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Transcript
Qualitative Analysis of Biomolecules 1.1. Learning objectives and experimental aims The aim of this first unit is the successful application of experimental laboratory techniques which allow the qualitative analysis of biomolecules. These include experimental procedures for the analysis of carbohydrates, amino acids, proteins and nucleic acids. Your results should enable you to identify the unknown samples provided to your group. 1.2. Theoretical background 1.2.1. Qualitative detection of carbohydrates Carbohydrates are biochemical molecules consisting of carbon (C), hydrogen (H) and oxygen (O). Apart from exceptions (like deoxyribose), all of them can be described by the empirical sum formula C m (H 2 O) n , in which m can be different from n. In the living organism, they fulfil important physiological functions such as being energy sources, structural elements and storage molecules. Carbohydrates can be divided into several groups based on their properties, e.g. • the number of monomers they consist of o monosaccharides (e.g. glucose, fructose) o disaccharides (e.g. sucrose) o oligosaccharides (e.g. raffinose) o polysaccharides (e.g. starch, amylose, cellulose) • the number of C-atoms in monomers (e.g. pentoses or hexoses) • their functional groups: aldehydes or ketones (aldoses or ketoses) • their reducing or non-reducing properties These properties are used in the following methods for the detection of carbohydrates: MOLISCH’s test MOLISCH’s test is a sensitive chemical test for the presence of carbohydrates, which is based on the dehydration of the carbohydrate by a concentrated acid (usually hydrochloric acid or sulphuric acid) to produce an aldehyde, which condenses with two molecules of phenol, resulting in a red- or purple-coloured compound. FEHLING’s test Fehling's can be used to distinguish aldehyde vs ketone functional groups. The compound to be tested is added to the Fehling's solution (deep blue) and the mixture is heated. Aldehydes are oxidized, giving a positive result (red precipitate), but ketones do not react, unless they are alpha-hydroxy-ketones. Fehling's test can be used as a generic test for monosaccharides and other reducing sugars (e.g., maltose). It will give a positive result for aldose monosaccharides (due to the oxidisable aldehyde group) but also for ketose monosaccharides, as they are converted to aldoses by the base in the reagent, and then give a positive result. IODINE Iodine solution (iodine dissolved in an aqueous solution of potassium iodide) reacts with starch producing a purple black colour. test positive test negative test MOLISCH FEHLING IODINE 1.2.2. Qualitative detection methods for amino acids, peptides and protein Amino acids are biologically important organic compounds containing amine (-NH 2 ) and carboxylic acid (-COOH) functional groups, usually along with a side-chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids. Proteins are biological macromolecules that are built up of proteinogenic amino acids linked by peptide bounds. Most of the proteinogenic amino acids can be formed by the cellular machinery, but nine of them are called “essential” amino acids for humans. These essential amino acids cannot be produced from other compounds by the human body and so must be taken in as food. Other amino acids are essential under certain special circumstances (marked with *). Essential amino acids Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine Non-essential amino acids Alanine, Arginine*, Aspartic acid, Cysteine*, Glutamic acid, Glutamine*, Glycine*, Proline*, Serine*, Tyrosine*, Asparagine*, Selenocysteine Protein functions vary widely and include catalysing reaction (enzymes), DNA replication, cell signalling, membrane transport etc. The proteins differ mainly in the sequence of the amino acids they are made of. This sequence of amino acids is determined by the sequence of nucleotides in the gene encoding for a certain protein. The sequence of the amino acids determines the folding and three-dimensional structure of a protein resulting in a certain protein activity. The detection of amino acids in the experiments is based on reactions of their different functional side chains or by formation of coloured complexes with the peptide bonds. ELLMAN’s reagent Ellman’s reagent or 5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB (IUPAC-name: 5-(3Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid) is used to quantify the number or concentration of thiol groups in a sample. Figure 1Structural formula of ELLMAN’s reagent The thiol present in cysteine reacts with the reagent, cleaving the disulphide bond to give 2-nitro-5-thiobenzoate (TNB−). This ionizes to the TNB2− di-anion in water at neutral and alkaline pH and has a yellow colour. Figure 2 Reaction of DTNB with a thiol (R-SH) BIURET (alternative: LOWRY assay) 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. The Biuret reagent is made of sodium hydroxide (NaOH) and hydrated copper(II)sulphate, together with potassium sodium tartrate. Potassium sodium tartrate is added to complex and to stabilize the cupric ions. The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the displacement of the peptide hydrogen atoms under the alkaline conditions. A tri or tetra dentate chelation with the peptide nitrogen produces the violet colour. The Biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the colour, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law. LOWRY assay This method combines the reactions of copper ions with the peptide bonds under alkaline conditions (the Biuret test) with the oxidation of aromatic protein residues. The Lowry method is best used with protein concentrations of 0.01–1.0 mg/mL and is based on the reaction of Cu+, produced by the oxidation of peptide bonds, with Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid in the Folin–Ciocalteu reaction). The reaction mechanism is not well understood, but involves reduction of the Folin–Ciocalteu reagent and oxidation of aromatic residues (mainly tryptophan, also tyrosine). Experiments have shown that cysteine is also reactive to the reagent. Therefore, cysteine residues in protein probably also contribute to the absorbance seen in the Lowry Assay. The concentration of the reduced Folin reagent is measured by absorbance at 750 nm. As a result, the total concentration of protein in the sample can be deduced from the concentration of Trp and Tyr residues that reduce the Folin–Ciocalteu reagent. 