The effects of potassium permanganate on lecithin and choleterol monolayers

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between two aqueous phases as well as for studies of t h e i n t e r a c t i o n of a d s o r b a t e s with such films. Tocopherol m e m b r a n e s will, therefore, help define t h e forces in t h e more c o m p l e x l i p i d - l i p i d or l i p i d - p r o t e i n i n t e r a c t i o n of biological m e m b r a n e s . W e t h a n k t h e Medical R e s e a r c h Council of C a n a d a for financial support. W . D . S . holds a scholarship, M.B. a research s t u d e n t s h i p of the Medical R e s e a r c h Council.

Ddpartement de Biophysique, Facultd de Mddecine, Universitd de Sherbrooke, Sherbrooke, Qudbec (Canada) I 2 3 4 5 6 7 8 9 io

WOLF D. SEUFERT GAETAN BEAUCHESNE MARCEL BI~LANGER

P. MUELLER, D. O. RUDIN, H. T. TIEN AND W. C. WESCOTT,J. Phys. Chem., 67 (1963) 534W. D. SEUFERT, Nature, 207 (1965) 174. P. LX.UGER,W. LESSLAUER,E. MARTIAND J. RICHTER, Biochim. Biopkys. Acta, 135 (1967) 2o. A. M. LIQUORIAND C. BOTR~, J. Phys. Chem., 71 (1967) 3765 . D. O. SHAH AND J. H. SCHULMAN,J. Lipid Res., 8 (1967) 215. M. M. STANDISHAND B. A. PETHICA, Biochim. Biophys. Acta, 144 (1967) 659. A. FINKELSTEINAND A. CASS, Nature, 216 (1967) 717 . M. TAKAGI,K. AZUMIAND U. KISHIMOTO,Ann. Rept. Biol. Osaka Univ., 13 (1965) lO7. H. T. TIEN AND S. CARBONE, Nature, 212 (1966) 718. H. T. TIEN AND E. A. DAVlDOWlCZ,J. Colloid Interface Sci., 22 (1966) 438.

R e c e i v e d F e b r u a r y 2oth, 197o

Biochim. Biophys. Acta, 211 (197o) 356-358

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The effect of potassium permanganate on lecithin and cholesterol monolayers* Salts of h e a v y m e t a l s h a v e been used e x t e n s i v e l y as stains in electron micros c o p y of cell organelles a n d m e m b r a n e s . However, little is k n o w n a b o u t t h e intera c t i o n of these h e a v y m e t a l ions with lipids a n d p r o t e i n s in m e m b r a n e s . Therefore, we h a v e u n d e r t a k e n a s t u d y on t h e i n t e r a c t i o n of h e a v y m e t a l ions with p h o s p h o l i p i d a n d cholesterol m o n o l a y e r s as a m o d e l lipid m e m b r a n e . The i n t e r a c t i o n of OsO 4 a n d u r a n y l a c e t a t e w i t h lecithin a n d cholesterol m o n o l a y e r s a n d l i q u i d c r y s t a l s has been r e p o r t e d p r e v i o u s l y 1-3. C h r o m a t o g r a p h i c a l l y p u r e L - a - d i p a l m i t o y l lecithin was p u r c h a s e d from Mann Research L a b o r a t o r y (New York, N.Y.); egg lecithin was s u p p l i e d b y the S y l v a n a Chemical Co. (Orange, N . J . ) . B o t h lecithins were c h r o m a t o g r a p h i c a l l y pure a n d gave single s p o t s on a t h i n l a y e r c h r o m a t o g r a p h i c p l a t e w i t h a c h l o r o f o r m - m e t h a n o l w a t e r (60:35:5, b y vol.) solvent system. T h e f a t t y acid composition of egg lecithin, which c o n t a i n s a p p r o x i m a t e l y equal a m o u n t s of s a t u r a t e d a n d u n s a t u r a t e d f a t t y acids, has been r e p o r t e d p r e v i o u s l y 4. H i g h p u r i t y cholesterol was s u p p l i e d b y A p p l i e d * Presented at the i2th Annual Meeting of the Biophysical Society, Pittsburgh, Pa., February, i968.

