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Bioconjugate Chem. 2000, 11, 8−13
ARTICLES Hydrogen Gas Evolution and Carbon Dioxide Fixation with Visible Light by Chlorophyllin Coupled with Polyethylene Glycol Tetsuji Itoh, Hideharu Asada, Kazunori Tobioka, Yoh Kodera, Ayako Matsushima, Misao Hiroto, Hiroyuki Nishimura, Toshiaki Kamachi,† Ichiro Okura,† and Yuji Inada* Toin Human Science and Technology Center, Department of Biomedical Engineering, Toin University of Yokohama, Kurogane-cho, Aoba-ku, Yokohama 225-8502 Japan, and Department of Bioengineering, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Received April 21, 1999; Revised Manuscript Received September 20, 1999
Chlorophyllin a was conjugated with R-(3-aminopropyl)-ω-methoxypoly(oxyethylene), PEG-NH2, to form the PEG-chlorophyllin conjugate through acid-amide bonds. The PEG-chlorophyllin conjugate was stable toward light illumination under anaerobic condition in comparison with chlorophyllin a. The conjugate catalyzed the reduction of methyl viologen in the presence of 2-mercaptoethanol and the evolution of hydrogen gas in the presence of methyl viologen (an electron carrier), 2-mercaptoethanol (an electron donor) and hydrogenase (Scheme 1). Furthermore, the PEG-chlorophyllin conjugate catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme. Utilizing the reducing power of NADPH generated by the PEG-chlorophyllin conjugate under the illumination, CO2 fixation was accomplished by the synthesis of malate (C4) from pyruvate (C3) and CO2 in the presence of malic enzyme (Scheme 2). These reactions mentioned above did never proceed in dark or without each emzyme.
INTRODUCTION
Chemical modification of proteins with poly(ethylene glycol) (PEG), nontoxic, nonimmunogenic and amphiphathic polymer has been extensively studied for the purpose of applying proteins to biomedical and biotechnological processes (1, 2). Exogenous enzymes such as L-asparaginase with antitumor activity and adenosine deaminase (ADA) were successfully modified with PEG to reduce their immunoreactivity and immunogenicity (3, 4). In the biotechnological field, PEG-modified hydrolases became soluble and active in hydrophobic media and catalyzed the reverse reactions of hydrolysis, ester synthesis, and ester exchange reactions (5, 6). Along this line of investigations, Itoh et al. (7) reported that chlorophyllin a was conjugated with R-(3-aminopropyl)-ω-methoxypoly(oxyethylene), PEG-NH2,1 to form PEG-chlorophyllin through acid-amide bonds. The PEG-chlorophyllin conjugate was soluble not only in aqueous solution but also in organic solvents and possessed a potent photosensitizing activity to produce superoxide anion during the illumination, although chlorophyllin immediately lost its activity by light illumination. * To whom correspondence should be addressed. Phone: +8145-974-5060. Fax: +81-45-972-5972. † Tokyo Institute of Technology. 1 Abbreviations: PEG-NH , R-(3-aminopropyl)-ω-methoxy2 poly(oxyethylene); PEG, polyethylene glycol; ADA, adenosine deaminase; Ru(bpy)32+, ruthenium(II)-tris-bipyridine; Zn-TMPyP4+, Zn(II)-meso-(N-tetramethylpyridinium)porphyrin; ZnTPPS3, meso-tetraphenylporphyrintrisulfonate; MV2+, methyl viologen; MV+, methyl viologen cation radical.
