Electrophoretic study of conformational changes of a human soluble beta-D-galactoside-binding lectin upon storage.
Human brain lectin (HBL), a beta-galactoside specific soluble lectin, was purified by affinity chromatography. An alkylated derivative of this lectin was also prepared. Both native and modified molecules were conserved at -20 degrees C in the presence or absence of beta-mercaptoethanol, a reducing agent which was described to maintain the lectin activity in vitro or in the presence of beta-mercaptoethanol and lactose. The impact of storage conditions, over one year, on the native and derivated lectins, was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and titration curve, using the PhastSystem (Pharmacia). Western-blot analysis using an anti-HBL antibody and size-exclusion high performance liquid chromatography were used to complete the study. The subunit M(r)s were estimated before freezing (T0) and after three and twelve months (T3, T12). They were comparable for all preparations. In all samples tested, isoelectric focusing demonstrated the existence of at least three acidic proteins, with the pI ranging between 4.7-4.9. Titration curves clearly showed pH-dependent conformational changes, resulting in a panel of differently charged molecular species, some of which may be related to different oxidative states of the cysteine residues. We concluded that lectin can be stored at -20 degrees C for at least one year before use as a reagent since the modifications revealed by electrophoretic analysis do not alter the hemagglutination activity and carbohydrate binding properties. The immunoreactivity also remained unchanged.
Avellana-Adalid
V
V
Laboratoire de Biochimie et Technologie des Protéines, U.F.R. Santé, Médecine et Biologie Humaine, Université Paris-Nord, Bobigny, France.
Joubert-Caron
R
R
Caron
M
M
Bladier
D
D
D015153:Blotting, Western; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D037161:Galectins; D006385:Hemagglutination Inhibition Tests; D006388:Hemagglutinins / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007785:Lactose / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D008970:Molecular Weight; D011487:Protein Conformation
D037161:Galectins; D006388:Hemagglutinins; D008623:Mercaptoethanol; D007785:Lactose