Sei sulla pagina 1di 7

Zo Bradshaw Lower Science I Biology Lab (Biuret Test)

Date: 21st November, 2012 Title: Semi-Qualitative Biuret Test Aim: To estimate concentration range of unknown samples of protein solution using the colour standards established. Apparatus and materials: Test tubes, test tube rack, measuring cylinders, syringes, 0.1% protein sample, 2% protein sample, potassium hydroxide, copper sulphate, 3 unknown samples Introduction: Proteins are marcomolecules of a high relative mass. They can appear very different and perform diverse functions and are made from one or more polypeptide chains. Polypeptides are made up of unbranched chains of the 20 different amino acids that cells use to make proteins. These amino acids always contain the elements carbon, hydrogen, oxygen and nitrogen and sometimes sulphur. Even though there are 20 various amino acids which behave in very different ways because they have different side groups, they all share the same molecular formula. The amino acids that make up a protein are linked together during protein synthesis which happens on the ribosomes in a cell. Every amino acid has a central carbon atom, an amino group (NH2) and a carboxylic acid group (COOH). Attached to the central carbon atom are a hydrogen atom (H) and a residual group (R) a group that is specific to the type of amino acid. This R group is a different molecule in different amino acids which can make them neutral, acidic, alkaline, aromatic (has a ring structure) or sulphur-containing. When 2 amino acids are joined together (condensation) the amino group from one and the acid group from another produce one molecule of water and form a peptide bond which links the carbon (C) of the carboxylic group of one amino acid with the nitrogen (N) of the amino group of the other. Due to the bonding and the shape and chemical nature of the 20 various amino acids, the shape of a whole chain of amino acids (a polypeptide or protein) is very specific. Every protein possesses a characteristic three dimensional shape its conformation. In this, there are four separate levels of structure and oganisation.

The first one is typically called the primary structure. This is the sequence of amino acids in a polypeptide chain which dictates it biological function. This sequence is in turn then rigidly controlled by the gene that codes for the protein, i.e. the DNA. The primary structure depends on the order and number of amino acids in a particular protein. The secondary structure of proteins is the basic shape that the chain of amino acids takes on. The 2 most common structures are the a-helix and the 13-pleated sheet these structures are maintained by hydrogen bonds that form between different amino acids. The a-helix has a structure which is like an extended spiral string with the R groups pointing towards the outside of the helix. In it, the hydrogen bonds form between the oxygen of the CO group of one amino acid and the hydrogen of the NH2 group of the amino acid four places ahead of it in the chain. Hydrogen (H) bonds are relatively weak but because there are so many, the total binding effect is strong and stable. The helix is flexible and elastic. 13-pleated sheets are composed of side by side chains, which are more extended than those in a-helixes. They are arranged in a parallel fashion and are connected by hydrogen bonds formed between the C=O and NH groups of one chain and the NH and C~O groups on adjacent chains. The tertiary structure of a protein is described as when the polypeptide chain bends and folds extensively forming a precise, compact, complex globular or fibrous 3D shape held together mainly by hydrogen bonds. Three other bonds also help to maintain this shape they are ionic bonds, disuiphide bonds and hydrophobic interactions between R groups. As with the structure of the -helix, the hydrogen bonds form between some of the polar R groups (side chains) such as the dipolar-NH and CO groups. Ionic bonds are formed between positively and negatively charged or ionised side chains such as the amine and carboxylic acid groups. These are weak interactions, but together they help give the protein a stable shape. Disulphide bonds then help to reinforce the protein with strong covalent bonds which form between two amino acids with sulphur groups on their side chains. Hydrophobic interactions are formed between non-polar side chains. Fibrous proteins are made of long molecules arranged to form fibres. Their primary structures can be more variable then the globular proteins and can have a limited range of various amino acids that can be joined together to form chains of varying length. Fibrous proteins are insoluble in water and usually have a structural role instead of being metabolically active. Globular proteins are made of chains folded into a compact structure. Although these folds are less regular than in a helix, they are highly specific and a particular protein will always be folded in the same way, composed of exactly the same

