2009_Book_FoodChemistry

44 1 Amino Acids, Peptides, Proteins
Separation of peptide fragments is achieved by
gel and ion-exchange column chromatography
using a volatile buffer as eluent (pyridine,
morpholine acetate) which can be removed
by freeze-drying of the fractions collected.
The separation of peptides and proteins by
reversed-phase HPLC has gained great importance,
using volatile buffers mixed with
organic, water-soluble solvents as the mobile
phase.
The fragmentation of the protein is performed by
different enzymic and/or chemical techniques,
at least by two enzymes of different specifity.
The arrangement of the obtained peptides in the
same order as they occur in the intact protein
is accomplished with the aid of overlapping
sequences. The principle of this method is
illustrated for subtilisin BPN ′ as an example in
Fig. 1.11.
(1.86)
N → O-acyl migration via the oxazoline and subsequent
hydrolysis of the ester bond:
1.4.1.4 Sequence Analysis
The classical method is the Edman degradation
reaction. It involves stepwise degradation of
peptides with phenylisothiocyanate (cf. 1.2.4.2.3)
or suitable derivatives, e. g. dimethylaminoazobenzene
isothiocyanate (DABITC). The
resultant phenylthiohydantoin is either identified
directly or the amino acid is recovered.
The stepwise reactions are performed in solution
or on peptide bound to a carrier, i. e.
to a solid phase. Both approaches have been
automated (“sequencer”). Carriers used include
resins containing amino groups (e. g. amino
polystyrene) or glass beads treated with amino
alkylsiloxane:
(1.88)
(1.87)
Hydrolysis of proteins with dilute acids preferentially
cleaves aspartyl-X-bonds.
The peptides are then attached to the carrier by
carboxyl groups (activation with carbodiimide
or carbonyl diimidazole, as in peptide synthesis)
or by amino groups. For example, a peptide