Biochemical tests using Benedict’s reagent for reducing sugars and non-reducing sugars. Iodine/potassium iodide solution for starch.

Add Benedicts solution to the sample and heat:

• If it turns orange it is a reducing sugar
• If its blue then do the following:

Add hydrochloric acid and heat for a further 5 minutes, after this add hydrogen carbonate to neutralise it. Now repeat the process from the begining of the test and add benedicts solution and then heat:

• If it turns orange then it is a non-reducing sugar
• If it stays blue then it is not a sugar at all.

Acid is added to hydrolyse the non-reducing sugars breaking them down into monomers- reducing sugars.
The solution is neutralised as acid would prevent the Benedicts test from working.

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To test for starch pipette a couple of drops of iodine on to the sample you are testing.

• If it stays red then there is no starch
• If it goes blue/black then there is starch.

Biological molecules such as carbohydrates and proteins are often polymers and are based on a small number of chemical elements. Monosaccharides are the basic molecular units (monomers) of which carbohydrates are composed. The structure of a-glucose as... and the linking of a-glucose by glycosidic bonds formed by condensation to form maltose and starch.

Carbohydrates are biological molecules (this means they are produced by living things) they contain Carbon, Hydrogen and Oxygen. They have the empirical formula CH20.

Carbohydrates are often polymers made up of monomers; polysaccharides made up of monosaccharides.

Glucose is a monosaccharide, it is a hexose- which means it contains 6 carbons- so its molecular formula is C6H12O6. There are other hexose which will be made up of the same components, but they are different molecules due to their structure, the structure of alpha-glucose is:

Structure of alpha-glucose

Two monosaccharides join together by a condensation reaction, to make a disaccharide; when more are joined it becomes a polysaccharide. In the condensation reaction between two glucose molecules, a glycosidic bond is formed (bond between the two sugars) creating a disaccharide and water:

Products of a condensation reaction between two glucoses

Two glucoses are joined they form the disaccharide maltose. Many glucoses joined together form the polysaccharide starch,

Starch, the role of salivary and pancreatic amylases and of maltase located in the intestinal epithelium; disaccharides, sucrase and lactase; Sucrose is a disaccharide formed by condensation of glucose and fructose. Lactose is a disaccharide formed by condensation of glucose and galactose. Lactose intolerance.

Starch is a polysaccharide, it is broken down by the body into monosaccharides, in two stages:

• Amylase (released from the salivary and pancreatic glands) breaks starch down into maltose,
• Maltase (found in the intestines) breaks maltose down into glucose.

Sucrose is a disaccharide, it is digested in to two monosaccharides:

• Sucrase breaks sucrose down into glucose and fructose.

Lactose is a disaccharide, it is digested in to two monosaccharides:

• Lactase breaks lactose down into glucose and galactose.

Some peoples bodies do not produce enough lactase, this means there will be undigested lactase in their digestive system (after eating a product containing this sugar, like milk.) This lactase is fermented by bacteria which produces methane. The symptom of this is painful wind and the name of this condition is lactose intolerance.

Having lactose in the intestine decreases the water potential, this causes water to move in by osmosis, diluting the faeces and giving diarrhoea to the sufferer.

Candidates should be able to use the lock and key model to explain the properties of enzymes. They should also recognise its limitations and be able to explain why the induced fit model provides a better explanation of specific enzyme properties

The lock and key theory helps us to understand that an enzyme has an active site that bonds with one kind of substrate.

However it suggests that the structure is rigid and that bonds can only be made to the active site.

Induced fit theory shows us that the structure is flexible and can be changed by the substrate or by bonds at an allosteric site (not the active site.) This helps us see how reactions are sped up- because the substrate does not have to fit into the enzyme in exactly the right way as the enzyme moulds around it.

Description and explanation of the effects of temperature, competitive and non-competitive inhibitors, pH and substrate concentration.

Temperature
At low temperatures there is a less energy, the substrates will move around more slowly making them less likely to reach the active site.
At optimum temperature there is a lot of energy and as substrates move around more they more frequently end up in the active site.
Past optimum temperature there is too much energy for an enzyme, and the bonds holding it together will break: this is called denaturing.

PH
Different types of enzymes have very different optimum PHs.
Above and below this PH the bonds holding the enzyme together will break, and the amino acids will have their charges changed, preventing them from forming bonds with the substrate.

Substrate concentration
At low concentration, substrates get into the active site less frequently meaning they react little.
At medium concentrations, substrates will get into the active site a lot meaning they are constantly reacting.
At high concentrations there will be a substrate in the active site all the time: after this, adding more substrate will not speed up the reaction, because the enzymes are always busy anyway (this is why at a certain point increasing the substrate concentration makes no difference to the rate of reaction.)

Competitive inhibitors
These compete with substrates to bond with the active site of an enzyme.
Once they have bonded with the active site they block it so substrates can't bond with it.
The bonds are usually weak hydrogen bonds, however, and will soon break (reversible.)
The effects of competitive inhibitors can be reduced by adding more substrate because that means it has more substrates to compete with, so a lower chance of getting into the active site.

Non-competitive inhibitors
These bind to an allosteric site (away from the active site) which distorts the active site to make it less complimentary to its substrate. This is usually an irreversible strong covalent bond

The properties of enzymes relating to their tertiary structure.

Enzymes have a globular structure- these are suited to metabolic reactions. Globular structures are ball-like and tend to be flexible.

A small area of an enzyme (3-12 amino acids long) will form a depression on the surface of the enzyme due to how the polypeptide chain has been folded- this is the active site. In this area will be R groups which can form bonds with substrates to make the enzyme substrate complex.

The lock and key and induced fit models of enzyme action.

The lock and key theory
An enzyme has a active site which compliments (fits with) a specific substrate exactly (before binding). It states that the enzyme has a fixed shape. A substrate will go into an enzyme like a key goes into a lock and form an enzyme-substrate complex; this then changes the bonds in the substrate to form the products.

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The induced fit theory
This states that an enzyme has an active site which is not perfect for a substrate, but that it is flexible.
A substrate will alter the shape of the active site to make it complimentary, the changed shape will alter the bonds in the substrate creating the products.

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