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Tuesday, August 13, 2013



AMINO ACID ANALYSIS Presentation Transcript:

2.What is amino acid?
 What is amino acid?
Amino Acid: aminated carboxylic acid (R-COOH)

3.Classification of Amino Acid 1. By the location of Amino-group :? /? / ?-AA
2. By its acidity: neutral/ acidic/ basic AA
   ratio of Amino-group to carboxylic group
3. By whether containing phenyl group
   aromatic / non aromatic AA
4. By its occurrence in protein
    Protein / non protein AA
5. By polarity of R group :
   polar / apolar side chain AA
6. By its nutrient value to human:
   Essential AA and non-essential AA

The amino acid analysis is a technique used to determine the amino acid composition of a protein or peptide – not the sequence of amino acid residues

It is based on two distinct processes:
     1.  Separation of amino acids from amino acid mixture     obtained from protein hydrolysis
     2.  Detection of the separated amino acid components

5.Amino Acid Analysis
Amino acid analysis involve four basic steps:
Sample preparation including purification and Hydrolysis
Separation by chromatographic method
Derivatization (labeling of AA with a detectable UV absorbing or fluorescent marker
Data interpretation

6.Purification of Protein Sample:
Protein sample must be purified by any suitable chromatographic technique
Buffer components (e.g. salt, urea, detergent) may be removed from the sample before analysis, particularly when RP-HPLC is used for separation

Acid hydrolysis is the most common method used for protein hydrolysis.
Proteins are hydrolyzed using 6N HCl containing 0.1-1% Phenol preceding AA analysis.
Addition of phenol prevents the halogination of tyrosine

A known amount of internal standard (norleucine) is added to the protein sample to be hydrolyzed
Since norleucine does not naturally occur in proteins, is stable to acid hydrolysis and can be chromatographically separated from other protein amino acids, it makes an excellent internal standard

9.Norleucine vs Leucine

10.Acid hydrolysis   The acid hydrolysis may be carried out either in
   Liquid or Vapor Phase
Liquid Phase:
Place the protein sample in hydrolyzing tube and dry
Add 200 µl 6 N HCl per 500 µg of lyophilyzed solution
Add internal standard of Norleucine
Freeze the sample tube in dry ice and flame seal in vacuum
Keep at 110ºC for 24 h in vacuum or inert atmosphere to prevent oxidation

11.Vapor Phase Hydrolysis:
The molar amount of internal standard should be added in the sample approximately equal to that of most of the amino acids in the sample (~5 nmoles of each amino acid (i.e. 10 µg of protein)

The sample is transferred to a hydrolysis tube and dried under vacuum. The tube is placed in a vial containing 6 N HCl, a small amount of phenol and the protein is hydrolyzed by the HCl vapors under vacuum

The hydrolysis is carried out for 65 minutes at
   150 oC

12.Hydrolysis Effects

13.After hydrolysis residual HCl is removed in a rotary evaporator
Dissolved in water or buffer depending on whether the separation method is ion exchange or RP-HPLC

14.Separation by chromatographic method The common method involve are:
Ion Exchange Chromatography
Reverse-Phase HPLC

15.Automatic AA analyzer (Ion exchange Chromatography)

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