Affinity Chromatography - Department of Molecular and Cellular ...

Ligands containing amino groups can be attached to Thiopropyl Sepharose 6B or Activated
Thiol-Sepharose 4B by multi-point attachment or coupling through a small number of groups
using the heterobifunctional thiolating reagent, SPDP. The coupled molecules may be recovered
by eluting with a reducing agent. This may be extremely useful when elution is difficult
using other methods. The entire ligand-protein complex is eluted from the medium.
Coupling other functional groups
EAH Sepharose 4B may be used as a starting material for coupling via alternative functional
groups (Figure 71). Phenolic groups may be attached via diazonium derivatives (VII) or via
the bromoacetamidoalkyl derivative (V) prepared by treating EAH Sepharose 4B with
O-bromoacetyl-N-hydroxysuccinimide. This derivative also couples via primary amino groups.
The spacer arm of EAH Sepharose 4B may be extended by reaction with succinic anhydride
at pH 6 (VI) to form a derivative to which amino groups can be coupled by carbodiimide
reaction. Carboxyl groups are coupled to EAH Sepharose 4B by the carbodiimide reaction
(III). Thiol derivatives, prepared by reaction (IV), couple carboxyl groups in the presence of
carbodiimide and the thiol ester bond may be cleaved specifically using hydroxylamine,
thus providing a simple and gentle method for eluting the intact ligand-protein complex.
NH(CH 2)
5COOH
ECH Sepharose 4B
II
carbodiimide
RNH 2
NH(CH 2)
5CONHR
CNBr-Activated Sepharose
I
RNH 2
NHR
III carbodiimide
RCOOH
NH(CH 2)
6NHCOR
NH(CH 2) 6NH2
EAH Sepharose 4B
V
NH
IV
2
NH(CH
CH S
2)
6NHCOCH(CH 22 ) SH
2
CH2
NH 2 CH CO
(homocysteine thiolactone)
(1)
(2)
(3)
VII
O
=
O 2N C N 3
(p-nitrobenzoylazide)
Na 2 SO 2 4
(sodium dithionite)
HNO2
(nitrous acid)
VI
NH(CH 2)
6NHCOCH2Br
O
O
BrCH 2CON
O
(O-bromoacetyl-N-hydroxysuccinimide)
O O O
(succinic anhydride)
NH(CH 2)
6 NHCO N+
N
NH(CH 2)
6NHCO(CH 22 ) COOH
Fig. 71. Reactions used to couple ligands to Sepharose.
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