Chapter 27: Amino Acids, Peptides and Proteins
Sequencing Peptides
In order to determine which amino acids are present in a protein, the protein has to be broken apart.
This requires that the amide bonds are
hydrolysed
to the component amino acids (
review including mechanism
?)
Chemical hydrolysis using 6M HCl / heat will break
all
the amide bonds (
i.e.
it is not selective).
The amino acids are then separated using chromatography (this tells us which amino acids are present)
Reaction with
ninhydrin
allows the molar ratios of the amino acids to be determined. (
i.e.
how much is present)
Enzymatic hydrolysis using
proteases
is more selective and can be used to break the protein into fragments rather than individual amino acids.
As examples:
Trypsin
cleaves amides where the
C=O
is part of
lysine
or
arginine
.
Chymotrypsin
cleaves amides where the
C=O
unit has an aromatic side chain :
phenylalanine
,
tyrosine
,
tryptophan
.
Carboxypeptidases
cleaves only at the C-terminus (this allows the C-terminus to be identified)
End group analysis
means that a "molecular flag" is attached to the N-terminus so allowing it to be recognised.
Once this information has been collected, the pieces needed to be analysed and the sequence carefully deduced.
©
Dr. Ian Hunt
, Department of Chemistry