Date: EXERCISE 4. Figure 1. Amino acid structure.

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1 Student s name: Date: Points: Assistant s signature: Index numer: /6 EXERISE 4 AMIN AIDS AND PRTEINS. Amino acids are structural units (monomers) of proteins. There are 20 different amino acids coded for in DNA that are used to build proteins (after post-transcriptional modifications over 20). The shape and biological properties of each protein is determined by the sequence of amino acids building it. Each amino acid consists of an alpha () carbon atom having following attachments: a hydrogen atom an amino group (hence "amino" acid) a carboxyl group (-). This gives up a proton and is thus an acid (hence amino "acid") one of 20 different "R" groups (different side chains). It is the structure of the R group that determines its unique properties. The general amino acid structure is shown in Fig. 1. Figure 1. Amino acid structure. A (-) carbon atom with 4 different constituents is said to be chiral. The only amino acid not exhibiting chirality is glycine since its '"R-group" is a hydrogen atom. All of the amino acids in proteins exhibit the same absolute steric configuration as L-glyceraldehyde (Fig. 2.). Therefore, they are all L--amino acids. D-amino acids are never found in proteins, although they exist in nature. D-amino acids are often found in polypeptide antibiotics and bacterial cell walls. + 3 N N L - glyceraldehyde 3 L - alanine 3 D - alanine Figure 2. Steric configuration of glyceraldehyde and alanine. 1

2 ommon names, structural formulas and standard three-letter and one-letter abbreviations for the 20 L-amino acids are given below. Table 1. -amino acids found in proteins. Amino Acid Symbol Structure Amino Acids with Aliphatic R-Groups Glycine Gly - G Alanine Ala - A Valine Val - V Leucine Leu - L Isoleucine Ile - I Non-Aromatic Amino Acids with ydroxyl R-Groups Serine Ser - S Threonine Thr - T Amino Acids with Sulfur-ontaining R-Groups ysteine ys - Methionine Met-M 2

3 Amino Acid Symbol Structure Acidic Amino Acids and their Amides Aspartic Acid Asp - D Asparagine Asn - N Glutamic Acid Glu - E Glutamine Gln - Q Basic Amino Acids Arginine Arg - R Lysine Lys - K istidine is - Amino Acids with Aromatic Rings Phenylalanine Phe - F Tyrosine Tyr - Y Tryptophan Trp-W Imino Acids Proline Pro - P N 3

4 There are many classifications of amino acids properties. Figure 3. shows one of the most common grouping of amino acids according to their properties. 10 of the 20 amino acids are called "essential" because their source is a proper protein diet and they cannot be produced in our bodies. The remaining are called "nonessential" because they are synthesized by cells. The essential amino acids of human protein are: tryptophan, arginine, phenylalanine, lysine, threonine, valine, methionine, leucine, histidine and isoleucine. The nonessential amino acids found in human protein are glycine, alanine, serine, cysteine (cystine), aspartic acid (aspartate), asparagine, glutamic acid (glutamate), glutamine, tyrosine, proline (and hydroxyproline). Figure 3. Properties of amino acids. Isoelectric point (pi) of a given amino acid is the p at which the majority of molecules in solution have a net charge of zero. At this p the acidic (-) and basic (-N 2) groups react with each other to form a dipolar ion or internal salt. The internal salt of an amino acid is given the special name zwitterion (dipolar ion). p < pi p = pi p > pi In strongly acidic medium (at p < pi) amino acid molecules carry a positive charge. In basic solution (p > pi) amino acid molecules carry a negative charge. Both carboxyl and amino groups, undergo all the reactions typical for them: salt formation, esterification and decarboxylation (for - group), deamination (for -N 3 group), formation of lactams (only for γ-amino acids). 4

5 Polypeptides and proteins are large-molecule compounds composed of many amino acids. The amino acids are linked together in long chains through a union between amino and carboxyl groups, by elimination of water. This union is known as the peptide linkage (bond). The reaction below illustrates the peptide linkage between two molecules of alanine: 3 N 2 + N N 2 N peptide bond 3 N - Ala - Ala - 2 (Alanylalanine) Ala Ala (an example of a dipeptide) forms during condensation reaction of two alanine molecules. The presence of the carbonyl group within a peptide group allows for electron resonance stabilization to occur, so that the peptide bond exhibits rigidity, but not like the typical = double bond. The peptide bond is, therefore, said to have partial double-bond character. N N + (1) (2) onsidering structure (1) shows a carbon-oxygen double bond and structure (2) shows a carbon-nitrogen double bond. As the real structure of the peptide group is the hybrid of these two structures (it s between 1 and 2), the six atom group is planar. Two configurations are possible for the atoms of a planar peptide bond: in one, the two α-carbons (R-groups) are cis in relation to each other, in the other, they are trans. The trans configuration is more favorable, because the α-carbons with the bulky groups attached to them are farther from each other than they are in the cis isomer: + N + N cis trans 5

