130327SCH4U_biochem April 09, 2013

Transcription

1 Option B: B1.1 ENERGY Human Biochemistry If more energy is taken in from food than is used up, weight gain will follow. Similarly if more energy is used than we supply our body with, weight loss will occur. It is important to know how much energy is available from a particular food, so processed foods are labelled with energy content, as well as percentages of carbohydrates, fats and protein. Calorific value (energy content) of foods is measured in kj g 1, kj 100 g 1 or even kj mol 1 if the food is a pure substance, such as glucose. An accurately weighed sample of the food is burnt in oxygen in a bomb calorimeter. This is a sealed metal container or bomb containing a crucible in which the food is held. A sparking device is used to start the combustion of the food in the oxygen-rich atmosphere. Heat released during the combustion of the food is conducted through the metal bomb to a measured amount of water in an insulated container surrounding it. The temperature of the water increases and the temperature change is measured with a thermometer. c= 4.18 for water

2 2.2 PROTEINS Proteins are natural polymers (biopolymers) that are essential to life. All organisms, even the tiniest viruses, contain proteins. Proteins have many different functions in living things. For example, they act as biological catalysts (enzymes), they give structure (hair, muscle, feathers and nails), they provide energy and, in some cases, they are hormones. Proteins are polymers of 2-amino acids. All 20 of the amino acids are required for protein synthesis, but humans are thought to be able to synthesize only 10 of them. The remaining 10 are called essential amino acids and must be obtained from the diet.

3 In a neutral solution an amino acid may both lose an H+ ion from the carboxyl group and gain an H+ on the amino group. Such an ion with both a negative and positive charge on it at the same time is known as a zwitterion (from the German, zwei meaning two). The ph at which an amino acid occurs as a zwitterion is called its isoelectric point. At this ph the amino acid carries no net electrical charge.

4 The reaction of many amino acids produces a condensation polymer called a polypeptide, in which hundreds or even thousands of amino acids are joined. There are many millions of different combinations of sequences of the 20 different amino acids. If there is a large enough number of amino acids involved, the polymer produced is called a protein rather than a polypeptide. Each protein contains a fixed number of amino acids in a particular sequence. This order, number and type of amino acids making up the polymer is called the primary structure of the protein. The presence of oxygen and nitrogen atoms in a protein results in significant polarity within the polymer chains. Dipole dipole bonding occurs at regular intervals between parts of the polypeptide chains, resulting in two major structures: α-helices, which are coils of protein, like springs; and β-pleated sheets, which have a corrugated appearance. These helices and sheets are referred to as the secondary structure of the protein. If the coiled protein molecules fold over themselves to form a particular threedimensional shape, the protein is said to have a tertiary structure. The unique, tertiary structure of a protein is responsible for the unique function of that protein. Separate polypeptide chains may interact with each other to give further complexity to the structure. This is known as the quaternary structure. This is the overall arrangement of the polypeptide chain to form the working shape of the protein. Hemoglobin has a quaternary structure that involves four polypeptide chains grouped together around four iron ions. These interactions may be due to hydrogen bonds, van der Waals forces between non-polar side groups, ionic attractions between ionized side groups, ion dipole attractions or covalent bonds (disulfide bridges) that form when sulfurcontaining side groups react.

5 Physical and chemical agents may destroy these three-dimensional protein shapes, changing the nature of the protein. Such changes are called denaturation. For example, heat breaks hydrogen bonds, so heating a protein strongly destroys the helical structure. In some proteins, heat causes the unfolding of the polypeptide chains, followed by a more random re-forming of bonds, resulting in precipitation or coagulation. This is what happens when an egg is boiled. Proteins perform a wide range of functions in the body. They are used for structure, movement, transport, catalysis of chemical reactions, energy transformation and storage, protection, control and buffering. Electrophoresis is an analytical technique that separates charged substances according to the different rates at which they move (depending on their size and electrical charge) when subjected to a potential difference. In preparing a protein sample for electrophoresis, the protein is denatured and hydrolysed, when it is heated in a solution of hydrochloric acid for a period of time. This separates the amino acids from each other. In PAGE (polyacrylamide gel electrophoresis) a tiny sample of protein solution is placed into a well in the centre of a polyacrylamide gel. The gel is moistened with buffer solution and a voltage is applied. Depending on the ph of the buffer solution, the amino acids separate according to their charge and their mass Let us consider a mixture of amino acids being separated by PAGE. If the ph of the buffer solution is equal to the isoelectric point of one of the amino acids in the mixture, that amino acid will not be charged in the buffer solution and it will not move beyond the starting position. Amino acids with isoelectric points that are greater than the ph of the buffer solution will be positively charged and will migrate towards the negative terminal. The greater the mass of the amino acid, the slower will be its progress through the gel. The gel may be developed by spraying with ninhydrin, an organic dye that changes the colour of the amino acids and the amino acids will appear as bands along the length of the gel.

6 Negative Terminal Positive Terminal ph=6.0 Proteins can also be analysed by gel electrophoresis without being hydrolysed into individual amino acids. The technique known as SDS PAGE involves the denaturing of the proteins in the presence of a detergent called sodium dodecyl sulfate (SDS) that coats the proteins with a negative charge. The protein mixture is placed at one end of the gel and a potential difference is applied across the gel. The proteins migrate towards the positive terminal and separate according to their molecular masses, with the lower molecular mass proteins moving at a greater rate. Marker proteins are run as standards to calibrate the process. These proteins have known molecular masses, so the unknowns can be compared to them. To release the amino acids from the protein chain, the peptide bonds must be hydrolysed. The solution of amino acids is spotted onto the paper which is then placed in a developing tank containing the mobile phase which will be a solvent or a mixture of solvents. When the mobile phase has moved almost the full length of the paper, the different amino acids will have moved different distances due to the relative attraction of their different side groups to the stationary and mobile phases. Since the amino acids are colourless, their positions on the chromatogram cannot be distinguished until the finished chromatogram is sprayed with a solution of ninhydrin. Chromatography separates components of a mixture according to their relative attraction to a stationary phase and a mobile phase. In paper chromatography, the stationary phase is a strip of filter paper.

7 The amino acids that have separated from the mixture can be identified by comparing their positions on the chromatogram with those of pure samples of amino acids that have been run on a chromatogram under the same conditions. The calculation of a retention factor (Rf) for each amino acid will give a more definite identification. The Rf is determined by measuring the distances moved by each amino acid and by the solvent: A more sophisticated chromatographic method for the separation of proteins, such as the proteins in milk and those found in blood, is high performance liquid chromatography (HPLC). In this technique the mixture in solution is swept along a column containing the stationary phase under high pressure. Separation of the components of the mixture occurs due to their relative attractions to the stationary and mobile phases. The components are identified by a UV detector since these colourless compounds absorb in the ultraviolet region of the electromagnetic spectrum.