Bioconjugate Chem. 1998, 9, 409−412
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Photostable Chlorophyll a Conjugated with Poly(vinylpyrrolidone)-Smectite Catalyzes Photoreduction and Hydrogen Gas Evolution by Visible Light Tetsuji Itoh,† Asako Ishii,† Yoh Kodera,† Ayako Matsushima,† Misao Hiroto,† Hiroyuki Nishimura,† Toshi Tsuzuki,† Toshiaki Kamachi,‡ Ichiro Okura,‡ and Yuji Inada*,† Toin Human Science and Technology Center, Department of Materials Science and Technology, Toin University of Yokohama, 1614 Kurogane-cho, Aoba-ku, Yokohama 225-8502, Japan, and Department of Bioengineering, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Received September 12, 1997; Revised Manuscript Received January 14, 1998
Chlorophyll a was adsorbed to a synthetic smectite intercalated by poly(vinylpyrrolidone) (PVP) to form the chlorophyll-PVP-smectite conjugate (Chl-PVP-SME) having an absorption maximum at 677 nm. The conjugate was found to be stable toward light illumination in comparison with chlorophyll-smectite, chlorophyll-PVP, and free chlorophyll a. Chl-PVP-SME had a photoinduced activity for catalyzing the reduction of methyl viologen. Furthermore, the evolution of hydrogen gas was observed when an aqueous suspension containing Chl-PVP-SME, methyl viologen (an electron carrier), 2-mercaptoethanol (an electron donor), and hydrogenase was illuminated by visible light.
INTRODUCTION
Chlorophyll molecules bind to proteins to form chlorophyll-protein conjugates in the layered thylakoid membrane of chloroplasts and exhibit physiological functions: the photolysis of water and the reduction of NADP (1) under visible light illumination. A large number of investigations on photosensitization have been published using photocatalytic inorganic compounds such as titanium oxide in the presence of ultraviolet ray (2). To enhance the efficacy of the photocatalytic reactions, Domen et al. (3, 4) demonstrated that a series of layered metal oxide, perouvskite, catalyzes overall water splitting. In addition, Miyata et al. (5) prepared viologens intercalated into the layered space of bentonites in relation to their photochromic behavior. From the viewpoint of solar energy conversion, however, photocatalysts such as chlorophyll and porphyllin derivatives which work under visible light illumination are indispensable. Okura et al. (6, 7) observed photoinduced hydrogen evolution using zinc meso-tris(sulfonatophenyl)porphyrin as a photosensitizer in the presence of an electron donor and an electron carrier under visible light illumination. Chlorophyll, however, has been seldom used as a photocatalyst because the natural pigment loses its photostability by extraction with an organic solvent. We prepared chlorophyll a adsorbed onto bentonite (8) and onto smectite (9). Each conjugate was found to be photostable, and its adsorption maximum was shifted to a longer wavelength by increasing the amount of chlorophyll a adsorbed onto clay minerals. Bentonite and smectite minerals, which consist of two-dimensional silicate layers separated by hydrated exchangeable cations, swell with a variety of molecules and form inter* Author to whom correspondence should be addressed. Phone: +81-45-974-5060. Fax: +81-45-972-5972. † Toin University of Yokohama. ‡ Tokyo Institute of Technology.
calated complexes (10). Recently, we reported that the chlorophyll-bentonite conjugate exhibited a high photosensitizing activity for reducing nitro blue tetrazolium, through the formation of superoxide anion (11). This paper deals with preparation of a chlorophyllPVP-smectite conjugate having high photostability against light. Furthermore, the conjugate evolves hydrogen gas effectively in the presence of an electron donor, an electron carrier, and a catalyst under visible light illumination. EXPERIMENTAL PROCEDURES
General. Chlorophyll a purified from Spirulina, poly(vinylpyrrolidone) (PVP)1 with an average molecular weight of 10 000, and methyl viologen (1,1-dimethyl-4,4bipyridinium chloride) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Smectite powder (hectorite), which was hydrothermally synthesized, was obtained from Co-op Chemical Co., Ltd. (Tokyo, Japan). Its properties are as follows: elemental composition Si 8.00, Mg 5.65, Li 0.70, Na 1.05; transmittance 95% in 1% aqueous solution at 500 nm; and methylene blue adsorption 101 mequiv per 100 g. Hydrogenase was isolated from Desulfovibrio vulgaris according to Yagi’s method (12). Other reagents were of analytical grade. Chlorophyll-PVP-Smectite. The chlorophyllPVP-smectite conjugate (Chl-PVP-SME) was prepared to test the photostability of chlorophyll a as follows. At first, smectite was suspended in a 3% NaCl aqueous solution for 24 h three times to make Na-smectite. Nasmectite was then stirred in a PVP aqueous solution (5%) with a 3:2 weight ratio of PVP and Na-smectite, and the mixture was freeze-dried to form a PVP-smectite con1Abbreviations: PVP, poly(vinylpyrrolidone); Chl-PVPSME, chlorophyll-poly(vinylpyrrolidone)-smectite conjugate; PVP-SME, poly(vinylpyrrolidone)-smectite conjugate; ChlPVP, chlorophyll-poly(vinylpyrrolidone) conjugate; Chl-SME, chlorophyll-smectite conjugate.
