SBM 1013 BIOCHEMISTRY1

LAB PRACTICAL 6

PROPERTIES OF PROTEINS AND AMINO ACIDS - QUALITATIVE TESTS

Name Matric No. Section

: Siti Fatimah Azzahrah bt Roslan : 0616996 :2

Date of Submission : 24 August 2006

Siti Fatimah Azzahrah bt Roslan BSBM

0616996

Section 2

Lab Report Experiment 6 Properties of Proteins and Amino Acids Qualitative Tests Introduction Proteins are extraordinarily complex molecules. Complete models depicting even the smallest of the polypeptide chains are almost impossible to comprehend. Biochemists have distinguished several level of the structural organization of proteins. Primary, structure, the amino acid sequence, is specified by genetic information. As the polypeptide chain folds, it forms certain localized arrangements of adjacent amino acids that constitute secondary structure. The overall three-dimensional shape that a polypeptide assumes of called the tertiary structure. Protein that consists of two or more polypeptide chains (or subunits) are said to have a quaternary structure. On the basis of composition, proteins are classified as simple or conjugated. Simple proteins, such as serum albumin and keratin, contain only amino acids. In contrast, each conjugated protein consists of a simple protein combined with anon-protein component. The non-protein component is called prosthetic group. ( A protein without its prosthetic group is called an apoprotein. A protein molecule combined with its prosthetic group is referred to as a haloprotein). Prosthetic groups typically play an important, even crucial, role in the function of proteins. Conjugated proteins are classified according to the nature of their prosthetic groups. The objectives of this experiment are to identify proteins from the samples given, to investigate the properties of proteins and amino acid, and also to perform the qualitative tests of proteins and amino acids accordingly. Method To perform this experiment, we have been provided with five samples, labelled A,B,C,D and E, and we were required to perform a total of seven tests on each sample. First, all the test tubes were labelled as A, B,C,D and E, and 0.5ml of each sample was transferred to its appropriate test tube. For Biuret test, 0.5ml of Biuret reagent was added to samples and the observations were recorded. Next, in Xanthoproteic acid test, 5 drops of concentrated nitric acid was added to each sample. Then, the test tubes were heated in boiling water bath for 3-4 minutes and the observations were recorded. After that, the tubes were cooled and 25 drops of 3M NaOH solution were added to change the pH to slightly alkaline. Any changes were again recorded. This test must be performed in the fume hood. The third test which was Ninhydrin test was conducted by first adding 0.1ml (2-3 drops) of 2% ninhydrin solution to samples. The tubes were then swirled and observations were recorded. Next,the test tubes were heated in a boiling water bath for about 2 minutes and any changes that occurred were recorded. Cyteine test was done by adding 5 drops of 3M NaOH and 2 drops of 1% lead nitrate. After that, the tubes were heated for 5 minutes in a boiling water bath and the observations were recorded. In protein

Siti Fatimah Azzahrah bt Roslan BSBM

0616996

Section 2

denaturation test, 5% sodium chloride was added to each sample and the tubes were heated in boiling water for 10 minutes and the observations were recorded. The sixth test was precipitation with acid anions. For this test, 15 drops of 2% trichloroacetic acid (TCA) was added to samples and the observations were recorded. The last test was precipitation with heavy metal ions. 15 drops of 2% copper sulphate solution was added to each sample, then the contents were swirled and the observations were recoded. Lastly, 3 drops of 0.1M sodium hydroxide was added to tubes, swirled, and any changes were recoded. It was difficult sometimes to identify the precipitation as its formation was in very small amount. Results (Please refer Table 1) Discussion This experiment was done by performing seven tests on five different samples which were labelled as A,B,C,D and E. We started with Biuret test which is the general test for the presence of protein. The Biuret reagent from this test consists of CuSO4 in NaOH solution. If the Biuret reagent is added to protein under the copper (II) it will form a colored complex with peptide bonds from the protein. The blue reagent turns violet in the presence of protein, and changes to pink when combined with short-chain polypeptides. From the results, sample C and D turned the blue solution into violet. Therefore, it is known that only sample C and D are proteins among all the five samples. These samples are thus probably BSA or gelatine. Secondly, Xanthoproteic acid test was conducted to test for the presence of specific functional groups in proteins such as aromatic rings. Concentrated nitric acid is used to nitrate the aromatic rings of proteins or amino acids, which in return show a yellow-orange or orange-red colour solution. Because nearly all proteins contain aromatic rings, it is also taken as protein-test. Sample B and C showed positive results with this test, which were the formation of orange-red and yellow-orange solution respectively. So, we know that sample B and C contain aromatic rings, thus they should be either phenol or BSA. As sample C is also a protein, so it can be determined that C is BSA. Next, Ninhydrin test, which is meant to test the presence of ammonia or primary amines, was done. Ninhydrin reacts with primary amino group on both protein molecules and amino acids to yield a coloured complex. A blue to blue-violet colour is given by aamino acids and constitutes a positive test. Other colours (yellow, orange, red) are negative. Sample A and C gave positive results, both turned the solution into blue-violet colour. A and C should be either Glycine or BSA. Since sample C is already known as BSA, so sample A is Glycine. During cysteine test, all samples gave negative results by not changing the colourless solutions, except for sample C which formed black precipitate. This result tells

