Amino Acid Composition of Proteins

1- amino acid analysis
Gives relative quantities of each amino acid, but nothing about order.
First must boil protein in 6N HCl for 24 hours to break peptide bonds.
Subject hydrolysate to ion-exchange chromatography at different pH’s OR use
phenylisothiocyanate (PITC) to generate derivations and subject result to HPLC.
Problems:
1- acid hydrolysis converts asparagine to aspartic acid and glutamine to glutamic acid
2- also lose some serine, threonine, and tyrosine.
3- side chain of tryptophan is destroyed.
Amino Acid Residue Sequence
Use Edman degradation
Uses PITC reagent that reacts with the free N-terminus to form a PTC-peptide --->
treat with trifluoroacetic acid ---> last amino acid is cleaved ---> extract with organic
solvent (butyl chloride)---> PTH-amino acid ---> analyzed by chromatography.
Then take remaining peptide, remove next to last amino acid, etc.
Is now automated --> sequenator.
Good for small peptide of < 50 amino acid residues.
If protein is greater than 50 amino acid residues, must use proteases or chemical reagents
to cleave some of the peptide bonds.
1- cyanogen bromide - reacts with methionine residues - cuts on COOH side
2- proteases
trypsin - cleaves to carboxyl side of lysine and arginine
chymotrypsin - cleaves at aromatic and bulks nonpolar side chains
(phenylalanine, tyrosine, tryptophan)
Staphylococcus aureus V8 protease - cleaves to carboxyl side of
glutamate and aspartate
Need to use at least 2 different cleavage techniques to obtain overlapping sequences using
Edman degradation.