ESR study of proton transport across phospholipid vesicle membranes

Journal of Biochemical and Biophysical Methods, 18 (1989) 237-246

237

Elsevier

BBM 00736

ESR study of proton transport across phospholipid vesicle membranes * V.V. Khramtsov, M.V. Panteleev and L.M. Weiner Institute of Chemical Kinetics and Combustion, Novosibirsk 630090, U.S.S.R.

(Received 1 August 1988) (Accepted 24 January 1989)

Summary A new method for measuring the rates of proton transfer through bilayer phospholipid membranes using pH-sensitive nitroxyl radicals is suggested. The pH-sensitive alkylating radical was covalently b o u n d to glutathione. This modified glutathione is p H sensitive at p H 1.5-4.5 and does not penetrate across phospliolipid membranes. Using ESR this probe was applied to register the kinetics of p H variations inside large unilamellar phospholipid vesicles after creation of a transmembrane proton gradient. In the acidic region (pH - 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of an undissociated acid. The m e m b r a n e permeability coefficients have been determined for a series of acids (HC1, HC104, HNO3, upper estimate for H2SO4). Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as p H probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media. Key words: ESR; p H probe; Nitroxyl radical; Liposome; T r a n s m e m b r a n e proton transport

Introduction

Proton transport across vesicles and natural membranes is of crucial importance for bioenergetics and plays a significant role in the regulation of cell functions [1,2]. Molecular pH probes are widely used [3-5] for studying proton transmembrane transport. In most cases, the application of molecular pH probes is based on the Correspondence address: V.V. Khramtsov, Institute of Chemical Kinetics and Combustion, Novosibirsk 630090, U.S.S.R. * Preliminary results of this work were presented at the XII International Conference on Magnetic Resonance in Biological Systems, Todtmoos (1986). 0165-022X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

238

possibility of their incorporation into the inner volume of liposomes and their recognition of the inner p H (PHin). By measuring the characteristics of a p H probe such as fluorescent intensity [3] or chemical shifts in 31p N M R signals [4] after the creation of a transmembrane p H gradient, one can determine the kinetics of P[-[in yariations and, thus, the value of proton flux across the membrane. However, the application of fluorescent p H probes is restricted by a high and often unidentified sensitivity of the fluorescence intensity to the probe-membrane interaction, to the ionic strength of the solution and to the type of buffer employed. The low sensitivity of the N M R method requiring high probe concentrations, essentially limits its application. Hence, it seems promising to use stable nitroxyl radicals as p H probes due to the high sensitivity of the ESR method, well developed ESR spectra theory [51 and achievements in organic chemistry of nitroxides [7]. In this connection Cafiso et al. [51 have used the distribution sensitivity of a few spin-labelled amphiphiles between water and lipid phases to study the transmembrane p H gradient or transmembrane potential Recently, papers have appeared wNch report on the ESR spectral sensitivity of a number of nitroxyl radicals to absolute p H media values [8-12]. The most pronounced pH-dependent spectral changes are observed as a result of the protonation of imidazoline (I) and imidazolidine (It) radicals at the N3 atom. Changes in a N constants are 0.8-1.3 G and in g factors. Ag = (2 3) × 10 - 4 [10]. The p K values of the radicals of type t and [l are determined to a great extent by substituents at C4(X1, X 2, X) [10,11]. The radicals studied can be used as spin p H probes witNn the range of p H from 0 to 14. the accuracy of p H assessment being up to 0.05 p H units. Eartier, we have reported [10] the synthesis of the pH-sensitive macromolecular p H probe (pH range 1.7-4.77, namely, the spin labelled human serum albumin which can be applied m many biological objects. This work is devoted m the develooment of a method for measuring the proton transfer rates across biological membranes using pH-sensitive nitroxyl radicals. To this end, the imidazolidine radicat was covalently bound m glutathione with the formation of a spin probe, which is p H sensitive at p H 1.5-4.5 and does not penetrate across the membrane. This probe was used to follow by ESR the kinetics of p H variations inside large unilamellar phospholipid vesicles after the creation of a transmembrane proton gradient. As shown, in the acidic region (pH - 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of a nondissociated acid. The membrane permeability coefficients have been determined for a series of acids (HCI, HC104, H N O 3).

5x~ , N \

~

I,

Xl X2 N/

I

0

O'

(I)

(Z) S c h e m e 1.