Biochim. Biophys. Acta, 211 (197o) 358-361

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Science Laboratories (State College, Pa.). Inorganic chemicals of reagent grade and twice distilled water were used in all experiments. Surface pressure and sin-face potential were measured by methods described previously 5. Surface measurements were taken on subsolutions of 0.02 M NaC1 at p H 6.0 and 25 °. Monolayers were first compressed to a desired surface pressure and then I ml of KMn04 solution (conch. 12 mg/ml) was injected into the subsolution (400 ml). While the subsolution was agitated by a magnetic stirrer, similar to that employed in the enzymic hydrolysis of lecithin monolayers 4, changes in surface pressure and potential were recorded at time intervals of I or 1. 5 min. All films were studied in duplicate and the results were not significantly different. However, without the stirring device the reproducibility was poor. Fig. I shows the changes in surface pressure of cholesterol, dipalmitoyl and egg lecithin monolayers after injection of KMnO 4 in the subsolution. It is evident that cholesterol and dipalmitoyl lecithin monolayers are not influenced by KMn04 at any surface pressure, whereas egg lecithin monolayers show an increase in the surface pressure. However, after an initial rapid increase, the surface pressure showed a gradual decrease. The increase in surface pressure is due to interaction of the double bonds of f a t t y acyl chains with KMnO 4 in the subsolution. The rate of increase of surface pressure depends upon the initial state of compression of egg lecithin monolayers. A greater rate of change of surface pressure at low initial surface pressures suggests that the double bonds ill the f a t t y acyl chains are more easily pulled towards the aqueous phase at low surface pressures than at high surface pressures. I t is interesting to note that even at a surface pressure of 37 dynes/cm, the reaction between the f a t t y acyl chains of egg lecithin and KMnO 4 occurs. HUGHES AND RIDEAL6 have similarly shown that the rate of oxidation of oleic acid monolayers b y KMnO 4 decreases with increasing surface pressure of the monolayers.

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Fig. I. Effect of K M n O 4 on the surface pressure of egg lecithin monolayers initially compressed to different surface pressures. F o r cholesterol and dipalmitoyl lecithin monolayers, t h e r e were no changes in surface pressure w h e n K M n O 4 was injected u n d e r the monolayers m a i n t a i n e d at different surface pressures. Fig. 2. Effect of KMnO 4 on surface potentials of egg lecithin monolayers initially compressed to different surface pressures. F o r cholesterol a n d dipaimitoyl lecithin monolayers there were no changes in the surface potential w h e n K M n O 4 was injected u n d e r the monolayers m a i n t a i n e d a t different surface pressures. (i2 m g K M n O l in 40o ml of o.o2 M NaC1 subsolution at p H 6.o.)

Biochim. Biophys..4cta, 211 (197 o) 358-361

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MARSDEN AND RIDEAL7 have further shown that at high surface pressmes the rate of oxidation by KMnO 4 is greater for fatty acid chains having cis- rather than trans- double bonds. The mechanism of reaction between KMnO 4 and a double bond is not yet well understood. However, GILBY AND ALEXANDER8 have attributed the initial increases in surface pressure to the formation of the dihydroxy acidL The hydroxy groups added on the double bond are pulled towards the aqueous phase and thereby increase the surface pressure. Subsequently, further oxidation occurs at this point with ultimate fission and formation of two carboxyl groups. The resulting sholtchain molecules are soluble in the subsolution and hence leave the monolayers causing a decrease in the surface pressure s. This explains why there is an initial increase and subsequent slow decrease in the surface pressure of egg lecithin monolayers. The results shown in Fig. i also suggest that the rate of dissolution of short-chain molecules from oxidized monolayers depends upon the initial state of compression of the monolayers. Fig. 2 shows the corresponding changes in surface potentials of cholestelol, dipalmitoyl and egg lecithin monolayers when KMnO 4 was injected in the subsolution. Cholesterol and dipalmitoyl lecithin monolayers are not influenced by KMn04, whereas egg lecithin monolayers exhibit a decrease in surface potentials. In contrast to surface pressure, the rate of change of potential incleases with the surface pressure. This can be explained as follows: The increase in the surface pressure is presumably due to fatty acyl chains being pulled down at the air-water interface upon oxidation, whereas the decrease in the surface potential is mainly due to oxidation of the double bonds. The fall of surface potentials at high surface pressures, therefore, suggests that the fatty acyl chains are oxidized at high surface pressure but not pulled down at the interface to the same extent as that at low surface pressures. The greater rate of change of surface potential at high surface pressures is probably the result of a greater surface concentration of molecules in the monolayer (i.e. the number of double bonds per cm 2 is greater at high surface pressures). We should point out that at pH's near neutrality, the double bonds in the fatty acyl chains are oxidized by KMn04, whereas those in cholesterol molecules are not. However, at pH 2-3, the double bond of cholesterol is oxidized by KMnO 4 resulting in an increase of I7O-I75 mV in the surface potential of cholesterol monolayers. A comparison of the previous works on the effect of OsO 4 and uranyl acetate on lecithin and cholesterol monolayers 1-8 with the present results indicates that both KMnO 4 and OsO 4 react with the double bonds of fatty acyt chains but not with phosphorylcholine groups in lecithin monolayers, since they do not influence the surface pressure or potential of dipalmitoyl lecithin monolayers. If there had been ionic or ion dipole interaction between phosphorylcholine group and KMnO, or Os04, it would have changed the surface potential of dipalmitoyl lecithin monolayers. The presence of double bonds is necessary for the interaction of OsO 4 or KMnO 4 with lecithin monolayers. In contrast, uranyl ions react with the phosphate gxoups of both saturated or unsaturated lecithins, increase the surface potential by 200-250 mV and tend to solidify lecithin monolayers *. The interaction of OsO 4 with the double bond of cholesterol decreases the surface potential by I I 5 mV (ref. I), whereas KMn04 and uranyl acetate do not influence the surface potential of cholesterol monolayers 2. However, at acidic pH, KMn04 reacts with cholesterol monolayers and increases the surface potential by I7o-I75 mV. BiocMm. Biophys. Acta, 211 (197o) 358-36i