6 Peptide is the name given to a short polymer of amino acids. Peptides are classified by the number of amino acid units in the chain: dipeptide is a molecule containing 2 amino acids joined by an amide bond (peptide linkage) those containing 3, 4 and 5 amino acids are called tripeptides, tetrapeptides, pentapeptides and so on molecules containing 2 to 10 amino acids are called oligopeptides those containing more than 10 amino acids are called polypeptides polymers of more than 100 amino acid residues are termed proteins. + By convention, polypeptides are written from the left, beginning with the amino acid having the free -N 3 group and proceeding to the right toward the amino acid with the free - group. Terminal amino acid with + the free -N 3 group is called the N-terminal amino acid and that with the free - group is called -terminal amino acid. Proteins and polypeptides exist in four main structural forms: 1. Primary structure: refers to the sequence of amino acids in its polypeptide chain. This structure is stabilized by peptide bonds. Protein primary sequences can be written with a 3-letter abbreviation for the 20 amino acids (e.g. Gly-Ile-Val-..) a 1-letter abbreviation (e.g. GIVEQTSISLYQLENYN) 2. Secondary structure: refers to regular, repeated patterns of folding of the protein backbone. The two most common folding patterns are the alpha helix and the antiparallel beta sheet. This structure is stabilized by hydrogen bonds. In the first pattern polypeptide chain has a shape of a right handed spiral. The second one consist of extended polypeptide chains with neighboring chains running in opposite (antiparallel) directions (Fig. 4.). In alpha helix polypeptide chain spirals around a central "helix axis" with a clockwise twist. In beta pleated sheet polypeptide chain is nearly fully extended. Figure 4. Secondary structure of protein. (opywright Pearson Education) 6

7 3. Tertiary structure: refers to overall folding pattern and arrangements in space of all atoms in a single polypeptide chain. Among the most important factors in maintaining this structure are disulfide bonds, hydrophobic interactions, ionic forces and hydrogen bonding (Fig. 5, 6.). Figure 5. Interactions holding the tertiary structure of protein. (opywright Pearson Education) Figure 6. 3D shape of protein. 4. Quaternary structure: is the arrangement of the individual subunits of a protein with multiple polypeptide subunits (e.g. hemoglobin has 2 alpha and 2 beta subunits). nly proteins with multiple polypeptide subunits have quaternary structure (Fig. 7.). Figure 7. Quaternary structure of collagen (a) and haemoglobin (b) molecules. (opywright Pearson Education) 7

8 There are two major classes of proteins: 1. Simple proteins: albumins, globulins, glutelins, prolamins, histones, protamines. 2. onjugated proteins: nucleoproteins, glycoproteins, phosphoproteins, chromoproteins. Properties of proteins 1. Proteins are amphoteric; due to the fact that their molecules contains free amino and carboxyl groups, they react either with acids or bases, forming a protein salts. 2. Proteins are denatured by some of the heavy metals ions, such as mercury, lead and silver, also by heat, extremes of p, alcohols, concentrated acids and detergents. Denaturation is the loss of correct 3-dimensional structure. nly non-covalent bonds are destroyed during denaturation. When proteins are denatured, their enzymatic activities no longer work and they often precipitate out of solution this is what happens when you boil an egg. 3. When boiled with dilute acids or alkalis or treated with digestive enzymes, proteins are hydrolyzed to amino acids. 4. Peptides undergo the biuret reaction the reaction for detecting the presence of peptide bond. This reaction is positive for peptides beginning from tripeptides and for biuret. N 2 N N 2 T + u() 2 purple complex biuret 5. Peptides (containing aromatic amino acids) undergo xanthoproteic reaction. 8

9 Experiment 1 Detection of amino acids and polypeptides (proteins) A. Millon s test Millon s test is specific to phenol containing structures (tyrosine is the only common phenolic amino acid). Millon s reagent is concentrated N 3, in which mercury is dissolved. As a result of the reaction a red solution is considered as positive test. A white precipitate (or red after heating) appears in the presence of proteins. Procedure: Add 1 cm 3 of solution (tyrosine, tryptophan, glycine, gelatin, casein and unknown solution) to six labeled test tubes. To all test tubes add 2-3 drops of Millon s reagent and place them into a hot water bath for 5 minutes. Type the observation in Table (on page 11). B. Adamkiewicz-opkins test The compounds that have indole ring can condense with aldehydes (more readily with formic aldehyde) to form colourful ring at the juncture of the two liquids (water and concentrated 2S 4). Among protein amino acids, only tryptophan undergoes this reaction. Procedure: Add 1 cm 3 of solution (tyrosine, tryptophan, glycine, gelatin, casein and unknown solution) to six labeled test tubes. To all test tubes add 1 ml of glyoxylic acid. Mix well and then introduce 0.5 ml of concentrated 2S 4 (add carefully drop by drop on the wall of test tube). Type the observation in Table. 9