239

Materials and Methods The synthesis of radicals R1 and R2 are described in [10,13] and [14], respectively. The radicals R1 and R2 have given satisfactory elemental analysis data: 43.1% C (calculated 42.8%), 7.2% H (7.2%), 11.3% N (11.2%), 31.7% Br (32.0%) for radical R1 [13] and 61.0% C (calculated 61.2%), 11.1% H (10.8%) and 17.6% N (17.8%) for radical R2 [14]. 21.7 mg of glutathione (Sigma) and 28 mg of R1 radical were dissolved in 2 ml of 0.1 M K-phosphate buffer (pH 7.6) and then incubated for an hour with stirring at 37 o C. Then 2 ml of chloroform were added to the reaction mixture, the latter was shaken and the lower phase removed. The procedure was repeated - 6 times until the ESR signal in chloroform disappeared. The product yield was - 7 0 % . The ESR spectrum of the resulting radical has a different pH-dependence (Fig. 1) than R1 and a substantially higher water solubility. The most probable structure of a modified glutathione (given in Scheme 2 as R-Glut) is based on the possibility of covalent bond formation upon interaction between the alkylating radical group (-CH:Br) and the glutathione SH-group [10,15]. The water R-Glut solution was kept at 2 ° C over a period of 3 - 4 days. Prior to R-Glut application, the invariability of properties able to affect the experimental results was controlled: firstly, the intensity, shape and pH-dependence of the ESR spectrum were preserved, and secondly, the impermeability o f R-Glut through the membrane of phosphatidylcholine liposomes was preserved (see Results and Discussion). Egg phosphatidylcholine (PC) isolated and purified according to [16] was kindly donated by Dr. V.G. Budker (Novosibirsk Institute of Bioorganic Chemistry). Large unilamellar liposomes from egg PC were prepared according to [17] in 0.1 M K-citrate buffer: lipid concentration was 20 mg/ml. Chloroform solutions of phosphatidic acid (Sigma), cetyl-trimethyl-ammonium bromide (Chemapol) and cholesterol (Sojuzkhimreaktiv) and the water solution of the R-Glut were added to PC chloroform solution at the stage of liposome preparation. T h e final concentration of the radical was = 1 0 - 3 M. The radical, not present in the inner liposomal volume, was removed as follows. The liposomes were centrifuged at 15 000 × g for 15 min. Then the supernatant was removed and the liposome solution was diluted with 0.1 M K-citrate buffer to the same volume. The procedure described was repeated 3 - 4 times, as a result the radical concentration in the supernatant was _< 10 -7 M, while in the liposome solution it was = 1 0 - 4 M. The liposome radius measured by the quasielastic laser light scattering technique, using the instrument described in [18], was = 2000 A. Note that the internal volume of the liposome at a

CH2Br"-T~HN/

"~---~N/H

CH2--C--NH--CH--CH2--S\[ [i 1 C~21H / c.2 I

•

"

R1

R2

CH--NH2I

COON Scheme 2.

.° I H2~ HOOC

,Y /~N/X 0'}

R-Glut

240

o®

o @ ®o

15,5 - -

g

ol v

...........

-e~ @o

1 5 --

o~ o O @

o~o I 2

iI II

',

I I I

! pK I 3 pH

Fig. 1. pH dependence of the a N constant for radical R-Glut in H20 (o) and in 0.1 M K-citrate buffer (0). Radical pK measured by the ESR method is 34- 0.1. lipid concentration 20 m g / m l was equal to about 10% of the total Iiposome solution volume. The transmembrane p H gradient was established by mixing th~ liposome solution with an equal volume of 0.1 M potassium citrate buffer of a given p H in a self-constructed mixer with a mixing time of ~< 1 s. To produce an acid gradient (HX, where X - = C1-, C104-, NO3-) in the absence of the X - gradient, the mixed volumes contained 0.3 M salt (K_X)o The required pH in these volumes was attained by adding H2SO 4. ESR spectra were recorded on an ER-200D-SRC (Bruker) spectrometer in a flat cell or in a mixer. The hyperfine coupling constants, aN, were measured as the distance between the low field and the central fine of the triplet and are accurate to +0.02 G. The magnetic field corresponding to the center line was evaluated by comparison with ~,d-diphenyl-fl-picrylhydrazyl ( g = 2.0037). p H was measured using a pH meter OP-205/1 (Hungary) to + 0.05 pH units. Where the effect of the buffer concentration on the ESR spectrum of the R2 radical was studied, argon was bubbled through the sample for 20 rain to remove oxygen.