sequence of amino acids because this determines their function. If the structure is disrupted, the protein ceases to function properly and is said to be denatured. Globular proteins are metabolically active and soluble in water with hydrophilic R groups on the surface forming hydrogen bonds with water molecules and hydrophobic R groups internally that exclude water. In the quarternary structure two or more polypeptide chains associate together to form a protein, the said polypeptide chains can either be the same of different and this structure stabtises and holds the polypeptide chains together. These chains are connected by hydrophobic interactions and hydrogen and ionic bonds. There are also several different types of proteins each with various functions. Almost all enzymes are proteins, there are also structural proteins, for example, collagen and elastin in connective tissue, keratin in skin, hair and nails. Actin and myosin in muscles are contractile proteins which allow contraction and therefore movement. There are also hormonal proteins for instance, insulin which helps to regulate glucose metabolism, glucagon and growth hormone. Transport proteins are another type for example, haemoglobin facilitates the transport of oxygen around the body and a type of albumin in the blood transports fatty acids and lipids. Proteins such as carrier and channel proteins in the cell membrane regulate movement across it. In addition, defence proteins such as immunoglobulins (antibodies) protect the body against foreign invaders; fibrinogen in the blood is vital for the clotting process. Procedure: The Biuret test was conducted on known concentrations of 2cm3 protein solutions to establish a colour standard. For this test 2cm3 of potassium hydroxide and 0.5 cm3 of copper sulphate were used. The Biuret test was then conducted on the unknown samples provided and the exact same procedure for making the colour standard was used. The known colour standard was then used to determine the concentration range of the unknown samples. The results were recorded in a suitable table.

Results: A TABLE ILLUSTRATION THE RESULTS FOUND FROM CONDUCTING THE BIURET TEST ON KNOWN CONCENTRATIONS OF PROTEIN SAMPLES. Concentration of protein sample 4% Observations The mixture turned dark purple in colour. Colour Intensity *** Deductions A dark purple complex was formed meaning that a high concentration of peptide bonds were present. A purple complex was formed meaning that peptide bonds were present.



The mixture turned a little lighter purple than the 4% solution. However, with time the purple in the mixture deepened to give the same dark purple colour as the 4% solution. The mixture turned light blue in colour.

** which turned to *** over time.

No complex was formed and therefore there were no peptide bonds present.

Key: *** - Very dark purple **- Lighter purple *- Lightest purple

A TABLE ILLUSTRATING THE RESULTS FOUND FROM CONDUCTING THE BIURET TEST ON UNKNOWN CONCENTRATIONS OF PROTEIN SAMPLES. Concentration of protein sample A Observations The mixture turned dark purple in colour. Colour Intensity *** Deductions Protein concentration is equal to/less than that of the 4% solution. Protein concentration is more than/equal to that of the 0.1% solution. Protein concentration is less than that of the 2% solution.

The mixture turned light blue in colour.

The mixture turned a little lighter purple than the 4% solution. However, with time the purple in the mixture deepened to give the same dark purple colour as the 4% solution.

** which turned to *** over time.

Key: *** - Very dark purple **- Lighter purple *- Lightest purple

Discussion: The Biuret test is one which is used to identify the presence of the proteins in a substance. The reagent used is Biuret solution, which is a solution of copper sulphate and sodium of potassium hydroxide. The bond between the amino group and the carboxyl acid group on adjacent amino acids in a protein is a peptide linkage. All proteins have several peptide linkages and it is the nitrogen atoms in these linkages that react with the copper (II) ions in the reagent to form a purple complex. This purple colour is a positive test for the presence of protein therefore if the solution turns purple there are proteins present. On the other hand, if the solution remains blue, there are no proteins present. Biuret is a compound derived from urea which also contains the CONH- group and gives a positive result. The Biuret test is used for the quantitative determination of total protein concentration. The intensity of the colour produced in the biuret reaction is proportional to the number of peptide linkages participating in the reaction. The greater the protein concentration, the deeper the purple. In the test, the protein sample of 4% concentration gave the highest purple colour intensity. This suggests that there was a high concentration of peptide linkages whose nitrogen atoms combined with the copper (II) ions to form the deep purple complex that was seen in the observation. The purple colour in this sample was the most intense seen when compared to the other samples. In the unknown solutions, Sample A showed the same result, this would suggest that the concentration of Sample A is close to 4%. The results for the protein sample of 2% concentration showed that the initial colour of the mixture was a light purple, then with time, the mixture changed to a darker purple on reaction with the reagent. This indicates that this reaction happened at a slower rate than the 4% reaction because the concentration of protein was lower and therefore, took a longer time to attain the same result. These results were mirrored in the Sample E solution suggesting that the concentration was close to that of the 2% sample. In comparison to the other samples, the colour intensity of Sample E was not as intense as Sample A but it was more intense than Sample C. The 0.1% concentration was the final protein sample, when the Biuret test was performed this gave a light blue solution. This suggests that there were no peptide bonds present or that the concentration of peptide bonds present was so negligible that the reagent was not able to react with them. This result was similar to that of Sample C and so it can be determined that the sample C was close to that of the 0.1% concentration. When compared with Samples A and E, this reaction was found to be the least intense. Precautions: To reduce parallax error the measuring cylinders were read at eye-level. The test tubes were labeled to prevent the mixing up of the test-tubes.

Limitations: Sources of error: The measuring cylinder that was used may have been tainted with previous samples which could have led to inaccurate results. Conclusion: The experiments results showed that Sample A was closest to the 4% protein concentration, that Sample C was similar to the 0.1% protein sample and that Sample E identified closest with the 2% protein sample.