1.2.3. Qualitative detection of nucleic acids Nucleic acids are organic molecules that serve as the subunits (or monomers), of nucleic acids like DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). A nucleotide is made of a nucleobase (also termed a nitrogenous base), a five-carbon sugar (ribose for RNA; 2deoxyribose for DNA) and at least one phosphate group. In figure 3, the deoxyribose and adenine make up a nucleoside (specifically, a deoxyribonucleoside) called deoxyadenosine. With the one phosphate group included, the whole structure is considered a deoxyribonucleotide (a nucleotide constituent of DNA) with the name deoxyadenosine monophosphate. Figure 3 This nucleotide contains the five-carbon sugar deoxyribose, a nitrogenous base called adenine, and one phosphate group. The nitrogenous bases are divided into purine bases and pyrimidine bases based on their chemical properties. In DNA, the purine bases are adenine and guanine, while the pyrimidines are thymine and cytosine. RNA uses uracil in place of thymine. Adenine always pairs with thymine by 2 hydrogen bonds, while guanine pairs with cytosine through 3 hydrogen bonds, in each case because of the unique structures of the bases. There are several ways to detect nucleic acids, of which FEULGEN stain and DISCHE stain will be used. FEULGEN stain FEULGEN stain is based on the reaction with Schiff’s reagent after initial hydrolysis of the nucleotides. The nucleotides present are red after the staining. DISCHE stain DISCHE’s reagent is a solution of diphenylamine and concentrated sulphuric acid in glacial acetic acid. When a solution containing deoxyribose is heated under its presence, the solution turns bright blue. The actual structure of the dye is hitherto unknown, but the deoxyribose initially reacts to 4-Oxo-5-hydroxy-pentanal which reacts with diphenylamine giving the blue colour. 1.3. Procedures At the end of the practical course, each group should be able to identify the provided unknown samples. Use all experimental protocols with three different samples • Positive sample (a known biomolecule) • Negative sample (distilled water) • Unknown sample For all experiments, you need the following materials: • • • • • pipettes pipette tips reaction tubes (1.5 mL and 2 mL) heat block (at 99 °C) disposable plastics 1.3.1. Detection of carbohydrates Chemicals: • α-naphthol solution (10 %) • concentrated sulphuric acid • FEHLING solutions I & II • LUGOL solution (0.5 g Iodine & 1 g KI in 100 mL dist. water) MOLISCH test 1. pipette 300 µL sample into a test tube 2. add 2 drops of 10% α-naphthol solution 3. carefully add 500 µL H 2 SO 4 to the solution; this will form a layer underneath the other solutions 4. observe changes. Note ring formation at the border of the two layers. FEHLING test 1. mix 3 drops of Fehling I reagent and 3 drops of Fehling II reagent in a test tube 2. add 0.5 mL sample 3. heat up in a heat block for approx. 10 minutes 4. observe changes Iodine test 1. pipette 1 mL sample into a test tube 2. add 1 drop of LUGOL solution to the sample at room temperature 3. observe the colour change 4. heat up in a heat block and cool the sample afterwards 5. observe the changes after heating and while cooling MOLISCH negative unknown glucose fructose saccharose/ sucrose starch FEHLING Iodine 1.3.2. Detection of amino acids Chemicals: • Tris buffer (1 M, pH 8) • Urea in water (9 M) • 5,5‘-Dithio-bis-2-nitrobenzoic-acid) solution (DTNB, pH 7) • copper(II)sulphate (1 %) • NaOH solution (0.1 M) ELLMAN test 1. mix 200 µL sample, 200 µL Tris buffer and 500 µL urea solution (mix gently) 2. add 200 µL of DTNB 3. observe changes BIURET test 1. add 500 µL sample to a test tube 2. add 1 mL 0,1 M NaOH solution 3. add 2 drops copper(II)sulphate (1%) 4. observe changes (use white background!) ELLMAN BIURET negative unknown cysteine histidine tyrosine protein 1.3.3. Detection of nucleic acids Chemicals: • NaOH solution (2 M) • HCl (5 M) • SCHIFF reagent • Diphenylamine reagent (1 g Diphenylamine in 2.5 mL H 2 SO 4 ) • pH indicator paper FEULGEN test 1. add 500 µL sample to a test tube 2. add 5 drops HCl (5M) 3. heat for 15 minutes in heat block 4. neutralize with 10-15 drops of NaOH (2M) (check pH with indicator paper!) 5. Add 3 drops SCHIFF reagent 6. Observe changes DISCHE test 1. pipette 500 µL sample into a test tube 2. add 1 mL diphenylamine solution 3. heat in heat block 4. observe changes FEULGEN negative unknown guanosine deoxyguanosine DNA DISCHE Hazards and precautionary phrases 10 % α-naphthol-solution (in ethanol) 96% ethanol Fehling solution I Fehling solution II Sulphuric acid (H 2 SO 4 ) LUGOL solution Tris buffer (1M, pH 8) DTNB (300 µM in 50 mM KH 2 PO 4 ) 50 mM KH 2 PO 4 Urea (9 M) NaOH solution (0.1 M) Copper(II)sulphate (1%) NaOH solution (2 M) HCl solution (5 M) SCHIFF reagent Diphenylamine solution glucose Fructose Sucrose Starch solution bovine serum albumin (BSA) Tyrosine Histidine Cysteine Guanosine Deoxyguanosine DNA MOLISCH test FEHLING test Iodine test ELLMAN test BIURET test FEULGEN test DISCHE test Disposal Due to the minor quantities, no special disposal is required; dispose of via sink under running water Contains heavy metals, neutralize and dispose of in inorganic waste container Either dispose of in sink under running water; or inorganic waste container Dispose of in inorganic waste container Dispose of in inorganic waste container Dispose of in inorganic waste container Dispose of in inorganic waste container