**Author details**

A hydrophobic environmental sensitive fluorophore, badan, labeling LukF, has also been

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

Groulx et al. [31] measured the stoichiometry of oligomerization of another pore-forming toxin using SMI. Monomers of *Bacillus thuringiensis* toxin Cry1Aa were labeled with a fluo‐ rophore at a cysteine residue. After the complex formation, molecules were attached on a coverslip to observe the photobleaching process. Counting the step number during the pho‐

Nabika et al. [32] used SMI for observation of lateral diffusion of cholera toxin B subunit (CTX) on the artificial lipid bilayer containing GM1, which is the receptor of CTX. The diffu‐ sion coefficient was one order smaller than that of lipid molecules in the membrane, and

On the contrary, toxin has been used for single-molecule measurement. Since direct fixation of molecules to a substrate possibly induces artifacts in the measurements, single molecules are sometimes entrapped into fixed tiny liposomes in which the molecules can move more freely. In this case, however, the solution around the molecules cannot be changed during the experiment, limiting experimental conditions. Okumus et al. [33] used liposomes, recon‐

As shown in this chapter, SMI can be used to detect molecular interactions between proteins and other biological molecules. In addition to detections of static oligomerization states, SMI allows characterization and analysis of dynamic reaction processes, including association-

Kinetic analyses based on SMI measurements have several advantages over analyses using conventional biochemical and ensemble-molecule imaging measurements: SMI allows quan‐ titative measurements with minimal disruption of the system integrity. Actually, SMI is ap‐ plicable to complex systems, like living cells, and avoids perturbations for synchronization. Measurements in complex systems are useful in analyses of the reaction kinetics between unknown elements, as shown in the case of RAF and the undetermined kinase(s). SMI meas‐ urements have often found novel reaction intermediates. This is because virtual synchroni‐ zation at the reaction steps and complete separation between the forward and backward

These advantages of SMI measurements make them effective in quantitative analysis of bio‐ logical reaction kinetics, providing basic information required in system-level analyses in re‐ cent molecular cell biology. In the near future, SMI measurements will be expanded to be used in pharmacology to provide novel drug screening methods and analyses of the sites of action for medical drugs, in pathology to detect currently undetermined dysfunctions of

/s) diffusive fractions. This observation

/s) and lower (< 0.1 μm2

was explained by assuming multivalent binding between CTX and GM1 molecules.

used to detect complex formation with HS in single molecules [30].

stituting pore-forming toxin, to allow exchange of inside solutions.

dissociation kinetics and enzymatic reactions.

tobleach, it was concluded that the toxin forms a tetramer.

there were higher (0.4 μm2

Applications

450

**5. Conclusions**

reactions are allowed.

Kayo Hibino1 , Michio Hiroshima1,2, Yuki Nakamura2 and Yasushi Sako2\*

\*Address all correspondence to: sako@riken.jp

1 Laboratory for Cell Signaling Dynamics, RIKEN QBiC, Japan

2 Cellular Informatics Laboratory, RIKEN. 2-1 Hirosawa, Japan

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Applications

452


**Chapter 18**

**Molecular Recognition of Glycopolymer Interface**

Saccharides on the cell surfaces play important roles in the living systems. For example, it mediate the cell-cell adhesion, fertilization, protein transportation, infection of pathogens and cancer metastasis etc [1, 2]. The saccharide-protein interactions also involve the various biological events (Table 1). Actualy, the saccharides are the model compounds of some of the medicines like oseltamivir [3]. The interaction between galactose and asialoglycoprotein receptor is a possible mechanism for the hepatocyte-specific drug delivery systems [4]. Therefore, it has been pointed out that the saccharide-protein interaction can be utilized for the novel bio-functional mateials such as cell cultivation, medicine target, and drug deliver‐

**Target Saccharide structure**

Cholera toxin GM1:Galβ1-3(NeuAcα2-3)GalNAcβ1-4Galβ1-4GlcCer

© 2013 Miura et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 Miura et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

distribution, and reproduction in any medium, provided the original work is properly cited.

Gb3: Gal1α-4Galβ1-4GlcCer

Neu5Acα2-6Galβ1-4(3)GlcNAcβ1-, Neu5Acα2-6Galβ1-3GalNAcβ1

Wheat germ agglutinin (WGA) GlcNAc, Neu5Ac

Lectin Concanavalin A (ConA) α-Man/α-Glc

Cell Hepatocyte β-Gal/β-GalNAc

Yoshiko Miura, Hirokazu Seto and

Additional information is available at the end of the chapter

Tomohiro Fukuda

**1. Introduction**

ly systems.

Pathogen Shiga toxin

(from E. coli O-157etc)

Influenza Type A for human

**Table 1.** The saccharide recognition of proteins, cells and pathogens.

http://dx.doi.org/10.5772/54156
