-linked at many points. They are generally insoluble and their chains form long, tough structures, so they have structural roles: collagen in tendons, bone and artery walls, keratin in hair, skin and nails, and elastin in the lungs. Solubility and role go together, and each follows from the amino acid sequence.

Haemoglobin is a globular protein with a quaternary structure built from four polypeptide chains: two alpha-globin chains and two beta-globin chains. Each chain is folded around a non-protein haem group, and at the centre of each haem group is an iron ion (Fe2+). The four haem groups give haemoglobin its dark red colour, and each iron ion can bind one molecule of oxygen, so one haemoglobin molecule can carry four molecules of oxygen. The importance of iron is that the oxygen binds reversibly to the Fe2+ ion: in the lungs, where the concentration of oxygen is high, oxygen joins to form oxyhaemoglobin, and in the tissues, where the concentration is low, the oxygen is released again. A person with iron deficiency anaemia has less iron available, so less haemoglobin is made and less oxygen can be carried, which is why they feel tired and short of breath on exertion. Because haemoglobin is globular and soluble, it can be packed into the cytoplasm of a red blood cell in very high concentration.

Collagen is the most abundant protein in a mammal and is a fibrous protein. Its primary structure is a repeating sequence of about a thousand amino acids in which every third amino acid is glycine, with a high proportion of proline and hydroxyproline in between. Glycine is the smallest amino acid and its single hydrogen atom can fit into the crowded centre of the molecule, which is why it must occur so regularly. Three of these chains wind round one another to form a triple helix, and the triple helices lie side by side and are cross-linked to each other by strong covalent bonds between their R groups, as well as by hydrogen bonds. The cross-linked bundles form collagen fibrils, and the fibrils are grouped into larger collagen fibres.

That structure explains every use of collagen. The covalent cross-links between the triple helices give collagen a very high tensile strength, so tendons, which join muscle to bone, can transmit the full force of a contracting muscle without stretching. The same strength, combined with a little flexibility, is why collagen is a major part of the matrix of bone, of cartilage at the joints and of the walls of arteries, which must stretch with every heartbeat and spring back without tearing. Collagen is insoluble, so it is not washed out of the tissue it strengthens, and its long fibres resist pulling but still allow the slight movement that a tendon, a joint or a blood vessel needs.

• Globular: spherical, polar R groups outside, soluble, physiological role - haemoglobin, enzymes, antibodies.
• Fibrous: long parallel chains, cross-linked, insoluble, structural role - collagen, keratin, elastin.
• Haemoglobin: 2 alpha-globin + 2 beta-globin chains, 4 haem groups, 4 Fe2+ ions, so 4 oxygen molecules per molecule of haemoglobin.
• Collagen: glycine every third residue, three chains wound into a triple helix, cross-linked into fibrils and then fibres.

Water: hydrogen bonding and why life depends on it

In a water molecule the oxygen atom is far more electronegative than the two hydrogen atoms, so it pulls the shared electrons towards itself. The oxygen therefore carries a small partial negative charge and each hydrogen carries a small partial positive charge, and the molecule as a whole is polar. A hydrogen bond is the weak attraction between the partially positive hydrogen of one water molecule and the partially negative oxygen of another. Each water molecule can form hydrogen bonds with up to four others, so liquid water is not a collection of separate molecules but one enormous, constantly rearranging network.

Solvent action. Because water is polar, it is attracted to other polar and charged particles. Water molecules surround ions such as sodium and chloride and the polar groups of sugars and amino acids, and pull them apart, so they dissolve. This is why water is the solvent of life: almost every metabolic reaction in a cell happens in solution in the cytoplasm, blood plasma and cell sap, and dissolved substances can be transported from one part of an organism to another. Non-polar molecules such as lipids and the hydrocarbon parts of membranes do not dissolve in water, which is exactly why membranes form a barrier and why hydrophobic interactions hold a globular protein in shape.

High specific heat capacity. The specific heat capacity of water is about 4.2 J per gram per °C, which is much higher than that of most liquids. Raising the temperature of water means making its molecules move faster, but much of the heat energy supplied is used instead to break hydrogen bonds, so a large amount of energy produces only a small rise in temperature. Water therefore acts as a thermal buffer: the temperature of the cytoplasm stays stable even when the reactions inside the cell release heat; the temperature of a pond, lake or ocean changes only slowly as the air temperature changes, so aquatic organisms avoid lethal temperature swings; and a large body of water warms and cools the air over it, which is why coastal climates are mild. It also means that organisms themselves change temperature only slowly and can keep a steady body temperature.

Latent heat of vaporisation. The latent heat of vaporisation of water is about 2.26 kJ per gram, and it is high for the same reason: before a water molecule can escape from the surface as a vapour, the hydrogen bonds holding it to its neighbours must be broken. Because so much energy is needed to evaporate a small mass of water, evaporating water removes a great deal of heat from the surface it leaves. Sweating in mammals and panting in dogs use this to cool the body, and transpiration from leaves cools a plant and prevents in some part the leaves overheating in bright sunshine. It is also why water does not evaporate away from a pond as quickly as a non-hydrogen-bonded liquid would, which matters because a pond that dried out quickly would kill everything in it.

• Water is polar: partial negative oxygen, partial positive hydrogens.
• A hydrogen bond joins the partial positive hydrogen of one molecule to the partial negative oxygen of another; each molecule can form up to four.
• Solvent action: polar and charged particles dissolve in water, so metabolic reactions take place in solution and substances can be transported.
• High specific heat capacity: much of the heat is used to break hydrogen bonds, so water resists changes in temperature.
• High latent heat of vaporisation: hydrogen bonds must be broken for molecules to escape, so evaporating water carries away a lot of heat.