It is well-known that chlorophyll molecules, in vivo, bind to proteins to form chlorophyll-protein conjugates in the layered thylakoid membrane of chloroplast and exhibit physiological functions: the photolysis of water and the reduction of NADP+ and CO2 fixation under visible light illumination (8). Photocatalytic reduction of carbon dioxide was reported using semiconductor photocatalysis such as titanium oxide (TiO2), silicon carbide (SiC) (9), and strontinum titanate (SrTiO3) (10) under the illumination of ultraviolet ray. Furthermore, Willner et al. (11-13, 25, 26) reported CO2 fixation and synthesis of amino acid via photoreduction of NAD(P)+ by ruthenium(II)-tris-bipyridine, Ru(bpy)32+, or Zn(II)-meso-(Ntetramethylpyridinium)porphyrin, Zn-TMPyP4+, as photosensitizer under visible light. Okura et al. (14, 15) observed the hydrogen evolution using zinc meso-tetraphenylporphyrintrisulfonate (Zn-TPPS3) as a photosensitizer in the presence of an electron donor and an electron carrier under visible light illumination. On the other hand, chlorophylls obtained by extracting with an organic solvent from living leaves have been seldom used as a photocatalyst because they are quite unstable on illumination. Quite recently, we reported a photostable chlorophyll a-poly(vinylpyrrolidone)-smectite conjugate, which catalyzes photoreduction and hydrogen gas evolution under visible light (16). The present paper deals with the photostability of the PEG-chlorophyllin conjugate under anaerobic condition and photochemical functions of the PEG-chlorophyllin conjugate: hydrogen gas evolution, photoreduction of
10.1021/bc990045t CCC: $19.00 © 2000 American Chemical Society Published on Web 12/16/1999
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NADP+ or NAD+, and CO2 fixation, that is formation of malate (C4) from pyruvate (C3) and CO2. EXPERIMENTAL PROCEDURES
General. Chlorophyll a purified from Spirulina and NADP+ were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ferredoxin-NADP+ reductase (EC 1.18.1.2) and malic enzyme, [(S)-malate, NADP+ oxidoreductase(oxaloacetate-decarboxylating), EC 1.1.1.40] were obtained from Sigma Chemical Co. (St. Louis, MO). R-(3-Aminopropyl)-ω-methoxypoly(oxyethylene) with an average molecular weight of 5050, PEGNH2, was kindly provided by Nippon Oil and Fats Co., Ltd. (Tokyo, Japan). Hydrogenase was isolated from Desulfovibrio vulgaris according to Yagi’s method (17). Other reagents used were of analytical grade. PEG-Chlorophyllin Conjugate. Purified chlorophyllin a was prepared by the method of Oster et al. (18) as follows: chlorophyll a (50 mg) dissolved in benzene (3 mL) was diluted with 47 mL of n-hexane and was saponified to chlorophyllin a by adding 1 mL of 7% KOH in methanol. After neutralizing excess alkali by bubbling carbon dioxide, purified chlorophyllin a was obtained by filtration, evaporation, and dialysis against water. Chlorophyllin a conjugated with PEG-NH2, the PEG-chlorophyllin conjugate, was prepared as follows (7). To chlorophyllin a (2.2 µmol) dissolved in 3 mL of 50 mM tris-HCl buffer (pH 7.8) was added 6.6 µmol of PEG-NH2, followed by adding stepwise 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (100 mg). The mixture was stirred for 6 h at room temperature to complete the reaction. After adding 50 mL of chloroform to the reaction mixture, the PEG-chlorophyllin conjugate was extracted into the chloroform layer followed by evaporation to obtain the PEG-chlorophyllin conjugate powder. Since the chlorophyllin a molecule has three carboxyl groups (18), three PEG chains bind to chlorophyllin a with acid-amide bonds to form the PEG-chlorophyllin conjugate (7). The amino group in tris, tris(hydroxymethyl)aminomethane, did not react with carboxyl groups in chlorophyllin, which is