Zoë 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.

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

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

Key: *** . Concentration of protein sample 4% Observations The mixture turned dark purple in colour.Very dark purple **.Results: A TABLE ILLUSTRATION THE RESULTS FOUND FROM CONDUCTING THE BIURET TEST ON KNOWN CONCENTRATIONS OF PROTEIN SAMPLES. The mixture turned light blue in colour. Colour Intensity *** Deductions A dark purple complex was formed meaning that a high concentration of peptide bonds were present. with time the purple in the mixture deepened to give the same dark purple colour as the 4% solution.1% The mixture turned a little lighter purple than the 4% solution. 2% 0.Lightest purple .Lighter purple *. * No complex was formed and therefore there were no peptide bonds present. A purple complex was formed meaning that peptide bonds were present. However. ** which turned to *** over time.

with time the purple in the mixture deepened to give the same dark purple colour as the 4% solution.Very dark purple **. C The mixture turned light blue in colour.A TABLE ILLUSTRATING THE RESULTS FOUND FROM CONDUCTING THE BIURET TEST ON UNKNOWN CONCENTRATIONS OF PROTEIN SAMPLES.Lighter purple *. Protein concentration is more than/equal to that of the 0. Colour Intensity *** Deductions Protein concentration is equal to/less than that of the 4% solution. * E The mixture turned a little lighter purple than the 4% solution. Protein concentration is less than that of the 2% solution.1% solution. Concentration of protein sample A Observations The mixture turned dark purple in colour. However.Lightest purple . ** which turned to *** over time. Key: *** .

which is a solution of copper sulphate and sodium of potassium hydroxide. this would suggest that the concentration of Sample A is close to 4%. when the Biuret test was performed this gave a light blue solution. Sample A showed the same result. the colour intensity of Sample E was not as intense as Sample A but it was more intense than Sample C. then with time. 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. The reagent used is Biuret solution. On the other hand. The results for the protein sample of 2% concentration showed that the initial colour of the mixture was a light purple. This indicates that this reaction happened at a slower rate than the 4% reaction because the concentration of protein was lower and therefore. this reaction was found to be the least intense. The greater the protein concentration. The Biuret test is used for the quantitative determination of total protein concentration. 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.1% concentration was the final protein sample. The intensity of the colour produced in the biuret reaction is proportional to the number of peptide linkages participating in the reaction. This purple colour is a positive test for the presence of protein therefore if the solution turns purple there are proteins present. 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. 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. the protein sample of 4% concentration gave the highest purple colour intensity. In the test. In comparison to the other samples.Discussion: The Biuret test is one which is used to identify the presence of the proteins in a substance. When compared with Samples A and E. The purple colour in this sample was the most intense seen when compared to the other samples. the deeper the purple. The test tubes were labeled to prevent the mixing up of the test-tubes. .group and gives a positive result. Precautions: To reduce parallax error the measuring cylinders were read at eye-level. took a longer time to attain the same result. Biuret is a compound derived from urea which also contains the –CONH. there are no proteins present. These results were mirrored in the Sample E solution suggesting that the concentration was close to that of the 2% sample. The 0. if the solution remains blue. The bond between the amino group and the carboxyl acid group on adjacent amino acids in a protein is a peptide linkage. In the unknown solutions. the mixture changed to a darker purple on reaction with the reagent.

1% protein sample and that Sample E identified closest with the 2% protein sample. that Sample C was similar to the 0. Conclusion: The experiment’s results showed that Sample A was closest to the 4% protein concentration.Limitations: Sources of error: The measuring cylinder that was used may have been tainted with previous samples which could have led to inaccurate results. .

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