~ e s ~ t s and Discussion Spin pH probes can be used to follow the kinetics of intraliposomal p H variations after creation of a transmembrane p H gradient and, thus the radicals may be useful for transmembrane proton transport studies. The following requirements for the radical used as a pH probe should be satisfied: firstly, it must be p H sensitive; secondly, it must not penetrate across the membrane. To obtain such a p H probe, a tripepdde glutathione was modified by a pH-sensidve [10] alkylating radical R1 (see Materials and Methods). The radical R-Glut, so obtained, is p H sensitive with p K = 3 _4-0.1 (Fig. 1) and can be used as a p H probe in the range

241

'E :3

lO

20

Time(s)

Fig. 2. The kinetics of R-Glut reduction with sodium ascorbate registered from the decrease in intensity ( I ) of the central component of the ESR spectrum of R-Glut. The concentration of the radical and sodium ascorbate was 10 . 4 M, the temperature was 23°C. (A) R-Glut in the inner volume of the liposome. The absence of ascorbic acid transport during the time about I rain allows one to conclude that its coefficient of permeability through PC membrane is << 10 . 7 c m / s which fits the permeability coefficient value about 10 - 10 c m / s [15]. (B) Radical R-Glut in the outer volume of the liposome.

1.5-4.5. R-Glut does not penetrate across the membrane of PC liposomes, as shown by the following experiments. The kinetic curves of R-Glut reduction with sodium ascorbate are illustrated in Fig. 2. It can be seen that when located in the inner liposome volume, R-Glut is not reduced with sodium ascorbate. However, when R-Glut is present outside the liposomes it is reduced in - 1 0 - 2 0 s. Note that R-Glut did not emerge from liposomes after creating p H gradients across the membrane (for - 5-10 h) which was judged from the rate of its reduction with sodium ascorbate. These experiments lead us to conclude that R-Glut can be used for studying the transmembrane proton transport. The shape of the high-field component of the ESR spectrum of R-Glut, which is most sensitive to the pH, is complicated because of the intermediate character of the frequency exchange (on the ESR time scale) between ESR signals of protonated (RH +) and non-protonated (R) radical forms, caused by the chemical reaction of proton transfer: R + H + ~ RH +

(1)

This fact hampers the choice of the spectral parameter to follow pHin variations. Therefore, to simplify the shape of the ESR spectrum of R-Glut, the samples were prepared in 0.1 M K-citrate buffer (pKbuff = 3.13 [201). It has been shown earlier that the increase in buffer concentration with a pKbufg value close to the p K of the radical, results in the acceleration of the frequency exchange in ESR spectra of short lifetime [21] and stable [22] radicals, induced by proton exchange between buffer molecules and radicals. Kf

R+BuH + ~RH ++Bu Kr

(2)

242

c j I

1

Fig. 3. The high-field component of the ESR spectra of 5 ×10 .5 M radical R2 in the KAc buffer of different concentrations at pH 4.35 (points were calculated [22]): 0 (A); 2.27 × 10 -~ M (B); 5.76 × 10 .2 M (C); 0.28 M (D). The temperature was 294 K. From the comparison of experimental and calculated ESR spectra the values of the rate constants of forward and reverse exchange reactions [2] /(-f = ( 2 - t - 0 . 1 5 ) ×108M-l-s -1, Kr= (5.3_+0.3)×108 M-l-s -1 were obtained [22].

