44 1 Amino Acids, Peptides, Proteins
(1.86)N→O-acyl migration via the oxazoline and sub-
sequent hydrolysis of the ester bond:
(1.87)Hydrolysis of proteins with dilute acids preferen-
tially cleaves aspartyl-X-bonds.
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 im-
portance, 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. 1.11.1.4.1.4 SequenceAnalysis
The classical method is theEdmandegradation
reaction. It involves stepwise degradation of
peptides with phenylisothiocyanate (cf. 1.2.4.2.3)
or suitable derivatives, e. g. dimethylaminoa-
zobenzene isothiocyanate (DABITC). The
resultant phenylthiohydantoin is either iden-
tified directly or the amino acid is recovered.
The stepwise reactions are performed in so-
lution 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)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