AMINO ACID COMPOSITION b.

PTC-AA selectively cleaved

DETERMINATION and treated with aqueous
acid phenylthiohydantoin
Steps: (PTH) AA derivative
c. PTH-AA identified by HPLC
1. Hydrolyze peptide bond
- Up to 50 AA
- Acid hydrolysis
- For shorter peptides, only step 3
- 6 M HCL at 110 C for 16-72
is done
hrs
If protein contains 1 cysteine
2. Analyze hydrosylate using
residues, disulfide bonds must be
chromatographic procedure
cleaved by:
(amino acid analysis)
a. Treatment with performic acid
a. Treatment with
oxidizes cysteine to 2
phenylisothiocyanate (PITC)
cysteic acid residues, OR

b. Reduction by dithiothreitol to
phenylcarbamoyl (PTC)-
form cystein residues
amino acid derivatives
treated with iodoacetate
b. PTC-AA subjected to HPLC
(prevents disulfide bond
c. Eluted PTC-AA detected and
reformation)
concentration is determined
by measuring absorbance at If protein is large selective
240nm (peak absorbance) cleaving
a. Use proteases or reagents to
- timing of peaks used to form shorter peptides
identify AA
Treatment Cleavage
- amount of AA proportional Site
to area under peak Trypsin Lys, Arg (C)*
Submaxillus Arg (C)
protease
AMINO ACID SEQUENCING Staphylococcus Asp, Glu (C)
aureus V8
Steps protease
Asp-N-protease Asp, Glu
1. Determine AA composition
2. Identify N-terminal residue (N)**
- Sangers (1-fluoro-2,4- Pepsin Phe, Trp, Tyr
dinitrobenzene) (N)
- Dabsyl chloride (highly Cyanogen Met (C)
fluorescent derivatives) bromide
- Dansyl chloride intensely * C-terminal side
colored and greater sensitivity) ** N-terminal side
These destroy the b. Use Edman degradation
polypeptide c. Infer AA sequence by
3. Use Edman degradation procedure locating overlapping
- Labels and removes N-terminal sequences
residue only; leaves the rest of The nucleotide sequence of the
the chain intact gene that codes fsor a protein
a. Polypeptide reacts with PITC can also be used to determine
PTC-AA AA sequence. However, this
cannot determine the disulfide
 bonds that occur and whether Horton, H.R., Moran, L.A., Ochs, R.S.,
modification of AA residues Rawn, J.D., Scrimgeour, K.G. 1993.
occurred after synthesis. Principles of Biochemistry. USA: Neil
Patterson Publishers/Prentice-Hall, Inc.

Lehninger, A.L. Nelsonm, D.L., Cox,

References: M.M. 1993. Principles of Biochemistry.
2nd ed. New York: Worth.