Bioconjugate Chem. 1995, 6,249-254
249
Synthesis and Characterization of Superoxide Dismutase-Deferoxamine Conjugate via Polyoxyethylene: A New Molecular Device for Removal of a Variety of Reactive Oxygen Species Haruya Sato,* Masayo Watanabe, and Yuji Iwashita Central Research Laboratories, Ajinomoto Company, Inc., Suzuki-cho, Kawasaki 210, Japan. Received July 27, 1994@
A conjugate of Cu,Zn-superoxide dismutase (SOD) with a strong iron chelating agent, deferoxamine (DFO), was synthesized (SOD-POE-DFO) via polyoxyethylene (POE) as a linking agent. N-terminal amino groups of lysine residues in SOD are modified with 1 : l binding products of polyoxyethylene and deferoxamine (POE-DFO) through a covalent amido bond. The mean number of the POE-DFO bound per one SOD molecule is calculated to be 3.3 by determining the C/N ratio after elemental analysis. The half-life of the SOD-POE-DFO is about 1.2 h in rats, whereas that of free SOD is about 5-10 min. POE plays the part not only of the linking agent but also of expanding the lifetime in the circulation. The SOD-POE-DFO possesses both the metal chelating ability (for DFO) and the ability of scavenging superoxide radicals (for SOD). Therefore, the SOD-POE-DFO of the present study can eliminate the superoxide radical and free iron simultaneously and in the same location, and thus, it would be a molecular device with multiple functions which prevents the damage to tissues by scavenging the variety of reactive oxygen species.
INTRODUCTION
The oxygen radical superoxide (02'-) and the nonradical hydrogen peroxide (HzOz)are produced in the body and play several important roles in the self-defense system. Excessive production of 02*and Hz02, however, can result in tissue damage. For example, highly reactive hydroxyl radical ( O H ) is ofZen generated by the HaberWeiss reaction (eq 11, and other oxidants may be produced in the presence of catalytic iron or copper ions.
+
02*-H,O,
-
0,
+ O H + OH-
(1)
A group of enzymes such as superoxide dismutase (SOD),' catalase, glutathione peroxidase, ceruloplasmin, etc. work for removal of these reactive oxygen species. Another important form of antioxidant defense is the storage and transport of iron and copper ions in inert forms that cannot catalyze the formation of reactive radicals. SOD is a typical antioxidant enzyme that scavenges 0 2 * - . SOD has been investigated as a therapeutic antioxidant for the treatment of ischemia-reperfusion injury in a wide variety of organs. For example, Bernier et al. have reported that SOD is effective on arrhythmia occurred immediately after reperfusion of ischemic heart (1). It has been further reported that pulmonary edema
* To whom correspondence should be addressed: Basic Research Department, Central Research Laboratories, Ajinomot0 Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan. Telephone: (044)244-5813 Fax: (044)244-9617. Abstract published in Advance ACS Abstracts, February 1, 1995. Abbreviations: SOD, Cu,Zn-superoxide dismutase; POE, a-(carboxymethyl)-w-(carboxymethoxy)polyoxyethylene; DFO, deferoxamine; POE-DFO, 1:l binding product of POE and DFO; DFO-POE-DFO, 1:2 binding product of POE and DFO; activated POE-DFO, activated ester of POE-DFO with N hydroxysuccinimide; SOD-POE-DFO, conjugate of SOD with POE -DFO. @
caused after occlusion and reperfusion of the pulmonary artery is prevented by SOD in the animal model (2). In cases where SOD is intravenously administered, however, SOD rapidly disappears from the circulation so that a time period to exhibit its activity in blood is very short. Therefore, SOD chemically modified with polyoxyethylene was prepared by Morimoto and his collaborators (3), and they reported the effective prevention of arrhythmia of rat caused by reperfusion also in vivo by the chemically modified SOD (4). On the other hand, some researchers have reported negative results on the SOD treatment of reactive hyperemia (5). It is also suggested that bell-shaped doseresponse curves and the potential toxicity of SOD have been suggested in a number of different myocardial ischemia-reperfusion models (6, 7). Furthermore, SOD behaves as a n enzyme that catalyzes the formation of free radicals in the presence of anionic scavengers and HzOz as substrates (8) and also produces HzOz in the superoxide dismutation reaction, which in the presence of iron results in the production of the very toxic O H via the Fenton reaction (9)(eq 2).
