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34 Vvayalakshmi
on histidine-ligand afftnity chromatography at or around the isoelec-
tric point of the protein/peptide (3).
2. Materials
2.1. Chemicals
1. Sepharose 4B or 6B (Pharmacia, Uppsala, Sweden).
2. Silica-Spherosil XOB30 (Rhone-Poulenc, France).
3. Lichrosorb 60 (Merck, France).
4. L. Histicline, epichlorhydrin, 1,4 butanediol diglycidyl ether, sodium
hydroxide (NaOH), sodium chloride (NaCI), sodium borohydride,
and all other reagents are obtained either from Sigma or from Merck
and are of Analar purity grade.
2.2. Apparatus
1. Stirring water bath or a shaft stirrer (OSI, France).
2. Normal laboratory vacuum filtration equipment.
3. Column, pump, detector, recorder, and fraction collector (LRB, Swe
den).
4. Radial flow column: Sepragen was a kind gift from Touzart et
Matignon, France.
3. Method
A typical gel is prepared by first introducing oxirane active groups
onto a polysaccharide based (e.g., Sepharose 4B@) or silica based, OH
containing insoluble matrices at basic pH (see Note 1). Then, the
active oxirane ring is opened and coupled to the primary amine group
of the amino acid histidine. The proposed structure of such an adsor-
bent is represented in Fig. 1.
1. Sepharose 4B is supplied as an aqueous suspension containing 20%
ethanol as a preservative. So, to start with, wash the gel as supplied by
the manufacturer with water to remove the ethanol and suction-dry
on a sintered glass. The surface cracking of the gel cake is taken as
the indication of the end of suction drying. To 10 g of suction-dried
gel contained in a reactor or an Erlenmeyer flask, add 5 mL of 2M
sodium hydroxide along with 0.5 mL of epichlorhydrin and 100 mg
sodium borohydride to avoid any oxidation of the primary alcohol
groups and keep under stirring. Avoid magnetic stirring, which will
disrupt the soft gel beads; use lateral or shaft stirring. Then, add pro
gressively another 5 mL of 2M sodium hydroxide and 2.5 mL of