K. Bauerarnaz et al., SALT-INDUCED IMMOBILIZATION OF SMALL AFFINITY LIGANDS ON AN EPOXIDE-ACTIVATED AFFINITY SUPPORT, Journal of chromatography, 803(1-2), 1998, pp. 73-82
Citations number
15
Categorie Soggetti
Chemistry Analytical","Biochemical Research Methods
The salt-induced immobilization of small ligands onto an epoxy-activat
ed HPLC stationary phase was found to be an effective route to the pre
paration of HPLC-compatible affinity supports. The ligands S-methylglu
tathione, S-butylglutathione and S-octylglutathione were immobilized a
t pH 10.5 under various conditions of salt type and concentration. In
potassium carbonate buffer the extent of reaction for the alkylglutath
ione ligands was insensitive to carbonate concentration (0.5-2 M) but
was substantially influenced by the hydrophobicity of the ligand. S-Oc
tylglutathione was immobilized with a 4-fold greater efficiency in 2 M
potassium carbonate than was the less hydrophobic S-methylglutathione
. By contrast, in potassium phosphate buffer the extent of the immobil
ization was strongly dependent on salt concentration. For the S-alkylg
lutathiones, a 2-fold increase in immobilization efficiency was found
when increasing the phosphate concentration from 0.5 to 2 M. Prior the
ory was extended to explain these results involving a salt-induced hyd
rophobic interaction between the ligand and the epoxy phase which in t
urn results in faster reaction rates between ligand and epoxy groups.
As predicted by this model, the immobilization of the very hydrophilic
ligand, nitrilotriacetic acid, was found to be affected minimally by
either salt type or salt concentration. Further, when the rate of hydr
olysis of the immobilized epoxy groups was studied, the maximum amount
of immobilized ligand was found, in some cases, to be limited by the
hydrolysis of the epoxy groups. Practical application of these princip
les allowed a nickel chelate HPLC-affinity phase to be produced using
the nitrilotriacetic acid as the ligand. The HPLC phase was found to b
e useful in multiple, small-scale purifications of a histidine-tagged
protein kinase C fragment. (C) 1998 Elsevier Science B.V.