10 . Detection of aromatic ring xanthoproteic reaction (reaction is positive for phenylalanine, tyrosine, tryptophan) The nitration reaction for tyrosine is the easiest one because of the presence of an - group in the aromatic ring. If the test is positive the yellow colour appears. 2 N 2 + 2N 3 N 2 2 S N 2 N 2 Procedure: Add 1 cm 3 of solution (tyrosine, tryptophan, glycine, gelatin, casein and unknown solution) to six labeled test tubes. To all test tubes add 5 drops of concentrated N 3 and place into a hot water bath for 5 minutes. Type the observation in Table. D. Ninhydrin reaction Ninhydrin reaction is used to detect free amino group the terminal amines of lysine residues in peptides and proteins. When reacting with these free amines, a deep blue or purple colour appears. (Exceptions: Proline gives a yellow colour and hydroxyproline pink colour 1 ). Procedure: Add 1 cm 3 of solution (tyrosine, tryptophan, glycine, gelatin, casein and unknown solution) to six labeled test tubes. To all test tube add 1 ml of acetone solution of ninhydrin and place into a hot water bath for 5 minutes. Type the observation in Table. 1 The products of ninhydrin reaction for proline and hydroxyproline are different, because these amino acids contain =N group, in comparison to other amino acids, having -N2 group. 10

11 Table for your observations. Millon s test Adamkiewicz- opkins test Xantoproteic reaction Ninhydrin reaction Glycine Tryptophan Tyrosine asein Gelatin Unknown solution nr Identification of unknown solution: onclusions: 11

12 Experiment 2 Thin layer chromatography Equipment: Eluent: methanol : chloroform : ammonia solution = 2 : 2 : 1 Solution A: 0.5% solution of ninhydrin Solution B: 1.0% u(n 3) 2 in acetone Solution of amino acids (0.5% solution): arginine proline tryptophan mix of arginine, proline, tryptophan (ratio 1 : 1 : 1) tested solution of unknown amino acids hromatography glass plate Procedure: Using a pencil (not a pen!) draw a line about 1 cm from the bottom edge of chromatographic plate. n the line mark 5 spots and label them (on the top of the plate), e.g. arginine A, proline P, tryptophan T, mix of tree amino acids M and unknown solution of amino acids S. Then, using capillary micropipette apply a small drop of the standard amino acid solutions (arginine, proline, tryptophan) on the first 3 marked spots of your plate. n the fourth spot apply the portion of mix of amino acid solution and on the last fifth point the portion of unknown solution of amino acids. Add the eluent to the chamber (about 0.5 cm from the bottom) and next put the plate inside it (Fig. 8.). Figure 8. hromatography chamber with a plate inside. Allow it stay until the solvent front reaches the top of the plate (1 cm from the top edge of the chromatogram). Remove the plate from the chromatographic chamber and draw, with your pencil, the front line of the solvent. Next dry it in the air and then in the dryer at the temperature of 100 (for 10 min.). 12

13 Spray the dried chromatographic plate with a first solution A and next with a solution B under a lab hood. opy the chromatographic plate using a semitransparent paper and glue it into the lab report. alculate R f for the corresponding amino-acids (Fig. 9.). Figure 9. Thin layer chromatogram calculating R f value. ere stick the chromatogram: R f calculations: Answer: Tested solution contained 13

14 Experiment 3 Biuret test The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper (II) ion forms violet-coloured coordination complexes in an alkaline solution. Reagents: Biuret reagent Solution of protein: 1 mg protein /ml 2 mg protein /ml 4 mg protein /ml 6 mg protein /ml 8 mg protein /ml unknown concentration of the protein pure water Procedure: Add 0.5 cm 3 of solutions (1 mg protein /ml, 2 mg protein /ml, 4 mg protein /ml, 6 mg protein /ml, 8 mg protein /ml and unknown concentration of the protein and pure water) into seven labelled test tube. Next add 2.5 ml of biuret reagent to all test tubes. Mix the content carefully and leave it for 30 minutes. After 30 minutes measure the extinction 2 of all prepared solutions of protein in comparison to water (mixed with biuret reagent). The measurement make in the spectrophotometer Marcel (λ = 540 nm). Type your data into the table: oncentration of the protein Extinction Water with biuret reagent 0 1 mg protein /ml 2 mg protein /ml 4 mg protein /ml 6 mg protein /ml 8 mg protein /ml Unknown concentration of protein 2 parameter defining how strongly a substance absorbs light at a given wavelength, per mass density (mass extinction coefficient) or per molar concentration (molar extinction coefficient). 14

15 Using the concentrations and extinction prepare the model curve and read the concentration of unknown solution of protein. Answer: Protein concentration in unknown solution is: 15