Biochemistry with elements of Chemistry Exercise 4zakladbiochemii-2wl.wum.edu.pl/sites/zakladbiochemii-2wl...Biochemistry with elements of Chemistry Exercise 4 Department of Biochemistry

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: __________________

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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.




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




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




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).




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)




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)




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)




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.




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:


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.




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:


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:


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.




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:




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.).




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 _____________________________________________________________




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).




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:____________________________________________

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Biochemistry with elements of Chemistry Exercise 4zakladbiochemii-2wl.wum.edu.pl/sites/zakladbiochemii-2wl...Biochemistry with elements of Chemistry Exercise 4 Department of Biochemistry