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Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 1
Student’s name: _____________________________
Index numer: _________________________
Date: __________________
Points:
/6
Assistant’s signature:
EXERCISE 4
AMINO ACIDS AND PROTEINS.
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 (-COOH). 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.
C
CH2OH
CHO
C
CH3
NH3
+
COOH
C NH3
+
CH3
COOH
OH HH
L - glyceraldehyde D - alanineL - alanine
H
Figure 2. Steric configuration of glyceraldehyde and alanine.
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 2
Common 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 Hydroxyl R-Groups
Serine Ser - S
Threonine Thr - T
Amino Acids with Sulfur-Containing R-Groups
Cysteine Cys - C
Methionine Met-M
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 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
Histidine His - H
Amino Acids with Aromatic Rings
Phenylalanine Phe - F
Tyrosine Tyr - Y
Tryptophan Trp-W
Imino Acids
Proline Pro - P N
H
COOH
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 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 pH at which the majority of molecules in solution have a net
charge of zero. At this pH the acidic (-COOH) and basic (-NH2) 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).
pH < pI pH = pI pH > pI
In strongly acidic medium (at pH < pI) amino acid molecules carry a positive charge. In basic solution (pH >
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 -COOH group), deamination (for -NH3 group), formation of lactams (only for γ-amino
acids).
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 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:
CH3
CH
NH2
C
O
N
H
CH
CH3
COOHCH3
CH
NH2
C
O
OH N
H
CH
CH3
COOHH+
peptide bond
- H O2
NH - Ala - Ala - COOH2
(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 –C=C– double bond. The peptide bond is,
therefore, said to have partial double-bond character.
Considering 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:
CN+
C
C
H
O
CN+
H
C
C
O
transcis
CNH
CC
O
CNH
+C
C
O
(1) (2)
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 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 -NH3+
group and proceeding to the right toward the amino acid with the free -COOH group. Terminal amino acid with
the free -NH3+ group is called the N-terminal amino acid and that with the free -COOH group is called
C-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. GIVEQCCTSICSLYQLENYCN)
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. (Copywright Pearson Education)
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 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 Figure 6. 3D shape of protein.
tertiary structure of protein.
(Copywright Pearson Education)
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). Only proteins with multiple
polypeptide subunits have quaternary structure (Fig. 7.).
Figure 7. Quaternary structure of collagen (a) and haemoglobin (b) molecules. (Copywright Pearson Education)
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 8
There are two major classes of proteins:
1. Simple proteins: albumins, globulins, glutelins, prolamins, histones, protamines.
2. Conjugated 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 pH, alcohols, concentrated acids and detergents. Denaturation is the loss of correct
3-dimensional structure. Only 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.
C
NH2
NH
C
O
NH2
O
T
+ Cu(OH) 2 purple complex
biuret
5. Peptides (containing aromatic amino acids) undergo xanthoproteic reaction.
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 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 HNO3, 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 cm3 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-Hopkins’ 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 H2SO4). Among protein
amino acids, only tryptophan undergoes this reaction.
Procedure:
Add 1 cm3 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
H2SO4 (add carefully drop by drop on the wall of test tube) . Type the observation in Table.
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 10
C. 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 -OH group in the
aromatic ring. If the test is positive the yellow colour appears.
HO CH2 CH COOH
NH2
+ HNO3
H2SO4
H2O2HO
O2N
O2N
CH2 CH
NH2
COOH2
Procedure:
Add 1 cm3 of solution (tyrosine, tryptophan, glycine, gelatin, casein and unknown solution) to six labeled
test tubes. To all test tubes add 5 drops of concentrated HNO3 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 colour1).
Procedure:
Add 1 cm3 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 =NH group, in comparison to other amino acids, having
-NH2 group.
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 11
Table for your observations.
Millon’s test Adamkiewicz-
Hopkins’ test
Xantoproteic
reaction
Ninhydrin
reaction
Glycine
Tryptophan
Tyrosine
Casein
Gelatin
Unknown
solution nr____
Identification of unknown solution: ___________________________________________________________
Conclusions:
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 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% Cu(NO3)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
Chromatography glass plate
Procedure:
Using a pencil (not a pen!) draw a line about 1 cm from the bottom edge of chromatographic plate. On 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. On 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. Chromatography 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°C (for 10 min.).
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 13
Spray the dried chromatographic plate with a first solution A and next with a solution B under a lab hood.
Copy the chromatographic plate using a semitransparent paper and glue it into the lab report.
Calculate Rf for the corresponding amino-acids (Fig. 9.).
Figure 9. Thin layer chromatogram – calculating Rf value.
Here stick the chromatogram: Rf calculations:
Answer: Tested solution contained _____________________________________________________________
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 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 cm3 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 extinction2 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:
Concentration 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).
Biochemistry with elements of Chemistry Exercise 4
Department of Biochemistry
Second Faculty of Medicine with the English Division and the Physiotherapy Division 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:____________________________________________