proved by analyzing with NMR and IR analyses. Photostability of chlorophyllin derivative under anaerobic condition was tested as follows: PEG-chlorophyllin conjugate (13 µM) and chlorophyllin a (14 µM) in 3 mL of 50 mM phosphate buffer (pH 7.4) were illuminated by a 60 W incandescent lamp at a distance of 10 cm (light intensity ) 200 J m-2 s-1) at 30 °C. Photoreduction of Methyl Viologen and Evolution of Hydrogen Gas. Photoreduction of methyl viologen by the PEG-chlorophyllin was tested in the presence of 2-mercaptoethanol. The PEG-chlorophyllin conjugate (13.8 nmol) was dissolved in 3 mL of 25 mM Tris-HCl buffer (pH 7.4) containing 0.6 µmol of methyl viologen (MV2+) and 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, light-intensity: 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. Hydrogen gas evolution by the PEG-chlorophyllin conjugate in the presence of methyl viologen (MV2+), 2-mercaptoethanol, and hydrogenase was tested according to the modified method of Okura et al. (14, 15). The reaction system (3 mL) consisted of PEG-chlorophyllin (4.2 or 15 nmol) dissolved in 25 mM Tris-HCl buffer (pH 7.4) in the presence of 0.6 µmol of methyl viologen (MV2+),
Figure 1. Photostability of chlorophyllin a and the PEGchlorophyllin conjugate under anaerobic condition. (A, B) The PEG-chlorophyllin conjugate (0.13 µM) and chlorophyllin a (0.14 µM) in aqueous solution, respectively. Samples were illuminated with a 60 W incandescent lamp at distance of 10 cm (light intensity of 200 J m-2 s-1) at 30 °C. The peak positions of the PEG-chlorophyllin conjugate and chlorophyllin a in aqueous solution were 657 and 654 nm, respectively. Scheme 1
Scheme 2
2-mercaptoethanol (1 mmol) and hydrogenase solution (40 µL). The sample mixture in a Pyrex cell (17.5 cm3) was deaerated by repeated freeze-pump-thaw cycles and the upper space of the cell (14.5 cm3) was filled up with argon gas. The sample in the cell equipped with a magnetic stirrer bar was illuminated with a 200 W tungsten lamp (light intensity, 200 J m-2 s-1). Ultraviolet ray with wavelength shorter than 390 nm was cut off by a Toshiba L-39 filter (Tokyo, Japan). The hydrogen gas evolved in sample cell was taken out through sampling valve and was analyzed by gas chromatography (14, 15). Photoreduction of NADP+ and NAD+. Photoreduction of NADP+ or NAD+ was performed as the method described by Vernon et al. (19) with some minor modification. The pH value in the sample mixture was maintained at 7.8 by 33 mM Tris-HCl buffer. The reaction mixture (3 mL) contained the PEG-chlorophyllin (4.5 µM), ascorbate (3.3 mM), NADP+ or NAD+ (0.33 mM), and ferredoxin-NADP+ reductase (2.5 units). The sample mixture was deaerated by repeated freezepump-thaw cycles and was illuminated with a 60 W incandescent lamp at a distance of 3.5 cm, light intensity: 1500 J m-2 s-1, at 30 °C. The photochemical
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Figure 2. Photoreduction of methyl viologen by the PEGchlorophyllin conjugate in the presence of 2-mercaptoethanol under illumination with a 60 W incandescent lamp at a distance of 3.5 cm (light intensity ) 1500 J m-2 s-1) at 30 °C. (A) PEGchlorophyllin conjugate (13.8 nmol), methyl viologen (0.6 µmol), and 2-mercaptoethanol (1 mmol) in 3 mL of 25 mM Tris-HCl buffer at pH 7.4.(B) In the absence of 2-mercaptoethanol.
Figure 3. Hydrogen gas evolution by the PEG-chlorophyllin conjugate in the presence of 2-mercaptoethanol, methyl viologen, and hydrogenase under illumination with visible light (light intensity of 200 J m-2 s-1) at 30 °C. (A, B) PEG-chlorophyllin conjugates of 15 and 4.2 nmol, respectively, in the presence of 2-mercaptoethanol (1 mmol), methyl viologen (0.6 µmol), and hydrogenase (40 µL) in 3 mL of 25 mM Tris-HCl buffer at pH 7.4. (C) In dark.
