Biochimica et Biophysica Acta, 423 (1976) 203-216 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 47039

A POSSIBLE MECHANISM OF THE GENERATION OF SINGLET MOLECULAR OXYGEN IN NADPH-DEPENDENT MICROSOMAL LIPID PEROXIDATION

K A T S U A K I S U G I O K A and M I N O R U N A K A N O

Department of Biochemistry, School of Medicine, Gunma University Maebashi, 371, Gunma (Japan) (Received May 30th, 1975)

SUMMARY

A simplified system, consisting of NADPH, Fe 3+-ADP, EDTA, liposomes, NADPH-cytochrome c reductase and Tris • HCI buffer (pH 6.8), has been employed in studies of the generation of singlet oxygen in NADPH-dependent microsomal lipid peroxidation. The light emitted by the system involves ZAgtype molecular oxygen identifiable by its characteristic emission spectrum and its behavior with fl-carotene. The generation of another excited species (a compound in the triplet state) could be demonstrated in this system by changes of light intensity and emission spectra which arise from photosensitizer (9, 10-dibromoanthracene sulfonate, eosin, Rose-Bengal)-mediated energy transfers. Chemiluminescence in the visible region was markedly quenched by various radical trappers and by an inhibitor of NADPH-cytochrome c reductase, but not by superoxide dismutase. During the early stage of lipid peroxidation, the intensity of chemiluminescence was proportional to the square of the concentration of lipid peroxide. These characteristics suggest that singlet oxygen and a compound in the triplet state (probably a carbonyl comPound ) are generated by a self-reaction of lipid peroxy radicals.

INTRODUCTION

The unsaturated fatty acids of liver microsomal phospholipids readily undergo peroxidation in the presence of NADPH and oxygen [1, 2]. The NADPH-dependent flavoprotein, NADPH-cytochrome c reductase, catalyzes the peroxidation of isolated lipids [3] or lipoprotein [4] under similar conditions, provided that EDTA is present in appropriate concentration in the reaction system. EDTA in such reconstructed systems appears to elevate the redox potential of Fe 3+ and of the iron complex, facilitating thereby the transfer of one electron from NADPH to tri-valent iron through the flavin moiety of the reductase [4]. Reduced iron, free in the solution,

204 plays an important role in the initiation and propagation of phospholipid peroxidation [4, 5]. NADPH-dependent lipid peroxidation is usually accompanied with an emission of ultra weak light [4, 6, 7]. Recently the emitter in the NADPH-dependent microsomal lipid peroxidation system has been spectrometrically confirmed to be lag type singlet molecular oxygen [7]. Little is known, however, of the relationship between lipid peroxidation and 10 2 generation. The present work was undertaken to identify the mechanism of 10 2 generation during peroxidative cleavage of phospholipid in microsomes, using a simplified NADPH-dependent lipid peroxidation system (NADPH-NADPH-cytochrome c reductase-Fe 3 ÷-ADP-EDTA-liposomes). MATERIALS AND METHODS

Reagents. ADP and 0c-tocopherol were obtained from Sigma. NADPH was purchased from Oriental Yeast Co. Ltd. The 9, 10-dibromoanthracene sulfonate (sodium salt) and fl-anthracene sulfonate (sodium salt) were kindly supplied by Professor G. Cilento. All other chemicals were of reagent grade, fl-Carotene, obtained from Tokyo Kasei Co., was purified by three crystallizations from a benzene/methanol mixture. The molar absorption at 4640 and 4940 A for crystalline fl-carotene in benzene was 11.0.104 and 9.4- 10'~ M -1 • cm -~, respectively. Enzyme preparation. Microsomal NADPH-cytochrome c reductase (spec. act., 18-25 pmol ferricytochrome c reduced/min per mg protein) was prepared from rat liver microsomes by established method [8]. Superoxide dismutase (spec. act., 2500 units/mg protein) was prepared from bovine red blood ceils and assayed in terms of its ability to inhibit (50 ~ ) the reduction of cytochrome c by milk xanthine oxidase by the method of McCord and Fridovich [9]. Preparation ofliposomes. The lipid was extracted from rat liver microsomes [2] by the method of Folch et al. [10] and the chloroform layer was then stored at --20 °C under anaerobic conditions. Just before the experiment, an aliquot of the chloroform solution was added to a I00 ml round bottomed flask and evaporated under reduced pressure. In some cases fl-carotene or ~-tocopherol in chloroform was mixed with the chloroform solution of the microsomal lipid, and evaporated to dryness as described above. Distilled water was then added to the flask and the lipids (with or without added chemicals) were thoroughly agitated by means of a Vortex mixer until the lipid film was no longer detectable on the sides of flask. The amount of lipid was measured as lipid phosphorous by a modification of the method of Bartlett [11 ]. Preparation of malondialdehyde. Malondialdehyde in free form was prepared by NADPH-dependent microsomal lipid peroxidation [2]. Malondialdehyde-bisbisulfate (sodium salt) was obtained by treating malondialdehyde-bis(dimethylacetal) with aqueous HC1 and mixing it with sodium meta-bisulfite [12]. The free form was then obtained by its hydrolysis in 1 M HCI for 20 min at room temperature. Both the enzymatically and chemically prepared malondialdehyde in free form was then purified by gel filtration on Sephadex G-10 column, using 0.1 M potassium phosphate/NaC1 buffer at pH 7.2 [2]. Incubation conditions. The standard reaction mixture consisted of liposomes (0.43 #tool phosphate per ml of incubation mixture), 1 • 10 -4 M Fe(NO3)3, 1.67 mM

205 A D P , 5 • 10- 5 M E D T A , 0.16 m M N A D P H , N A D P H - c y t o c h r o m e c reductase (0.5 unit) and 0.1 M T r i s . HC1 buffer ( p H 6.8) in a total volume o f 3 ml. In some experiments c o m p o n e n t s such as superoxide dismutase, radical scavengers and SH inhibitor were added to the standard incubation mixture to examine their effect on light emission or N A D P H consumption. Unless otherwise noted, incubation was carried out at 37 °C. Other methods. N A D P H disappearence and malortdialdehyde f o r m a t i o n were measured by previously described methods [4]. Lipid peroxide was determined by iodometry [13]. The excitation and fluorescence spectra o f dye in 0.1 M Tris • HCI buffer ( p H 6.8) and o f malondialdehyde in 0.1 M potassium p h o s p h a t e / N a C l buffer at p H 7.2 were determined by means o f a Hitachi Model 203 fluorescence spectrometer. The excitation energy (kcal/mol) of the dye was calculated f r o m its excitation wavelength (2 as A ) according to the equation (E = 2 . 8 5 8 9 . 1 0 8 c a l . 2 - 1 . m o l - 1 ) . Luminescence was measured by means o f a single photoelectron [14] and P a c k a r d Model-2311 liquid scintillation counter with the coincidence circuit off [15]. The emission spectrum was determined as previously described [7]. RESULTS

(1) Factors which promote light emission. To test the effect o f 02 concentrations on light emission, the reaction mixture containing N A D P H - c y t o c h r o m e c reductase, F e 3 + - A D P - E D T A , and liposomes was flushed with air or 100 % 0 2 for 30 s and chemiluminescence in both systems was c o m p a r e d after the addition o f N A D P H . In

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A possible mechanism of the generation of singlet molecular oxygen in nadph-dependent microsomal lipid peroxidation.

Biochimica et Biophysica Acta, 423 (1976) 203-216 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 47039 A POSSIBLE...
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