Eur. J. Biochem. 25 (1972) 64-70

Determination of ApH in Chloroplasts

2. Fluorescent Amines as a Probe for the Determination of ApH in Chloroplasts

Shimon SCHULDINER, Hagai ROTTENBERO, and Mordhay AVRON

Department of Biochemistry, The Weizmann Institute of Science, Rehovot

(Received August 31, 1971)

A method for the measurement of the internal pH of chloroplasts is described. The method is based on the uptake of a fluorescent amine by chloroplasts, which can be observed by the lower- ing of the fluorescence emitted from a suspension of chloroplasts.

It is shown that this uptake is dependent, as expected, on the dissociation constant of the amine and also on the number of ionisable amines in the molecule. Amines attached to two types of chromophores, acridine and naphthalene have been investigated. As previously shown for other amines, this uptake was found to be a prerequisite for their acting as uncouplers. The technique provides a simple way to continuously follow changes in ApH in chloroplasts.

A relation between energy generation and the quenching of the fluorescence of the uncoupler atebrin has been described by Kraayenhof [i]. He showed that the required energy could be provided by elec- tron transport, ATP hydrolysis or a pH gradient and it was therefore suggested that the extent of quenching may be used to measure the “energy state” of the chloroplast [1,2]. A similar effect was described in chromatophores and was suggested to be related to proton movements, rather than to the “energy state” [3].

We have recently shown that atebrin distributes between the inside of the chloroplast and the solution in accordance with the ratio of proton concentra- tions [4], in a manner similar to other amines [5,6]. The quenching was shown to be a consequence of its uptake and probably due to several factors [a]. Thus, as was shown with 5,5-dimethyl-2,4-oxazoli- dinadione in mitochondria [7] or methylamine [5,8] and NH, [9] in chloroplasts this uptake could be used as a measure of the H+ gradient across the membrane. The distribution of amine across the membrane is related to the proton distribution according to the following expressions where [A]T is the total amine concentration, [A] is the concentration of the un- charged amine and [AH+] is the concentration of the charged amine. Assuming that the uncharged amine is freely permeable across the membrane, and is the

Unusual Abbreviations. Atebrin, 3-chloro-9-(4-diethyl- amino-l-methylbutyl)-7-methoxy-acridine; tricine, N- tris (hydroxymethy1)-methylglycine.

only form that crosses the membrane, [A]in = [Aleut = [A] and since, K , = [A] [H+]/[AH+] we obtain

Two extreme cases are of interest. Firstly, where K , >> [H+]in, and [H+lout ( L e . p& << pHin, and PHout).

Thus, an amine of a very low pK will be equally distributed across the membrane independent of the ApH. The second extreme case, where Ka << [H’lin, and [H+Iout ( i .e . pK, > pHin, and PHout)

(3)

Thus, an amine of a high pK will be distributed across the membrane in accordance with the proton concen- tration gradient.

I n the case of diamine,

Vol. 25, No. 1, 1972 S. SCHULDINER, H. ROTTENBERG, and M. AVRON 65

8

z

!?

m In

3 ;;

.- r - B -

Four cases are of interest. The first is where K , and K , > [H+lin and [H+lout (PK, and P K ~ << pHin and PHout)

A pH = 3.1

t I on off

(5 )

Thus, as in the case of a monoamine when both pK values are very low, the amine will be equally distributed across the membrane independent of the ApH. The second case is where Kl > [H+]in and [H+lo,t > K , (pK1 Q pHin and pHout < pK2)

Thus, this is essentially the situation described for a monoamine of a high pK [Equation (3)]. The third case is where K , - [H+]in and [H+Iout > K , (pK, = PHin and pHout Q pK2)

From the above it is clear that from the dissocia- tion constants of the amine in question and its distri- bution across the membrane, one can calculate the proton distribution ratio, or the ApH.

In this report we show that the above relationships hold for several amines which we tested, whether attached to an acridine chromophore or to a naphthalene one. It is also shown that, as in the case of other amines [4-61, uptake is an essential pre- requisite for uncoupling of photophosphorylation. The technique provides an easier way to observe previous results [5,8,9] where movements of labeled methylamine [5,8] or NH,+ [9] were followed.