reduction of NADP+ or NAD+ was observed by measuring the absorbance increase at 340 nm, using the molar extinction coefficient of 6.22 × 103 M-1 cm-1. Carbon Dioxide Fixation via Photoreduction of NADP+. To the sample mixture of the PEG-chlorophyllin conjugate (15 µM), NADP+ (3.2 mM), pyruvate (0.8 mM), magnesium chloride (15 mM) and ascorbate (6 mM) dissolved in 10 mL of 50 mM phosphate buffer (pH 7.0) were added ferredoxin-NADP+ reductase (2.5 units) and malic enzyme (5 units). The sample mixture in a threenecked round-bottomed flask was deaerated by repeated freeze-pump-thaw cycles. Then sodium hydrogen carbonate (180 mM) was added to the sample mixture (the pH value shifted to 7.4) and the upper space of the cell was filled with argon gas. The sample solution was stirred with a magnetic stirrer and was illuminated with a 60 W incandescent lamp from a distance of 10 cm (light intensity, 200 J m-2 s-1) at 30 °C. The amount of malate formed by the coupling reaction of pyruvate and CO2 through NADPH (see Scheme 2) was determined by the method described by Ochoa et al. (20, 21).
Itoh et al.
Figure 4. Photoreduction of NADP+ or NAD+ by the PEGchlorophyllin conjugate under illumination with a 60 W incandescent lamp at a distance of 3.5 cm (light intensity ) 1500 J m-2 s-1) at 30 °C. (A) The PEG-chlorophyllin conjugate (4.5 µM), ascorbate (3.3 mM), monosodium NADP+ (0.33 mM), and ferredoxin-NADP+ reductase (2.5 U) in 3 mL of 33 mM TrisHCl buffer at pH 7.8. (B) Monosodium NAD+ (0.33 mM) was used instead of monosodium NADP+ in the above reaction system. (C) In dark.
Figure 5. Carbon dioxide fixation by the PEG-chlorophyllin conjugate in the presence of ascorbate, magnesium chloride, CO2, pyruvate, NADP+, ferredoxin-NADP+ reductase and malic enzyme under illumination with a 60 W incandescent lamp at distance of 10 cm (light intensity of 200 J m-2 s-1) at 30 °C. (A) The PEG-chlorophyllin conjugate (15 µM), ascorbate (6 mM), sodium hydrogen carbonate (180 mM), magnesium chloride (15 mM), pyruvate (0.8 mM), NADP+ (3.2 mM), ferredoxin-NADP+ reductase (2.5 U) and malic enzyme (5 U) in 10 mL of 50 mM phosphate buffer at pH 7.4. (B) In dark.
RESULTS AND DISCUSSION
Photostability of PEG-Chlorophyllin Conjugate under Anaerobic Conditions. It is well-known that chlorophylls extracted from living leaves are markedly breached by light illumination. Therefore, the stabilization of chlorophyll and its derivatives toward light illumination is essential to develop the artificial photosynthesis system in the future. In the present study, two kinds of chlorophyll derivatives, chlorophyllin a and the PEG-chlorophyllin conjugate, were subjected to photostability test under anaerobic condition. The results are shown in Figure 1; curves A and B show the photostabilization of the PEG-chlorophyllin conjugate and chlorophyllin a in aqueous solution under anaerobic conditions, respectively. The PEG-chlorophyllin conjugate (curve A) was more stable than chlorophyllin a (curve B). In aerobic conditions, the stability of each pigment was extensively reduced by light illumination in comparison with that in anaerobic conditions.
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Figure 6. Spectral change of chlorophyllin and the PEG-chlorophyllin conjugate by light illumination under anaerobic condition. (a, b) Chlorophyllin a (0.14 µM) and the PEG-chlorophyllin conjugate (0.13 µM) in aqueous solution, respectively. Samples were illuminated with a 60 W incandescent lamp at distance of 10 cm (light intensity of 200 J m-2 s-1) at 30 °C. Illumination time: 0, 5, 10, 20, 30, 40, 50, and 60 min.