Siti Fatimah Azzahrah bt Roslan BSBM

0616996

Section 2

us only sample C contains disulphide-bonded cysteine as the aim of this test is to detect the presence of disulphide-bonded cysteine in proteins. Most proteins are unstable at temperature of 50C or above. The heat disrupts weak interactions in proteins such as hydrogen bonds, thus protein unfolds and said to be denatured. Denaturation increases proteins solubility in water, which causes some proteins to precipitate in the form of large aggregates (coagulation), while others remain in solution. This phenomenon was shown by the results in which sample C formed coagulation. Other samples which might be proteins did not show positive results probably because some proteins are more resistant to heat denaturation. The positive result, the formation of white precipitate, was also shown only by sample C when reacted with acid anions. The anions of many organic acids like trichloroacetic precipitate proteins at acidic pH because they complex with the positively charged protein molecules forming insoluble salts. Since the protein will assume a positive charge only at pH below its isoelectric point, the protein solution is sometimes acidified prior to the addition of acid anion. As a protein approaches its isoelectric point, it becomes insoluble and precipitates from solution. This is a common method for removing proteins from biological fluid, as the precipitation can be removed by centrifugation or filtration to yield a protein-free solution. Finally, once again sample C showed positive result indicated by the formation of light blue precipitate, while other samples did not change the blue colour solution. At pH value above a proteins isoelectric point the protein will assume a net negative charge. It will the tend to form an insoluble salt after reacting with heavy metal cations. Based on the results obtained, it can be determined that sample E is water because it showed no positive results in any of the protein qualitative tests. Sample D is gelatine as it is a protein, and sample B is phenol as it contains aromatic rings. Glycine is the simplest, non-essential amino acid in the body and the only proteinforming amino acid that does not have chirality.Glycine functions in synthesis of many important compounds in the body and also as neurotransmitter in the central nervous system. Phenols serve as intermediate in the industrial synthesis of products as diverse as adhesives and antiseptics. Bovine Serum Albumin (BSA) is the most abundant protein plasma in the circulatory system, and it contributes 80% to colloid osmotic blood. It is responsible for the maintenance of blood pH. Gelatin contains large number of glycine, prolineand 4-hydroxyproline residues. It is primarily used as a gelling agent, and also in the making of whipped cream and chewable candy, and as a refining agent to clarify wine and fruit juice. Proteins make up about 15% of the mass of the average person. Protein molecules are essential to us in an enormous variety of different ways. Much of the fabric of our is constructed from protein molecules. Muscle, cartilage, ligaments, skin and hair these are all mainly protein materials. Smaller protein molecules play a vital role in keeping our body working properly. Haemoglobin, hormones, antibodies, and enzymes are all examples of these less obvious proteins. Generally proteins function in catalysis, structure, movement, defence, regulation, transport, storage, and stress response. Insufficient protein intake can cause diseases such as kwashiorkor and marasmus.

Siti Fatimah Azzahrah bt Roslan BSBM

0616996

Section 2

Conclusion Alhamdulillah, we have successfully conducted this experiment in which we managed to to identify each sample; sample A,B,C,D, and E are glycine, phenol, BSA, gelatine and water respectively. We were also able to investigate the properties of proteins and amino acid perform the qualitative tests of proteins and amino acids using the correct reagent. All has created everything with purpose, even very small amino acids play vital role in the function of our body. References 1. McKee, T. Biochemistry : The Molecular Basis of Life, 3rd edition. McGraw-Hill, 2003. 2. Murray, R.K. Harpers Biochemistry, 25th edition. McGraw-Hill, 2000. 3. Laboratory Manual Biochemistry Practical 6. 4. http://www.schoolscience.co.uk/content/5/chemistry/proteins/index.html 5. http://www.friedly.com/research/PhD/chapter5a.html 6. http://www.lsbu.ac.uk/water/hygel.html

Siti Fatimah Azzahrah bt Roslan BSBM

0616996

Section 2

Table 1

RESULTS QUALITATIVE PROTEIN TESTS

BIURET A (Glycine) B (Phenol) Blue solution not changed Blue solution not changed XANTHOPROTEIC Colourless solution not changed After heating : Yellow solution. + NaOH : Orange- red solution. After heating : Yellow precipitate. + NaOH : Yellow orange solution. Colourless solution not changed Colourless solution not NINHYDRIN Violet blue solution Orange solution. CYSTEINE Colourless solution not changed. Colourless solution not changed. Black precipitate. HEAT -ve -ve ACID ANIONS Colourless solution not changed. Colourless solution not changed. White precipitate HEAVY METAL ANIONS Blue solution not changed. Blue solution not changed

C (BSA)

Blue solution changed to violet. Blue solution changed to violet. Blue solution

Violet blue solution

Black precipitate.

Light blue precipitate.

D (Gelatin) E

Orange solution. Orange

Colourless solution not changed. Colourless

-ve -ve

Colourless solution not changed. Colourless

Blue solution not changed Blue solution

Siti Fatimah Azzahrah bt Roslan BSBM (Water) not changed

0616996

Section 2

changed

solution.

solution not changed.

solution not changed.

not changed