The buffer effect on the E S R signal is best shown b y the transition of a slow frequency exchange to a fast one. Fig. 3 depicts the high-field c o m p o n e n t of the E S R spectrum of the R2 radical ( p K = 4.35) at different K-acetate buffer concentrations ( p K = 4.75). Actually, by increasing the K-acetate buffer concentration, one can observe the transition from a slow frequency R ~ R H + exchange to a fast one. Thus the transformation to an exchange narrowed singlet considerably simplifies the spectrum. The R - G l u t E S R spectrum in 0.1 M K-citrate buffer also represents the exchange n a r r o w e d lines of the triplet which is characteristic for the transition f r o m intermediate to fast exchange with the radical p K remaining equal to 3.0 (Fig. 1). U p o n variation of the pH, the shift of the triplet line position is observed, their shapes being unchanged. The high-field c o m p o n e n t undergoes the most p r o n o u n c e d shift. Thus, the intensity of the E S R spectrum of R - G l u t at a frequency ~ ( p K ) , corresponding to the center of the high-field c o m p o n e n t at p H = p K = 3.0, is a convenient parameter with which to register the kinetics of intraliposomal pHin variations. W i t h the HC1 gradient across the m e m b r a n e of PC liposomes, we observed the kinetics of the intensity variations I(t) of the R - G l u t E S R spectrum at the frequency w ( p K ) (Fig. 4). The change of the HC1 gradient sign and the decrease in the z~HC1 value are shown to result in the change of the sign and the decrease in the rate of I ( t ) variation. F r o m the analysis of the initial portion of the kinetic curve I ( t ) , the ( d I / d t ) t = 0 derivative was found, allowing the estimation of the p r o t o n flux jH + across the m e m b r a n e surface unit at the initial m o m e n t of time after creating the p H gradient. Actually, taking into account that the n u m b e r of protons transferred across the liposome surface ( S ) during the period dr, is d H + = j H ÷ . S - d t and that the buffer capacity of solution is B = 1/V. d H + / d p H (V is the liposome

243

A 2 c

t=O

I

100

I

I

p.

3r" Time (s; 200

Fig. 4. The a m p l i t u d e of the E S R s p e c t r u m of R - G l u t at frequency ~0(pK) c o r r e s p o n d i n g to the center of the high-field c o m p o n e n t at p H = p K vs. time after c r e a t i o n of the t r a n s m e m b r a n e g r a d i e n t HC1. (A) pHin = 3.13, pHou t = 4.63; (B) p H i n = 3.13, pHou t = 1.63.

volume, d p H is the pHin change by transferring a number of protons d H + to liposomes), we obtain:

d~

=

,=o

dPHt

dH+

t

,=o

(3)

The value (dI/d~0),= 0 was determined from the shape of the high-field component of the R-Glut ESR spectrum I(o~) and (d~0/dpH)t= 0 was calculated from o~(pH) experimental dependence. The liposome radius r--- 3 • V/S and the buffer capacity B were estimated experimentally (see Materials and Methods). Table 1 lists the values of jH+ obtained by creating the same concentration gradient of different acids across the PC membranes. The considerable differences in the above values, when creating equal z~H + by various acids, allow one to

TABLE 1 VALUES OF THE PROTON FLUXES jH + UPON CREATION OF THE GRADIENT OF DIFFERE N T A C I D S A C R O S S T H E P C M E M B R A N E O F T H E L I P O S O M E S I N A 0.1 M K - C I T R A T E B U F F E R (pHou t = 1 . 6 3 , p H i n = 3.13) A N D T H E P E R M E A B I L I T Y C O E F F I C I E N T S O F C O R R E S P O N D I N G A C I D S A C R O S S P C M E M B R A N E S A T 23 ° C Acid JH + (M- c m . s - 1 ) P R x (crn. s - 1)

HC10 4 2 X 1 0 - lo (2.2 + 1)

HC1 1.8 × 10 - 9 (2 _+1)