METHODS

Chloroplasts from lettuce leaves were prepared essentially as previously described [ 101 except that they were finally washed and resuspended in a solution containing 0.2 M sucrose and 0.1 M KC1.

Fluorescence was measured in an Eppendorf fluorimeter. When the fluorescence of acridine derivatives was measured, the exciting light was filtered through a 4051436 filter. Emission was mea- sured through a Corning CS 4-96 filter, a Strand Electric Co. cinemoid filter No. 62 and a Wratten No. 58 filter. For naphthylamine fluorescence, the exciting light was filtered through a 3131366 filter. Emission was measured through Corning CS 4-96 and CS 3-73 filters. Actinic light was provided by a 24 V halogen lamp, filtered through a Schott RG 645 filter, which provided an incident light intensity of 5-7 x lo5 erg x cm-2 x sec-l (650-750 nm). Where 6 Eur. J. Biochem., Vol.25

pH and fluorescence changes were measured simul- taneously a Metrohm combined microglass electrode (type X) was introduced into the cuvette and the sig- nals were recorded on a multichannel Rikadenki recorder (Type B34). Photophosphorylation was determined by following the pH rise due to ATP formation using the values for the ATP/H+ ratio given in [ 1 I].

3-Chloro-9- (4-diethylamino- 1 -methylbutyl)-7-me- thoxy-acridine (atebrin) was obtained from Mann Laboratories, N-(1-naphthy1)ethylene diamine-dihy- drochloride, 1-naphthylamine and 9-aminoacridine derivatives from Fluka ; the other acridine derivatives were a gift of Dr. M. Shinitski from this Institute and they were prepared according to the methods described in [ 121.

A pH= 2. I. m I Off 1 -

ii t i

on off

A p H =3.0

, 1 min

-

t I on off

. A LAtebrin-- - Arninoacridine-9 - Acetamidoa~dine-

1 1 on off

1 rnin - B

V ( 1 - Naphthyl) ethylene-i-Naphthylamine diarnine

Fig. 1. Light-induced quenching of the fluorescence of difjeerent amines. The reaction mixture contained in a final volume of 3 ml; KCI, 40 mM; tricine-glycine, pH 8.0, 30 mM; pyo- cyanine, 15 pM, and chloroplasts containing 54 pg of chloro- phyll. Other details as described in Methods. (A) Acridine derivatives, (B) naphthalene derivatives. In (A) atebrin was added a t 0.2 pM, 9-aminoacridine a t 0.6 pM and 9-acetami- doacridine a t 8.0 pM. In (B) N-( 1-naphthy1)-ethylene diamine was added a t 0.7 pM, and I-naphthylamine a t 1.0 $I. The arrows indicate the addition of amine, and the switching

of the light

66 Fluorescent Amines and ApH in Chloroplasts Eur. J. Biocliem.

Table 1. The structure dissociation constants and distribution of fluorescent amines ~

Name Structure cln/cout at pH 8.0 pH

NHCH(CH,)CH,CH2CH,N(C,H,), 3-Chloro-9-(4-diethyl- amino- 1 -methylbutyl)- 7.9 10.5 - 2 40000 2.5 7-methoxy-acridine (ring) (diethylnmino)(9 amino) (atebrin)

NH,

9-Aminoacridine

9-Rcetamidoacridine

1-Naphthylamine

0

CH-C-NH Ji

NHCH2CH2NH, N-( 1-Naphthy1)-ethylene

diamine

9.99 (-2 1100 3.0 (ring) (9 amino)

4.2 very low - - (ring) (9 amino)

3.9 (1 -amino)

4.9 9.5 1300 3.1 (1-amino) (ethylamino)

RESULTS

We have tested three patterns of uptake, as expected from the dissociation constants of the amines, and their number: firstly, atebrin, containing two amines, one with a pK of 10.1, much higher than the external pH, and the second with a pK of 7.5, similar to the external pH [Eqn (7)]; secondly, 9-aminoacridine containing two amines, one with a p K of 10, much higher than the external pH and a second with a pK much lower than the internal pH (< - 2) [Eqn (6)] ; thirdly, 9-acetamidoacridine con- taining two amines, one with a pK of 4.2, lower than the external or internal pH, and a second one even lower than that [Eqn (5 ) ] .