+
Fe2+ H,O,
-
+
Fe3+ OH-
+O H
(2)
The exact mechanism of reactive oxygen damage, for example, what kind of oxygen is causing the damage, has not been clarified yet. Even in the studies up to now, neither SOD nor catalase has been enough effective to remove a variety of reactive oxygen species including singlet oxygen and hypochloride, and their pharmaceutical effects are not necessarily sufficient, either. One of the reasons must be that only one kind of reactive oxygen scavenger would not be enough to completely remove these oxygen species. Recently, the use of catalase conjugated to SOD has resulted in a n inhibition of the Fenton reaction in an in vitro system and offered much greater protection in a n isolated working heart model of ischemia-reperfusion injury (10).
1043-1802/95/2906-0249$09.00/00 1995 American Chemical Society
Sato et ai.
250 Bioconjugate Chem., Vol. 6,No. 3, 1995
On the basis of these observations, we have developed the hypothesis that a suitable arrangement of several antioxidants determines the physiological and pathophysiological effects of these antioxidants. A molecular device having multiple functions would be more effective for efficient prevention of damages caused by a number of reactive oxygen species. We have especially focused on the interaction and cooperation between SOD and a chelating agent. We choose deferoxamine (DFO) as the chelating agent, which chelates iron and inhibits iron-catalyzed formation of O H and lipid peroxidation in vitro (9). It is also reported to reduce reperfusion arrhythmia and reoxygenation-induced myocardial damage in isolated hearts (11). DFO has been used clinically in cases of acute iron intoxication (12). Nonetheless, acute and chronic toxicity of DFO is relatively high, and its plasma half-life is short (13).Recently, DFO has been conjugated to hydroxyethyl starch (HES), and improved toxicity of the conjugate has been reported (14, 15). The intent is to limit the distribution of DFO in the intravascular space, thereby increasing the duration of action and decreasing cellular toxicity (15). We report here the preparation, purification, and characterization of SOD covalently modified with 1:1 binding products of polyoxyethylene (POE) and DFO. This SOD-POE-DFO conjugate possesses both the metal chelating ability (DFO) and the ability of scavenging 0 2 ' - (SOD) and exhibits an increased circulatory halflife in vivo. These aspects of SOD-POE-DFO are expected to be especially effective for inhibition of the reactive oxygen formation and may offer much greater protection in oxygen free radical damage to tissues. The aim of our study is to create the molecular device to clarify the exact mechanism of the oxygen injury and to present a novel therapeutic means of oxygen induced damages. EXPERIMENTAL PROCEDURES
Chemicals. Deferoxamine mesylate was purchased from Sigma (U.S.A.). Human erythrocyte-derived superoxide dismutase (SOD) was prepared by the genetic engineering method. a-(Carboxymethyl)-w4carboxymethoxy)polyoxyethylene (POE, mean molecular weight of about 3000 Da) was purchased from Nippon Oils & Fats Co., Ltd. (Japan). Other chemicals were of reagent grade. General Procedures. IH-NMR spectra were measured as D2O (POE-DFO) or DMSO-& solution (activated POE-DFO) with a JNM GX-400 FT-NMR (Japan Electron Optics Laboratory, Japan). Chemical shifts were reported as parts per million (ppm) relative to tetramethylsilane or 2,2-dimethyl-2-silapentane5-sulfoxide. Liquid secondary ion mass spectra were recorded with a JMS-DX3OO spectrometer (Japan Electron Optics Laboratory, Japan) operating with a JMA3500 data system (Japan Electron Optics Laboratory, Japan). SDSPAGE was carried out using a gradient gel (SDS-PAGE mini, 8-16%, Tefco Co., Ltd., Japan). Aliquots of reaction mixtures were mixed with a SDS-gel loading buffer containing 2% SDS, 1%2-mercaptoethanol, 0.1% bromophenol blue, and 0.05% sodium phosphate buffer (pH7.0),heat-denatured a t 95 "C for 10 min, and loaded onto the gel. The finished gel was stained with Coomassie brilliant blue. Ion-exchange chromatography was carried out using a Hitachi HPLC system (Hitachi Ltd., Japan) for analytical work and a Biopilot (Pharmacia-LKB, Sweden) for preparative work. For analytical work, the HPLC measurement was carried out a t a flow rate of 1.0 m u m i n with an Asahipak ES-502N column (Asahi Chemical