Photoreduction of Methyl Viologen and Evolution of Hydrogen Gas. Photochemical redox system has been developed for the purpose of solar energy utilization (16), which is shown in Scheme 1. The system, including a photosensitizer (the PEG-chlorophyllin conjugate), an electron donor (2-mercaptoethanol), and electron carrier (methyl viologen) in Tris-HCl buffer (pH 7.4), has been able to evolve hydrogen gas when suitable catalyst (hydrogenase) is applied. A first series of experiments was conducted to test whether the PEG-chlorophyllin conjugate induces the reduction of methyl viologen by light illumination. Figure 2 represents the photoreduction of methyl viologen by the PEG-chlorophyllin conjugate. When a sample solution containing the PEG-chlorophyllin conjugate is illuminated in the presence of methyl viologen and 2-mercaptoethanol, the absorbance at 605 nm due to MV+ was sharply increased with illumination time. The reduction of methyl viologen ceased completely in the dark and the reduction proceeded again on light, which is shown by curve A. Without 2-mercaptoethanol (electron donor) in the reaction system, no photoreduction of methyl viologen took place even by light illumination (curve B). Since the reduction of methyl viologen proceeds in coupling reaction of the PEG-chlorophyllin conjugate and 2-mercaptoethanol by light illumination, the next experiment was conducted whether hydrogen gas is evolved or not in the presence of hydrogenase as shown in Scheme 1. Figure 3 shows the hydrogen gas evolution by PEG-chlorophyllin conjugate in the presence of 2-mercaptoethanol, methyl viologen and hydrogenase under illumination with visible light. The amount of hydrogen gas was linearly increased with illumination time depending upon the amount of the PEG-chlorophyllin conjugate. Curves A and B were the results obtained by 15 and 4.2 nmol of the PEG-chlorophyllin conjugate, respectively. No evolution of hydrogen gas was observed in the dark (curve C). In curve B, approximately 2.6 µmol of hydrogen gas was evolved for 3 h of illumination. Therefore, the amount of hydrogen ion reduced by the 3 h illumination is 5.2 µmol, which is 1200 times greater than 4.2 nmol of the PEG-chlorophyllin in the
reaction system. From the results obtained above, it can be concluded that PEG-chlorophyllin conjugate excited by illumination (Chl*-conj) releases an electron and changes to cationic PEG-chlorophyllin conjugate (Chl+conj). The released electron reduces methyl viologen dication (MV2+) to methyl viologen cation radical (MV+), which reduces hydrogen ion to form hydrogen gas by the catalytic action of hydrogenase. On the other hand, cationic Chl+-conj is reduced by 2-mercaptoethanol as electron donor to be reproduced as the PEG-chlorophyllin conjugate (Scheme 1). Carbon Dioxide Fixation via Photoreduction of NADP+. A next series of experiments was conducted to test whether the PEG-chlorophyllin conjugate induces the reduction of NADP+ or NAD+ followed by the carbon dioxide fixation, which is shown in Scheme 2. CO2 fixation via photoreduction of NADP by the PEGchlorophyllin conjugate was conducted, in which the formation of malate (C4) from pyruvate (C3) and CO2 proceeds with the generation of NADPH in the presence of malic enzyme. Figure 4 represents the photoreduction of NADP+ or NAD+ by the PEG-chlorophyllin in the presence of ascorbate (electron donor). The absorbance at 340 nm due to NADPH or NADH is increased with illumination-time, which is shown by curve A or B, respectively. However, no reduction of NADP+ or NAD+ was observed in the dark (curve C). In the absence of ascorbate or ferredoxin-NADP+ reductase in the reaction system, no photoreduction of NADP+ or NAD+ occurs, even by light illumination (data not shown). Since the reduction of NADP+ proceeds in coupling reaction of the PEG-chlorophyllin conjugate and ascorbate in the presence of ferredoxin-NADP+ reductase by light illumination, a next experiment was conducted to test whether CO2 fixation proceeds in the presence of pyruvate, carbon dioxide, and malic enzyme. Figure 5 shows the formation of malate (C4) from pyruvate (C3) and CO2 under illumination in the presence of ascorbate, the PEG-chlorophyllin conjugate, NADP+, ferredoxinNADP+ reductase and malic enzyme. The amount of malate is markedly increased with time after illumination (curve A). During illumination for 4 h, the amount
