Characterization of the carbohydrate binding specificity and kinetic parameters of lectins by using surface plasmon resonance.
An accurate, rapid, and sensitive method for characterizing the carbohydrate binding properties of lectins using a BIAcore apparatus and the detection method of surface plasmon resonance is described. As a model study, the sialic acid binding lectins from Sambucus nigra and Maackia amurensis, which are specific for the epitopes Neu5Ac(alpha2-6)Gal and Neu5Ac(alpha2-3)Gal, respectively, were chosen as suitable candidates. Two systems, one for the analysis of oligosaccharides and the other for glycoproteins, were developed after a rigorous analysis and evaluation of such parameters as binding conditions, buffers, and regeneration conditions. The systems take into account nonspecific binding, using the respective denatured lectin as negative blank, and avoid loss of activity: regeneration of the surface using either 10 mM NaOAc (pH 4.3) buffer (oligosaccharide system) or 20 mM HCl (glycoprotein system). The specificity of the lectins is well illustrated, while the kinetics parameters are shown to be sensitive to subtle changes in the recognized epitopes, and to be affected by steric hindrance. Surface plasmon resonance is a suitable technique for the analysis and characterization of lectins.
Haseley
S R
SR
Department of Bio-Organic Chemistry, Utrecht University, Utrecht, NL-3508 TB, The Netherlands. simon@boc.chem.uu.nl
Talaga
P
P
Kamerling
J P
JP
Vliegenthart
J F
JF
D000818:Animals; D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry*; D006736:Horses; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*
D002241:Carbohydrates; D037102:Lectins; D009844:Oligosaccharides