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These results indicate that the mechanism of interaction of KMn04 with cholesterol monolayers is different from that of 0s04, because these metal salts have opposite effects on surface potentials. The effects of KMnO4 on various lipid monolayers have been reviewed recently by SHAH1°. MORETZet al. 11 have shown, using the small-angle X-ray diffraction method, that both Os04 and KMn04 caused a significant rearrangement of molecules in the myelin membrane. The changes in surface pressures and potentials of lecithin monolayers reported here support the view that KMnO4 could significantly alter the arrangement of lipid molecules in the membrane due to its interaction with the double bonds in the fatty acyl chains of phospholipids. The author wishes to express sincere thanks to Drs. A. Chu and R. O. Anderson for critically reviewing this manuscript. This work was supported by a grant from the National Science Foundation (No. GB-5273).

Laboratory of Surface Chemistry, Biological Oceanography, Lamont Doherty Geological Observatory of Columbia University, Palisades, N. Y. Io 964 (U.S.A.)

D I N E S H O. SHAH*

I K. D. DREHER, J. H. SCHULMAN, O. R. ANDERSON AND O. A. ROELS, J. Ultrastruct. Res., I9 (1967) 586. 2 D. O. SHAH, J. Colloid Interface Sei., 29 (1969) 21o. 3 0 . R. ANDERSON, O. A. ROELS, K. D. DREHER AND J. H. SCHULMAN, J. Ultrastrua. Res., 19 (1967) 6oo. 4 D. O. SHAH AND J. H. SCHULMAN, J. Colloid Interfave Sei., 25 (1967) lO 7. 5 D. O. SHAH AND J. H. SCHULMAN, J. Lipid Res., 6 (1965) 341. 6 A. H. HUGHES ANn E. K. RIDEAL, Proc. Roy. Soc. London, Set. A, 14o (1933) 253. 7 J. MARSDEN AND E. K. RIDEAL, J. Chem. Soc., (1938) 1163. 8 A. R. GILBY AND A. E. ALEXANDER, Australian J. Chem., 9 (1956) 347. 9 T. P. HILDITCH, J. Chem. Sot., (1926) 1828. i o D. O. SHAH, in R. PAOLETTI AND D. KRITCHEVSKY, Advan. Lipid Res., (I97 o) in t h e press. I I R. C. MORETZ, C. K. AKERS AND D. F. PARSONS, Bioehim. Biophys. Acta, 193 (1969) I.

Received March 2oth, 197o * P r e s e n t a d d r e s s : D e p a r t m e n t of A n e s t h e s i o l o g y a n d D e p a r t m e n t of C h e m i c a l E n g i n e e r i n g , U n i v e r s i t y of Florida, Gainesville, Fla. 326Ol, U.S.A.

Biochim. Biophys. Acta, 211 (197 o) 358-361