Industry Co., Ltd., Japan) in a 0.02 M sodium formate buffered-solution (pH 8.0). A 1pL sample of the reaction mixture was directly loaded on the column. The detection of the POE, POE-DFO, and DFO-POE-DFO was performed by refractive index with a L3300 RI-detector and absorbance a t 229 nm with a 655A U V detector. For preparative work, HPLC measurement was carried out a t a flow rate of 13.0 mIJmin with a Q-Sepharose high performance column (60/100, Pharmacia-LKB, Sweden). Solvent A was prepared by adding a 0.01 M diethanolamine buffered solution (pH 8.8). Solvent B was prepared by adding 0.01 M diethanolamine buffered solution (pH 8.8) containing 0.3 M NaC1. Forty mililiters of the reaction mixture was injected directly into the column. The column was equilibrated with solvent A, and products were eluted by a linear gradient of NaCl from 0 to 0.3 M for 2 h. The detection of the POE-DFO and DFOPOE-DFO was performed by the absorbance at 229 nm. Gel-permeation chromatography of the conjugate was carried out using a Hitachi HPLC System (Hitachi Ltd., Japan). HPLC measurement was carried out a t a flow rate of 1.0 mIJmin with a TSK G3000SWXL column (Toyo Soda Co., Ltd., Tokyo, Japan) using a 0.1 M potassium phosphate buffered-solution (pH 6.8). SOD activity was determined by the method of McCord et al. (161, and the SOD protein concentration was determined by absorbance a t 672 nm (EIRlcm = 0.079). The remaining activity of the conjugate was defined in term of a ratio of remaining activity to the SOD activity prior to the reaction. A mean number of POE bound per one SOD molecule was calculated by determining the weight percent ratio of carbon to nitrogen after elemental analysis according to the following equation
C/N = 100[12(1358
+ 159n)/14(406 + 6n)l
where n is the number of POE-DFOs attached to one SOD molecule and C/N is the weight percent ratio of carbon to nitrogen in SOD-POE-DFO. Twelve and 14 are the atomic weights of carbon and nitrogen, respectively, and 1358 and 406 are the number of carbons and nitrogens in one SOD molecule, respectively. One hundred and fifty-nine is the number of carbons in one POEDFO which was obtained from the average molecular weight of 3000 Da of POE, and 6 is the number of nitrogens in one DFO molecule. Chelating ability of the immobilized DFO in SODPOE-DFO was determined according to the method of Emery (17). The displacement of iron from ferrioxamine (DFO/Fe2+complex) by gallium under reducing conditions was used to probe the change in the iron-binding characteristics of the conjugated chelator. Three mililiters of a solution including 2 mM ferrozine, 20 mM sodium ascorbate, 0.05 mM DFO, and 0.05 mM sodium acetate of pH 5.4 was pipetted into 4 mL cuvettes, and the absorbance a t 562 nm (25 "C) was monitored (molar absorptivity of 29000 M-I cm-l). After 5 min, 158 pL of 10 mM gallium nitrate were added to the DFO, yielding a final gallium concentration of 0.5 mM. The increase in absorbance, due to formation of the ferrous ironferrozine complex, was followed for a n additional 15 min spectrophotometrically (562 nm). Synthesis of POE-DFO. After 1.5 mmol of DFO was dissolved in 81 mL of a 0.05 M potassium phosphate buffered solution (pH 8.0) and 0.83 mmol of activated POE, which had been obtained by converting POE into activated ester of N-hydroxysuccinimide with 1,3-dicyclohexylcarbodiimide (DCC). The mixture was rapidly stirred homogeneously and allowed to stand a t room temperature for 1 h. Then the reaction solution was
Synthesis of Superoxide Dismutase-Deferoxamine
N-
Bioconjugate Chem., Vol. 6,No. 3, 1995 251
5
OOC-CH2-(OCH2CH3n-O-CH2-COO- N
1
(activated PO€) DFO
0
0
R-HN-OCCHy(OCH2CH2)n-OCH2CO-NH-R (DFQPOE-DFO) HOOCCH2+OCH2CH2)n-OCH2CO-NH-R (POE-DFO) HOOCCH2-(OCH2CH2)n-OCH2COOH
(PO€)
I
0-Sepharose column
HOOCCH2-(OCH2CH2)n-OCH2CO-"-R (POE-DFO)
N-
I
1) TMSCl/ Py 2) HOSu, DCC
OOCCH2
