Efficient wheat germ agglutinin purification with a chitosan-based affinity chromatographic matrix.
An efficient affinity chromatographic matrix based on chitosan for wheat germ agglutinin (WGA) purification was developed. The matrices assayed consisted of chitosan mini-spheres cross-linked with epichlorhydrin 45, 250 or 500 mM. The maximum adsorption capacity of pure WGA - calculated from the corresponding isotherms - was between 43.2 and 48.9 mg/g at pH 5.0 and between 16.6 and 27.6 mg/g at pH 8.5. However, the adsorption of agglutinin from wheat germ extract was higher at pH 8.5. In addition, 0.5 g of mini-spheres cross-linked with epichlorhydrin 250 mM adsorbed 94.5% of the WGA present in 5 mL of the concentrated extract. Acetic acid was able to elute 100% of the adsorbed WGA. The purity of the WGA obtained was greater than 95% and the purification factor was 56.8. The matrix was able to maintain an efficient performance of the purification process for three consecutive cycles. A new method to monitor the purification process by RP-HPLC was developed.
School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, Buenos Aires, Argentina.
María F
MF
Baieli
Nicolás
N
Urtasun
María V
MV
Miranda
Osvaldo
O
Cascone
Federico J
FJ
Wolman
D000327:Adsorption; D000345:Affinity Labels; D048271:Chitosan; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D017743:Equipment Reuse; D006384:Hemagglutination / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008855:Microscopy, Electron, Scanning; D014909:Wheat Germ Agglutinins / Q000302:isolation & purification* / Q000494:pharmacology
D000345:Affinity Labels; D014909:Wheat Germ Agglutinins; D048271:Chitosan
Affinity purification; Chitosan; Chromatographic matrix; Wheat germ agglutinin