S1043-1802(97)00165-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/16/1998
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jugate (PVP-SME) according to the method of Miyata et al. (5). Two milliliters of chlorophyll a dissolved in benzene (9.8 mg/mL) was added to PVP-SME (600 mg). The suspension was then shaken for 1 h at 25 °C to establish the adsorption equilibrium between chlorophyll a and PVP-SME. The Chl-PVP-SME thus prepared was collected by centrifugation and was dried under reduced pressure. The amount of chlorophyll a adsorbed to PVP-SME was spectrophotometrically determined by using the molar extinction coefficient of chlorophyll a (9.25 × 104 M-1 cm-1 at 661.6 nm in acetone) (13). One gram of smectite powder in the conjugate adsorbed 12.5 mg of chlorophyll a and 1.5 g of PVP. The absorption spectrum of Chl-PVP-SME in 50 mM Tris-HCl buffer (pH 7.8) was measured with a Shimadzu MPS-2000 multipurpose spectrophotometer (Kyoto, Japan) (14). The photostability of chlorophyll a was tested as follows. Chl-PVP-SME together with Chl-SME, Chl-PVP in water, and free chlorophyll a in benzene was illuminated with a 60 W incandescent lamp at a distance of 9.5 cm, with a light intensity of 200 J m-2 s-1, at 30 °C. Chlorophyll-PVP-Smectite Intercalated by Methyl Viologen. Chl-PVP-SME intercalated by methyl viologen was prepared to test whether the conjugate causes photoreduction of methyl viologen by visible light. First, an aqueous solution (17 mL) of methyl viologen (101 mg) was added to powdered PVP-SME (580 mg), and the mixture was stirred for 48 h. After centrifugation, the precipitate was washed with methanol and dried (the conjugate contained 21 mg of methyl viologen). PVP-SME intercalated by methyl viologen was then added to chlorophyll a (19.6 mg) dissolved in benzene (2 mL), and the suspension was shaken for 1 h at 25 °C to establish the adsorption equilibrium between chlorophyll a and PVP-SME intercalated by methyl viologen. The Chl-PVP-SME intercalated by methyl viologen was collected by centrifugation and was dried under reduced pressure. The conjugate was composed of Chl, PVP, SME, and methyl viologen at a weight ratio of 1:178:130: 16. Photoreduction of Methyl Viologen. Photoreduction of methyl viologen intercalated into Chl-PVP-SME was tested in the presence of 2-mercaptoethanol. The conjugate (17.3 mg; 0.06 µmol as chlorophyll a, 4.6 µmol as methyl viologen) was suspended in 3 mL of 25 mM Tris-HCl buffer (pH 7.4) containing 1 mmol of 2-mercaptoethanol. The sample mixture was deaerated by repeated freeze-pump-thaw cycles and was illuminated with a 60 W incandescent lamp at a distance of 3.5 cm, with a light intensity of 1500 J m-2 s-1, at 30 °C. The photochemical reduction of methyl viologen was observed by measuring the absorbance increase at 605 nm, using the molar extinction coefficient of 1.65 × 104 M-1 cm-1. Evolution of Hydrogen Gas. Hydrogen evolution by Chl-PVP-SME intercalated by methyl viologen in the presence of 2-mercaptoethanol and hydrogenase was tested according to the method of Okura et al. (6, 7). The reaction system (3 mL) consisted of 47.5 mg of ChlPVP-SME intercalated by 2.5 mg of methyl viologen (50 mg; 0.17 µmol as chlorophyll a, 3 µmol as methyl viologen) suspended in 25 mM Tris-HCl buffer (pH 7.4) in the presence of 2-mercaptoethanol (1 mmol) and a hydrogenase solution (40 µL). The sample mixture in a Pyrex cell (17.5 cm3) was deaerated by repeated freezepump-thaw cycles, and the upper space of the cell (14.5 cm3) was filled with argon gas. The sample in the cell equipped with a magnetic stirrer was illuminated with a 200 W tungsten lamp (light intensity of 200 J m-2 s-1). Light at wavelengths of