Fig.lA, shows the light-induced changes in the fluorescence of these three acridine derivatives and confirms the predictions for the three cases. Atebrin showed a very high light-induced quenching of the fluorescence which represents a ratio of concentra- tions of the amine of 40000 (ApH 2.5) ; 9-aminoaeridine showed a lower uptake representing a concentration ratio of 1100 (ApH 3.0) ; and 9-acetamidoacridine did not show any light-induced quenching. The latter, of course, does not mean that there is no H+-concen-

tration gradient, but that in accordance with its p K , the amine did not concentrate inside the chloroplast. It should be mentioned that the three compounds do not fluoresce to an identical extent. This is due to a shift of the absorption spectra to lower wavelengths, especially in the case of 9-acetamidoacridine. How- ever, shifting the excitation to a lower wavelength (366 nm) in the latter case produced more fluores- cence, but no quenching during illumination of chloroplasts. The same picture was revealed when the chromophore was naphthylamine instead of acridine (Fig. 1 B). Thus, when naphthylamine fluores- cence was followed, no uptake could be detected, in agreement with its low pK (3.9); when a basic ethylene diamine moiety (pK 9.5) was attached to this acidic amine, uptake similar to that observed in the case of 9-aminoacridine was seen.

Table 1 summarizes the structures, dissociation constants, distribution and the calculated ApH of several amines. Consistent results were also obtained when N-methyl-9-aminoacridine (“pseudo” pK 11.2, see [12]), and acridine (pK 6.6) were tested.

ApH values which were obtained with atebrin were consistently lower than those measured with the

Vol.26, No.1, 1972 S. SCHULDINER, H. ROTTENBERC, and M. AVRON

Q

t.0

z a

, .5

~ .O

.5 .O

011 -0

Fig.2. Graphic representation of A p H as a junction of the percentage quenching observed. The A p H refers to that cal-

culated for a monoamine of high p K [Eqn (3)]

" 0 10 100 1000 [ Amine] ( pM)

I . 1 - Naphtylamine 9

,-" 8 0 1 L I N ( 1- Naphtyl)

10 100 1000 [ Amine] ( FM)

Fig. 3. Inhibition of photophosphorylation by different amines. The reaction mixture contained in a total volume of 3 ml: KC1 40 mM; potassium phosphate, 5 mM; MgCl,, 2 mM; ADP, 0.65 mM and chloroplasts containing 40 pg of chloro- phyll pH 8.0. Other details as described in Methods. (A) Acridine derivatives. 0, atebrin; 0 , 9-aminoacridine; A , 9-acetamidoacridine. (B) Naphthalene derivatives. 0, N-( 1 -naphthyl) -ethylene diamine ; 0 , 1 aaphthylamine

5'

10000 t I I I I I L

1 5000

67

4 .O

3.5

3 .O

2.5 I, a

2.0

1.5

1.0

Fig.4. Inhibition of A p H by carbonyl cyanide p-trifluoro- methoxyphenylhydrazone (FCCP). Conditione were as in Fig. 1. 9-Aminoacridine concentration was 1.0 pM; N - ( l - naphthy1)ethylene diamine was 0.7 pM. 0, N-(1-naphthy1)-

ethylene diamine; A , 9-aminoacridine

other amines. However, it seems that the lowest concentration of atebrin necessary for A pH measure- ment is sufficiently high for some inhibition of ApH.

