Effect of Microtubules Hierarchy on Photoinduced Hydrogen Generation and Application to Artificial Photosynthesis
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Effect of Microtubules Hierarchy on Photoinduced Hydrogen Generation and Application to Artificial Photosynthesis Kosuke Okeyoshi1, 2, Kawamura Ryuzo1, Ryo Yoshida2, and Yoshihito Osada1 1 RIKEN Advanced Science Institute, 2-1 Hirosawa Wako-shi, Saitama 351-0198, Japan 2 Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
ABSTRACT Several strategies have been explored from viewpoint of biomimetics to accomplish artificial photosynthesis by using macromolecules as a medium such as liposomes, supramolecules, and hydrogels.1 Differing from disordered solution systems in which multiple components such as photosensitizer and catalytic nanoparticle are diffusively mixed, the photochemical reactions occur efficiently in medium due to maintenance of the dipersibility of the components and specific molecular arrangement. Here we attempt to clarify the effect of medium hierarchy for photoinduced electronic transmission among multiple components. By conjugating each component on tubulin and integrating them via self-assembly to microtubules, ideal component arrangements with optimum distance for the electronic transmission will be possible. INTRODUCTION We designed and fabricated Ru(bpy)32+-conjugated microtubules for photoinduced H2 generation. By conjugating Ru(bpy)32+ on microtubules, the structure enables to avoid aggregation among the Ru(bpy)32+ that cause loss of photoenergy in self-quenching process. The hierarchy of tubulin/microtubules is controlled by using 6-thioguanosine-5’-triphosphate (6-thioGTP) which maintains the structure as tubulin, or guanosine-5’-(β,γ-methylene)triphosphate (GpCpp) which maintains the structure as microtubules (Fig. 1). When the conjugated Ru(bpy)32+ is excited by photoenergy, it will effectively give electron to Pt nanoparticle and H2 generates.
EXPERIMENTAL Preparation of Tubulin. To remove microtubules-associated proteins, tubulin was purified from porcine brain using a high concentrations of PIPES buffer (1 M PIPES, 20 mM ethyleneglycolbis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 10 mM MgCl2; pH adjusted to 6.8 using HCl).2 The tubulin concentration was determined by measuring the absorbance at 280 nm using an extinction coefficient of 115,000. Ru(bpy)32+ Conjugation and Stoichiometric Estimation. Ru(bpy)32+-conjugated tubulin was prepared using Bis(2,2’-bipyridine)-4’-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) by amine coupling according to previous studies.3,4 The
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Ru(bpy)32+-tubulin stoichiometry, i.e., Ru(bpy)32+ molecules/tubulin was estimated to be 3.1 ± 0.1/2.0 on average. The efficiency of labeling by the amine coupling was 31%. The concentration of Ru(bpy)32+ was determined by measuring the peak absorbance at 460 nm. The conjugation of Ru(bpy)32+ on tubulin was confirmed by observing the loaded gel on an optical band-pass filter (= 450 ± 25 nm, BPB 45; Fujifilm). Tubulin concentration was determined by the contrast of the stain
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