Laboratory Manual for Practical Exercises Amino acids

Amino acids

Contents
Amino acids ....................................................................................................................................... 1
Exercise: Determination of amino acids with formaldehyde titration by Sörensen........................... 2
Exercise: Reaction of amino acids with ninhydrin .......................................................................... 3
Exercise: Individual amino acid tests.............................................................................................. 4
Exercise: Detection of peptide bond by biuret reaction ................................................................... 5

Proteins serve as a source of amino acids for organism. In organism, proteins undergo continuos
process of degradation to release individual amino acids. Thus, amino acids represent the building
blocks for the protein synthesis as well as the products of protein degradation.
The content of amino acids either in blood plasma or in urine mirrors their own dynamics. The
total concentration of amino acids in blood plasma (or serum) ranges from 2.4 to 4 mmol/L.
Approximately 1 g of free amino acids is eliminated from organism during 24 hours. This amount
corresponds to 140 mg of nitrogen.
The increased level of free amino acids in blood plasma/serum (hyperaminoacidemia) or
increased excretion of amino acids by urine (hyperaminoaciduria) is observed in either several inherited
disturbances of amino acids metabolism, such as alkaptonuria, cystinuria, tyrosinemia, or in transmitted
diseases, e.g. hepatitis and some renal diseases.
From the chemical point of view, amino acids can be aliphatic, aromatic or heterocyclic
carboxylic acids with an amino group on C-2 (-) carbon. Those polar, oppositely charged functional
groups give the amino acids an ampholytic character. Individual amino acids differ by chemical
structure of their side chain R.

The reactions of amino acids can be divided into 2 groups:

1. Reactions of the functional groups: they are based on the presence of amino or carboxylic group.
The reactions are common for all amino acids and are used for the determination of total content
of amino acids.

2. Reactions of the characteristic groups of side chains: they are specific reactions, which are used for
detection of individual amino acids.

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Laboratory Manual for Practical Exercises Amino acids

Determination of total content of amino acids

The presence of carboxylic group is used for the determination of amino acids by alkalimetric
titration.
The primary -amino group reacts with nihydrin forming a blue product. The reaction is
convenient for a test of amino acid presence and for the determination of total content of amino acids
by spectrophotometry. The -amino group reacts with some reagents (o-phtaladialdehyde,
fluorescamine) producing a very intensive fluorescent product.

Exercise: Determination of amino acids with formaldehyde titration by Sörensen

Amino acids are determined by alkalimetric titration. The amphoteric character of amino acids
does not allow direct titration therefore the amino group has to be blocked first with formaldehyde
forming a Schiff base. Then the carboxylic group remains free, the product is more acidic, and so it can
be titrated by volumetric sodium hydroxide solution.

Materials
Pipettes, a burrete, titrimetric flasks.
Formaldehyde, volumetric NaOH solution (c = 0.1 mol/L), phenolphtalein (pH range 8.2 -9.8)

Procedure
1. Pipette 5 ml of the amino acid sample into a titrimetric flask.
2. Add 2-3 drops of phenolphtalein
3. Add 10 ml of distilled water, 5 ml of formaldehyde, and stir up thoroughly.
4. Pipette 15 ml of distilled water into other titirimetric flask
5. Add 2-3 drops of phenolphtalein, 5 ml formaldehyde, and stir up rhotoughly. The titration serves
for comparison as a blank.
6. After 10 min, titrate both solutions with volumetric NaOH solution up to permanent pink colour.
7. Record both consumed volumes of NaOH solution.

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Laboratory Manual for Practical Exercises Amino acids

Calculation:

Sample Volumes of NaOH (ml) Sign N

(Nitrogen of amino acids)
(mg/l)
N = 140 . (a - b)

Amino acid (test tube 1) a

Blank (test tube 2) b

Conclusion:

Exercise: Reaction of amino acids with ninhydrin

The ninhydrin reaction is the most common reaction in analysis of amino acids. It is used for
detection and determination of amino acids, for example, after chromatographic separation.
At increased temperature ninhydrin quickly reacts with an amino acid by oxidative
deamination, releasing CO2, aldehyde and ammonia. Reduced ninhydrin binds ammonia released by
oxidation and other molecule of ninhydrin a blue coloured product is formed. Any primary amino group
will produce a blue colour and secondary amino group containing amino acid (proline) will give a
yellow/orange colour.

Materials
The test tubes, pipettes, filter paper, a water bath, a funnel.
Phospohate buffer pH 7.2, ninhydrin reagent, 96 % ethanol, sample (alanine)

Procedure

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Laboratory Manual for Practical Exercises Amino acids

Detection of amino acids

1. Pipette 0.3 ml of alanine into a test tube.
2. Pipette 0.3 ml of distilled water into the other one (blank).
3. Pipette 1 ml of buffer pH 7.2 and 0.3 ml of ninhydrin reagent into both test tubes.
4. Heat the test tubes in a boiling water bath exactly for 7 min.
5. Cool immediately afterwards.
6. The content of the test tube with the sample should be coloured to blue-violet or blue (proline and
hydroxyproline give yellow coloured product in the reaction with ninhydrin).

Observation:

Conclusion:

Exercise: Individual amino acid tests

For the detection of individual amino acids is utilized the chemical properties of the
characteristic groups of the side chain (aromatic ring, sulfur, etc.) which give specific reactions.

Xantoprotein reaction

Nitration of aromatic amino acids leads to yellow coloured nitroderivatives.

Materials
Test tubes, a test tube holder, a gas burner, pipettes.
Concentrated HNO3, 0.1 % amino acid solution (tyrosine), egg white solution, alanine solution.

Procedure

1. To 1 mL sample (use tyrosine solution, egg white solution, alanine solution) add 0.5 ml
concentrated HNO3.
2. Place the test tube into the boiling bath
3. The colour of the solution should change to yellow.
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Laboratory Manual for Practical Exercises Amino acids

Observation: Explain the differences caused by different samples used.

Sample Colouration

Tyrosine

Egg white solution

Alanine solution

Conclusions:
In the case of protein solution, the precipitate formed after addition of HNO3 is dissolved during heating.
By the reaction it si possible to detect the presence of tyrosine, phenylalanine, and tryptophan in the
tested solutions. The name of the reaction is by Greek xanthós = yellow.

Exercise: Detection of peptide bond by biuret reaction

The compounds having at least one peptide bond -CO-NH- in their molecule form a violet
complex species with Cu2+ in alkaline medium.

Materials
Test tubes, pipettes.
NaOH solution (2.5 M), CuSO4.5 H2O solution (0.5 M), protein solution.

Procedure
1. Pipette 1 ml of protein solution into a test tube.
2. Add 1 ml of NaOH solution and by dropping CuSO4.5 H2O solution A violet complex species is
formed.

The positive biuret reaction proves the presence of the peptide bond in a compound.

Observation:

Conclusions: