N-Terminal Amino Acid of a Protein
1. Objective :
Identification of N-terminal amino acid of a protein using dansyl chloride method.
2. Principle :
Crystals of amino acids are white and its solution appears colourless. Identification of amino acids depends on the amount of carbon, oxygen, nitrogen, sulfur, its solubility, melting point, colligative properties, etc. Specifically, dansyl chloride reacts with the free amino group of the N- terminal amino acid residues of a protein.
Upon acid hydrolysis it results in formation of dansylated amino acids and the other free amino acids are separated. They can be identified using ultra violet light. Under UV light dansyl amino acid appears greenish-yellow in colour.
3. Requirements :
i. Dansyl chloride solution (2.5 mg/ml in acetone) (prepare fresh) and keep at 4°C in a refrigerator.
ii. 0.2 M aqueous solution of sodium bicarbonate (keep at 4°C, when not in use).
iii. Ignition tubes/test tubes referred to as 'dansyl tubes' (5 cm x 2 mm).
iv. Dansyl plates [polyamide thin layer plates (5 cm x 5 cm coated both sides)] coated on both sides. Number the plates with a pencil in the top corner.
v. Mark the origin for loading with a pencil 0.3-0.5 cm from each edge in the lower left hand corner of the numbered side of the plate (the origin for loading on the reverse side of the plate should be immediately behind the loading position for the front of the plate i.e. 0.3-0.5 mm from each edge in the lower right hand corner).
vi. Prepare two chromatography solvents as below: Solvent 1: Formic acid : water (4.5: 100, v/v) Solvent 2: Toluene : acetic acid (8:1, v/v)
vii. Ultra violet source (254 |im or 265 |im), vacuum pump, dessicator.
A. For Amino Acids :
(i) Prepare the stock solution (lmg/ml) of standard dansyl amino acid in 0.2 M NaHC03. (standard dansyl amino acid are - Pro, Leu, Phe, Thr, Glu, Arg. Store the stock solution at 4°C when not in use).
(ii) Transfer 10 µl of each amino acid solution in a dansyl tube and mix gently. Add 30 |il of dansyl chloride solution (5 µl for each amino acid).
(iii) Add 10 µl of unknown amino acid and 5 µl of dansyl chlqride in a separate dansyl tube.
(iv) Seal the tube with parafilm and incubate at 37°C for 1 hour at room temperature for 3 hour).
(v) Dry the samples in vacuum because the small volume of liquid present will take about 5 minutes.
(vi) Dissolve the dried samples in 10 µl of acetone using a capillary tube. Load 1 |iil aliquots of the standard mixture at the origin (the diameter of the spot should not be allowed to exceed 1-2 mm).
(vii) Load 1 µl of unknown amino acid on the reverse side.
(viii) Place the plate in the first chromatography solvent when the loaded sample is fully dry. Allow the plate to develop and the solvent to reaches the top. Dry both sides of the plate.
(ix) Develop the dansyl plate in the second solvent at right angle to the direction of development in the first solvent. Dry the plate until the solvent front reaches the top.
(x) Observe the plate under UV light.
B. For Proteins
(.i) Transfer 10 µg of protein in 10 µl of 0.2 M NaHC03 in 1 ml stoppered tube.
(ii) Pour 10 µl of dansyl chloride solution into it.
(iii) Stopper the tube and incubate at 37°C for 1 hour (or at room temperature for 3 hours).
(iv) Dry the samples in vacuum.
(v) Transfer 20 µl of 6 N HC1 to 1 ml tube and close the tube tightly with stopper. Hydrolyse it keeping in an oven maintained at 105±10C for 24 hours.
(vi) Dry the samples in vacuum.
(vii) Dissolve the dried sample in 10 µl of acetone and load 1 µl aliquots of the standard mixture at the origin using a fine capillary tube. Diameter of the spot should not be allowed to exceed 1-2 mm.
(viii) Load 1 µl of protein hydrolysate containing the unknown dansyl amino acids on the reverse side.
(ix) Place the plate in the first chromatography solvent when the loaded samples are completely dry. Allow the plate to develop and reach the solvent to the top. Dry both sides of the plate.
(x) Develop the dansyl plate in the second solvent at right angle to the direction of development as found in the first solvent. Wait to reach the solvent to the top, and dry the plate.
(xi) Observe the plant under UV light.
5. Result :
You should identify three major fluorescent areas at the bottom of the plate. Dansyl chloride is hydrolysed into dansyl hydroxide. The hydrolysed product appears as blue fluorescent area. Dansyl amide (produced by side reaction with Nis3) shows blue-green fluorescence and is about one third of the way up plate.
These two spots are useful internal marker which appears on all dansyle plates. The third spot fluoresces green and will correspond to the dansyl derivative of the amino acid. Solvent 2 separates the dansyl derivatives of hydrophobic amino acids and some natural amino acids.
Derivatives of charged and other neutral amino acids remain at the lower end of the chromatogram. Chromatogram is compared with the standard provided to your in laboratory.