Download Qualitative Analysis of Biomolecules

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