Fluorescence Emission and Polarization for Analyzing Binding of

Jun 19, 2009 - Tomoko Okada , Chika Isobe , Takeaki Wada , Sumiyo Ezaki , and Norihiko Minoura. Bioconjugate Chemistry 2013 24 (6), 841-845...
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Bioconjugate Chem. 2009, 20, 1296–1298

COMMUNICATIONS Fluorescence Emission and Polarization for Analyzing Binding of Ruthenium Metalloglycocluster to Lectin and Tetanus Toxin C-Fragment Tomoko Okada,* Taro Makino, and Norihiko Minoura Graduate School of Bionics, Tokyo University of Technology, Katakura, Hachioji, Tokyo 192-0982, Japan. Received March 6, 2009; Revised Manuscript Received May 15, 2009

We have designed and synthesized ruthenium complexes bearing clustered galactose Ru(bpy-2Gal)3 and glucose Ru(bpy-2Glc)3. Changes in fluorescence emission (FE) and fluorescence polarization (FP) of the metalloglycoclusters were measured by adding each lectin (peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), concanaValin A (ConA), or wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA and ConA, the FE spectra of Ru(bpy-2Gal)3 and Ru(bpy-2Glc)3 showed new emission peaks, respectively. In addition, Ru(bpy-2Gal)3 and Ru(bpy-2Glc)3 exclusively increased the FP values by addition of PNA and ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were confirmed by the FE and FP measurement. From the FP analyses, the dissociation constants (Kd) of Ru(bpy-2Gal)3 to PNA and Ru(bpy-2Glc)3 to ConA were calculated to be ca. 6.1 × 10-6 M and 1.8 × 10-5 M. Furthermore, the FP analyses proved specific binding of Ru(bpy2Gal)3 to TCF.

Carbohydrates are well-known to play important roles in biological processes. Specific binding between carbohydrate and protein (lectin) can induce serious infections from virus and pathogenic toxins (1). Therefore, development of a useful method is desirable to investigate the affinity property of the carbohydrate. Fluorescence polarization (FP) analysis is an effective method to evaluate a binding event of a small carbohydrate molecule and large protein (2). Since the FP analysis requires a fluorescent-labeled carbohydrate probe, this study aimed to apply a fluorescent metalloglycocluster as a probe molecule. A metalloglycocluster, a group of a glycocluster containing a metal center, is an especially attractive carbohydrate probe, since the metal center gives characteristic photochemical and electrochemical properties that can be utilized as an indicator. Moreover, the clustered carbohydrate around the metal center would enhance the binding constant of generally weak interaction between carbohydrate and protein. Recently, many researchers reported the enhanced binding constant achieved by clustering the carbohydrate, such as dendrimers modified with carbohydrates at their branches, organic polymers possessing carbohydrate branches at each monomer unit, and ruthenium metalloglycoclusters (3-6). Since the ruthenium metalloglycocluster possesses characteristic fluorescence emission (FE) property, both the FE and FP analyses are applicable for the affinity evaluation. In this study, we propose the utility of FE and FP analyses using a ruthenium metalloglycocluster for evaluating the affinity property between carbohydrate and lectins. We designed metalloglycoclusters, Ru(bpy-2Gal)3 and Ru(bpy2Glc)3, as carbohydrate probes (Figure 1). Ru(bpy-2Gal)3 and Ru(bpy-2Glc)3 have a ruthenium metal center coordinated by three bpy-2Gal and bpy-2Glc. Since the ruthenium(II) complex * [email protected].

Figure 1. Schematic representation of the metalloglycoclusters Ru(bpy2Gal)3 and Ru(bpy-2Glc)3.

adopts octahedral geometry, six galactoses or glucoses are clustered around the metal center. These designed metalloglycoclusters were synthesized by chemical synthetic procedures. For the affinity evaluation, both ruthenium complexes were used without isolation of diastereomers (Λ- and ∆-forms).

10.1021/bc900101u CCC: $40.75  2009 American Chemical Society Published on Web 06/19/2009

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Figure 3. Changes in fluorescence polarization of the metalloglycoclusters (20 µM), (a) Ru(bpy-2Gal)3 and (b) Ru(bpy-2Glc)3, following addition of lectins (5 µM) (PNA (pink), RCA (orange), ConA (green), and WGA (blue)). Excitation wavelength was 468 nm, and polarization values were used at 580 nm.

Figure 2. Fluorescence emission spectra of the metalloglycoclusters (20 µM), (a) Ru(bpy-2Gal)3 and (b) Ru(bpy-2Glc)3, with lectin (5 µM) (PNA (pink), RCA (orange), ConA (green), and WGA (blue)) or without lectin (gray). Excitation wavelength was 468 nm.