HNO3 3 × 10 - 8 (1.4 + 0.7) × 1 0 - 4

H 2 SOn _< 1 0 - a2 ~<1 0 - v

244

conctude that the ] ( t ) kinetics observed cannot be explained in all cases by the transport of the same particle, H +, K + (as a substitute for H+), or of a molecule of citric acid. Moreover, the absence of the intensity variations of the I ( t ) signal (during - 2 h) in the case of the gradient of H 2 S Q indicates that the electrically uncompensated transport of a proton, the K+-dependent transport of a proton, and the H + transport in the form of undissociated citric acid are slow processes and their contribution to the I ( t ) kinetics observed in the case of AHCI, AHCtO 4 and z~HNO 3 can be neglected. Therefore, in these cases, the kinetics I ( t ) can be determined by anion (X-)-dependent transport of H+(X - = CI-, C[O~, N O 3 ) o r / a n d by proton transport in the form of undissociated acid. Further, to simplify the interpretation of kinetic dependence I(t), the p H gradient across the membrane was created in the absence of the X - gradient using H2SO 4 (see Materials and Methods). Table 1 shows that proton flex jH. calculated from the I ( t ) kinetics increases, creating an H + gradient by acids with a larger value of p K in the order HC104, HC1, H N O 3 ( p K a a o - - 8 , pKHcl -~ --7, pKHNo3 = --1.62 [20]). The foregoing favours the proton transport in the form of undissociated acid HX in the acidic medium. To prove the above conclusion independently, we have studied the membrane surface charge effect on the I ( t ) kinetics upon creating a HC1 gradient across the membrane. With the rate of proton transfer across the membrane being determined by a number of factors, such as the type of the buffer applied and the ionic strength of the solution, the variation of the membrane surface charge can result in considerable change of proton-hydroxyl permeability (3-4 times [23]). Table 2 lists the fluxes jH+ across the PC liposome with different composition calculated by [31 from the kinetic curves of I(t). In contrast to [23] almost the same variations in the membrane surface created by adding the negative phosphatidic acid (PA) or positive cetyl-3-methyl-ammonium bromide (C) virtually did not affect the values JH*, while increasing the m e m b r a n e ' rigidity' by the addition of cholesterol (Ch) considerably decreased j~+. Thus, the experiments on the influence of the TABLE 2 D E P E N D E N C E OF T H E P R O T O N F L U X JH+ ON T H E C R E A T I O N OF T H E HC1 G R A D I E N T ACROSS T H E M E M B R A N E (pHoto = 1.63, pHin = 3.13) O N T H E M E M B R A N E COMPOSITION. Membrane composition

jH +

(M.cm-s -1) 100% 99% 98% 95% 99% 98% 95% 90% 80% 70%

PC PC PC PC PC PC PC PC PC PC

+1% PA +2% PA +5% PA +1% C +2% C +5% C +10% Ch +20% Ch +30% Ch

1.8 x 10 -9 1.47<10 -9 1.3)<10 - 9 2×10 9 2 . 3 × 1 0 -9 1.5)<'10 - 9 1 . 4 x t 0 -9 8 × ! 0 -~° 6 × 1 0 -1° 6 × 1 0 -1°

245 m e m b r a n e s u r f a c e c h a r g e o n t h e I(t) k i n e t i c s p r o v i d e i n d e p e n d e n t e v i d e n c e t h a t in o u r c a s e t h e p r o t o n s a r e t r a n s p o r t e d in t h e f o r m of u n d i s s o c i a t e d acid. T h e a b o v e c o n c l u s i o n a l l o w s o n e to c a l c u l a t e t h e p e r m e a b i l i t y c o e f f i c i e n t of H X a c i d s a c r o s s t h e P C m e m b r a n e u s i n g t h e c o r r e s p o n d i n g v a l u e s of jH+ ( T a b l e 1): PHX

=JH+/AHX = J H + / (

all+" [X-])-K

a

(4)

w h e r e K a is t h e d i s s o c i a t i o n c o n s t a n t of t h e H X a c i d ( K a = 10-P~;), A H + is t h e v a l u e o f H + t r a n s m e m b r a n e g r a d i e n t , [ X - ] is t h e a n i o n c o n c e n t r a t i o n T a b l e 1 lists t h e p e r m e a b i l i t y c o e f f i c i e n t s PHX f o r HC1, H C 1 0 4, H N O 3 a n d t h e u p p e r e s t i m a t e f o r H 2 S O 4. N o t e t h a t t h e values, o b t a i n e d b y t h e E S R m e t h o d , f o r P H a a n d PHNO, a r e in a g r e e m e n t w i t h t h o s e m e a s u r e d in [23] o n t h e p l a n a r l i p i d b i l a y e r s ( P n a ~ 3 c m / s , PHNO~ ~ 1 0 - 3 C m / s , t h e t r a n s f e r of H 2 S O 4 u n o b s e r v e d ) . T h u s t h e a b o v e d a t a e x h i b i t t h e g r e a t p o t e n t i a l s o f the E S R t e c h n i q u e f o r studying the transmembrane proton transfer.

Simplified description of the method and its applications A new ESR method for measuring the rates of proton transfer through bilayer phospho!ipid membranes using pH-sensitive nitroxyl radicals is suggested. To this end, the imidazolidine radical was eovalently bound to glutathione with the formation of a spin probe, which is pH sensitive at pH 1.5-4.5 and dose not penetrate across the membrane. This probe was used to register by ESR the kinetics of pH variations for the pH range 1.5-4.5 inside large unilamellar phospholipid vesicles after the creation of a transmembrane proton gradient. Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as pH probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media.

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