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of malate synthesized from pyruvate and CO2 was 0.15 mM in which approximately 18.5% of pyruvate (0.8 mM) was converted to malate. No appreciable synthesis of malic acid from pyruvate and CO2 is observed in dark which is shown by curve B. The success of the present study is based on the finding of photostable chlorophyllin a conjugated with methoxypolyoxyethylene amine with the molecular weight of 5050, the PEG-chlorophyllin conjugate. Generally, chlorophyll derivatives including chlorophyllin and the PEGchlorophyllin are not photostable in aerobic conditions (7, 16). However, in anaerobic conditions, the PEGchlorophyllin conjugate is extensively stable in aqueous solution in comparison with chlorophyllin. Figure 6 represents their spectral changes of chlorophyllin and the PEG-chlorophyllin conjugate in aqueous solution under anaerobic conditions by light illumination. The absorption spectra of chlorophyllin and the PEG-chlorophyllin conjugate are shown in Figure 6, panels a and b, respectively. In the case of chlorophyllin, the absorption maximum at 654 nm is lowered with illumination followed by appearing a new band at 724 nm with the isobestic points at 684 and 588 nm. The Soret band at 404 nm is also lowered with illumination. On the other hand, the absorption spectrum of PEG-chlorophyllin conjugate with an absorption maximum at 657 nm with a shoulder at 689 nm is scarcely lowered by illumination. These phenomena are noteworthy facts, although the reason is unclear. A conceivable possibility of the phenomenon may be due to the interaction of tetrapyrrole ring having magnesium with long carbon chain (CH2CH2-O)n of poly(ethylene glycol). The changes of absorbance at 654 nm (peak position of chlorophyllin a) and at 657 nm (peak position of the PEG-chlorophyllin conjugate) with light illumination are shown in Figure 1. The chemical modification of bioactive substances with low molecular weight by polyethylene glycol derivatives has been extensively studied. Polyethylene glycol-modified hemin(PEG-hemin) with amphipathic properties exhibited peroxidase activity in organic solvents as well as in an aqueous solution (22). This technique became a good tool for determining peroxide in unsaturated fatty acids and lipids (23). PEG-hematoporphyrin with amphipathic properties acted as a photosensitizer; imidazole and indole were photooxidized in organic solvents, and uric acid was also photooxidized in neutral aqueous solution (24). The PEG-chlorophyllin conjugate, which is photostable in anaerobic conditions, catalyzed the CO2 fixation though formation of NADPH by light illumination. This finding may lead to not only the prevention of the Greenhouse effect due to accumulation of CO2 in terrestrial ecosystem but also photosynthesis in vitro. LITERATURE CITED (1) Kodera, Y., Matsushima, A., Hiroto, M., Nishimura, H., Ishii, A., Ueno, T., and Inada, Y. (1998) Pegylation of proteins and bioactive substances for medical and technical applications. Prog. Polym. Sci. 23, 1233-1271. (2) Inada, Y., Yoshimoto, T., Matsushima, A., and Saito, Y. (1986) Engineering physicochemical and biological properties of proteins by chemical modification. Trends Biotechnol. 4, 68-73. (3) Yoshimoto, T., Nishimura, H., Saito, Y., Sakurai, K., Kamisaki, Y., Wada, H., Sako, M., Tsujino, G., and Inada, Y. (1986) Characterization of polyethylene glycol-modified Lasparaginase from Escherichia coli and its application to therapy of leukemia. Jpn. J. Cancer Res. Gann. 77, 12641270.
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