To examine the binding properties of Ru(bpy-2Gal)3 and Ru(bpy-2Glc)3, fluorescence emission spectra were measured for each probe before and after the addition of each lectin (PNA, RCA, ConA, and WGA). Figure 2a shows the emission spectra of Ru(bpy-2Gal)3 with or without each lectin. Addition of PNA caused the most drastic change to the spectral shape compared with the other lectins (RCA, ConA, and WGA). In particular, a new emission peak appeared at ca. 580 nm, and this new peak disappeared following addition of galactose to the solution. The emission spectral change suggests that Ru(bpy-2Gal)3 specifically binds to PNA, and the local environment of Ru(bpy-2Gal)3 becomes more hydrophobic compared with when the Ru(bpy2Gal)3 presents alone in PBS (7). This assumption is supported by the emission spectrum of Ru(bpy-2Gal)3 in ethanol that is a more hydrophobic solvent than PBS. Following the addition of ethanol to the PBS solution of Ru(bpy-2Gal)3, a new emission peak was observed at 580 nm. Therefore, the new emission peak observed for Ru(bpy-2Gal)3 with PNA is evidence that Ru(bpy2Gal)3 specifically binds to the hydrophobic binding pocket of PNA. A similar spectral change was observed for Ru(bpy-2Glc)3 with ConA. Since the appearance of the new emission peak was not observed with other lectins (PNA, RCA, and WGA), the FE analysis confirms that Ru(bpy-2Glc)3 specifically binds to ConA. These FE measurements confirmed that galactose and glucose bind specifically to PNA and ConA, respectively. Such a drastic change in emission spectra is unusual during the course of the binding event between the ruthenium metalloglycocluster and lectin. The affinity evaluation method using the FE spectrum depends on the hydrophobicity of lectin. In contrast, the FP analysis allows direct investigation of the molecular motion (namely, molecular size) in the absence of contributions derived from the environmental conditions that the metalloglycoclusters experience. Therefore, the FP measurement was performed to confirm the affinity property of the metalloglycocluster to lectin.

Figure 3 shows the increased FP values when PNA, RCA, ConA, or WGA was added to each metalloglycocluster. Unlike the other lectins, FP value of Ru(bpy-2Gal)3 increased the most by the addition of PNA. In contrast, Ru(bpy-2Glc)3 showed the greatest increment when ConA was added to the solution. These drastic increments in FP values confirm that Ru(bpy-2Gal)3 and Ru(bpy-2Glc)3 specifically bind to PNA and ConA, respectively. Besides, electrostatic interaction between the divalent metalloglycoclusters and lectin is negligible because the emission spectrum of Ru(bpy)3 showed no changes following addition of each lectin. Therefore, the FE and FP analyses determined specific bindings of galactose to PNA and glucose to ConA, which are consistent with the previous reports showing high affinity of galactose to PNA and glucose to ConA. Changes in the FE and FP of Ru(bpy-2Gal)3 were unexpectedly negligible, suggesting poor affinity of Ru(bpy-2Gal)3 to RCA, despite the previous paper reports high affinity of galactose-bearing metalloglycocluster to RCA (3). Surface plasmon resonance analysis also proved that the RCA used in this study showed high affinity to galactose (4-aminophenyl-galactopyranoside) immobilized on a gold surface. Therefore, the low affinity is assumed to have a structural cause of the metalloglycocluster examined in this study, and molecular dynamics calculations are now underway to assess the possible reason. By varying the concentrations of PNA and ConA, FP values were collected to examine the detailed binding properties of Ru(bpy-2Gal)3 to PNA and Ru(bpy-2Glc)3 to ConA (Figure 4). The horizontal axis represents the concentration of the binding sites from each lectin, which possesses four independent binding sites for every lectin molecule. Ru(bpy-2Gal)3 showed a greater change in polarization than Ru(bpy-2Glc)3 signifying higher affinity of PNA to galactose than glucose. The FP value for Ru(bpy-2Gal)3 increased as the PNA increased and then plateaued. Nonlinear least-squares fitting determines the dissociation constant (Kd) of Ru(bpy-2Gal)3 and PNA as 6.1 × 10-6 M. In a similar manner, Figure 4b shows higher affinity of ConA to glucose than galactose, and the Kd value was estimated to be 1.8 × 10-5 M. These dissociation constants prove that the affinity of clustered galactose and glucose is higher than that of nonclustered galactose and glucose (Kd ≈ 10-3 to 10-4 M) (1). To confirm the clustering effect on the lectin binding event, Kd of a metalloglycocluster having two galactose residues, Ru(bpy)2(bpy-2Gal), was examined following the similar FP analysis with Ru(bpy-2Gal)3 (8). The Kd of Ru(bpy)2(bpy-2Gal) to PNA was estimated to be more than 10-4 M. Since highly clustered Ru(bpy-2Gal)3 showed lower Kd than Ru(bpy)2(bpy-2Gal), the clustered galactose of Ru(bpy-2Gal)3 was confirmed to raise its affinity to PNA. On the basis of the elucidated FP properties, affinity of the metalloglycoclusters (2 µM), Ru(bpy-2Gal)3 and Ru(bpy-2Glc)3, was examined using 0.05 µM of tetanus toxin c-fragment (TCF).

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properties between carbohydrate and lectins. Ru(bpy-2Gal)3 and Ru(bpy-2Glc)3 with the clustered galactose and glucose were confirmed to raise affinity to the lectin. Moreover, specific binding of Ru(bpy-2Gal)3 to TCF was proven by the FP analysis. These results ensure that the FP analysis using the ruthenium metalloglycocluster is useful for evaluating interactions between carbohydrates and toxins. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http:// pubs.acs.org.

LITERATURE CITED

Figure 4. Changes in fluorescence polarization of metalloglycoclusters (20 µM), Ru(bpy-2Gal)3 (green) or Ru(bpy-2Glc)3 (blue), following addition of (a) PNA and (b) ConA. Excitation wavelength was 468 nm and polarization values were used at 580 nm. The gray line represents a fitted curve obtained from nonlinear least-squares fitting.

The TCF reportedly has affinity for galactose (9, 10). Following the addition of TCF, Ru(bpy-2Gal)3 increased the polarization value by 47 mP; however, Ru(bpy-2Glc)3 increased only by 7 mP. Because of the distinctive increment of the FP value for Ru(bpy-2Gal)3, specific binding of galactose to TCF was confirmed. In summary, the FE and FP analyses showed that the synthesized novel metalloglycoclusters, Ru(bpy-2Gal)3 and Ru(bpy-2Glc)3, bind specifically to PNA and ConA, respectively. The results in this report proved that the combination of FE and FP analyses would empower evaluation of affinity

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