George Hirabayashi

Evaluation of concanavalin A-mannose interaction on the electrode covered with collagen film

An electrode covered with a lectin/collagen film was constructed to investigate whether the film was usable as a reaction field of binding between the lectin and sugar. The protein-sugar binding on cell surface plays an important role to various physiologic processes. The film is considered to be a cell surface, due to its biocompatibility. The immobilization of concanavalin A (Con A) which is one of proteins was attempted by an electrostatic interaction of the protonated functional groups of film to the negative charged Con A. The merit of this immobilization is that the interaction hardly causes any changes in the protein structure. Because Con A recognizes mannose moiety, the mannose was labeled with an electroactive compound. The binding was estimated from the changes of the electrode response based on the holding of electroactive moiety in the binding site of Con A to the mannose moiety. However, the electrode responses of glucose and galactose labeled with the same substance did not change. The result shows that Con A is immobilized on the film and combines with labeled mannose. Therefore, it is clear that the collagen film is suitable as the reaction field to evaluate the protein-sugar binding.

Voltammetric evaluation for the binding of wheat germ agglutinin to glucosamine-modified magnetic microbead

Binding of wheat germ agglutinin (WGA) on glucosamine-modified magnetic microbeads was investigated with voltammetry. A magnetic bead was considered as a cell, and the beads with amino groups were modified with the sugar by using a cross-linking reagent. To evaluate the binding, glucose labeled with an electroactive daunomycin was prepared as a probe. After WGA and the beads were mixed in 0.1M phosphate buffer (pH 7.0), the labeled glucose was added to the solution. The binding was monitored from the changes in the electrode response of labeled glucose because the labeled glucose was held to the binding site of WGA for the sugar. In contrast, other lectin not having the binding site to glucosamine or glucose was incubated with the glucosamine-modified beads. As a result, the change of peak current was not observed. Therefore, it is clear that the binding of WGA to glucosamine moiety on the bead surface selectively takes place. This method would be powerful for evaluation of interaction between protein and sugar chain existing at cell surface.

Voltammetric sensing of sugar by an electrode covered with wheat germ agglutinin/chitin film

The binding between wheat germ agglutinin (WGA) and N-acetylglucosamine at the electrode covered with chitin film was investigated with voltammetry. Chitin, beta-1,4-poly-N-acetylglucosamine, is one of the biolpolymers which have a high biocompatibility. WGA is immobilized to the surface of chitin film by the affinity of WGA to N-acetylglucosamine residue of chitin. To investigate the binding event of WGA on the chitin modified electrode, N-acetylglucosamine labeled with an electroactive compound was prepared. The binding causes the changes in the electrode response of labeled sugar. The peak current of labeled sugar decreased due to the specific binding with WGA on the chitin film modified at the electrode. N-Acetylglucosamine was successfully determined by using the competitive reaction with labeled sugar to WGA on the chitin film electrode.