The calculation of the ApH was done as follows. The concentration of the amine inside the osmotic compartment (tin) is equal to CinitQ/ V where Cinit = initial concentration, Q = the fraction of the total fluorescence that was quenched, V = the fraction of the volume of the osmotic compartment in the total volume. The new external concentration. (tout) is of course the difference between the initial concentration and the fraction of it that was taken in: tout = Cinit ( I - Q). The ratio between the internal and the external concentration will then be: CinlCout = [Q/(1 - Q)] x J/V. ApH can be calculated from these ratios, from equations (5 ) , (6) or (7), depending on the probe. Fig.2 is a graphic representation of the above function for different values of V . It can be seen that this type of function is most useful in a range of non-extreme values of Q. It is also clear that the accuracy of the values of ApH measured depend on the measurements of the osmotic volume. We deter- mined osmotic volume as previously described [5,8]. Nevertheless, it should be pointed out that the osmotic volume as determined for lettuce chloroplasts under specified conditions is reasonably constant. With KC1 as the osmotic agent at 50milliosmolar it is about 5Opl H,O per mg chlorophyll. I n addition, changes in the osmotic volume do not change drama-

Fluorescent Amines and ApH in Chloroplasts Eur. 3. Biochem. 68

1.0 l o ’ 6iO 7jO 8$ 9.0 l!O

PH

Fig.5. Dependence of ApH 0% external pH. Conditions as in Fig. 1, except for pH that was fixed either with tricine- glycine or with tricine-maleate buffers (30 mM). Chloroplasts contained 65 pg chlorophyll. 9-Aminoacridine concentration

was 1.0 pill

Light intensity (erg * cm-’* sec-’)

7.0 I a

4.01 I , , ,I 6.0 7.0 8.0 9.0 10.0

External pH

Fig.6. Dependence of internal pH on external pH. Conditions as in Fig.5

tically the values of ApH, thus for example if for 500/, quenching of the fluorescence with amino- acridine, a value of V of 0.5 p.l/ml is assumed, the ApH is 3.3. If the real value is 1.0 pl/ml, the actual d p H is 3.0.

Uncoupling by amines was previously suggested to be a direct consequence of their uptake [4,6,13]. It should, therefore, be appparent only by those amines that can be taken in. This indeed is the case for both types of amines checked (Fig. 3). Thus, again, only atebrin, 9-aminoacridine and N - ( 1-naphthyl)

Fig.7. Dependence of A p H and proton uptake on light intensity. The reaction mixture contained in a total volume of 3 ml: KCl, 30 mM; potassium tricinate, 2 mM; pyocyanine, 15 pM; chloroplasts containing 80 pg chlorophyll. (A) At pH 6.3, control values were dpH 1.8, proton uptake, 0.3 pmol H+ per mg chlorophyll and in (B) a t pH 8.0, the values were dpH 3.3 and 0.13 pmol/mg, respectively. 0, ApH; 0 , proton uptake. Wavelength of light wafl 650 to

750 nm

ethylene diamine were uncouplers in our system. Moreover, when atebrin and 9-aminoacridine are compared, atebrin which reaches higher internal concentrations a t lower external concentrations is indeed effective a t lower external concentrations. It should be pointed out that when these compounds were employed as probes for ApH, concentrations much lower than those required for uncoupling were always employed.

I n Fig.4 it can be seen that as shown with other techniques [5,8] the ApH was inhibited by un- couplers, like carbonyl cyanide p-trifluoromethoxy- phenylhydrazone (FCCP). The inhibition pattern was independent of whether the ApH was measured with 9-aminoacridine or with N-( 1-naphthy1)ethylene diamine.

The dependence of the ApH on the external pH is shown in Fig.5. It can be seen that the pattern is similar to that found with other techniques [8,9]. The absolute values are somewhat higher than those obtained by the centrifugation technique but very similar to those obtained with the NH,+ electrode [8,9]. If the internal pH measured is plotted against the external one (Fig.6) it becomes clear that for a wide range of external pH values the internal pH is rather constant, suggesting the existence of a natural buffering group around this internal pH (see also [S]).

If this is so, a t relatively low pH one should expect that the f i s t few protons transported into the osmotic volume will markedly shift the internal pH while most of the other protons will undergo buffering. This is clearly seen in Fig.7, where the number of protons

Vo1.25, No.1, 1972 S. SCHULDINER, H. ROTTENBERO, and M. AVRON 69

4.01 , , , 1 0 5 10 15

Protons added (nrnol)

Fig.8. Titration of the osmotic compartment of the chbroplasts. Conditions as in Fig.7. The following dark values of ApH (see [S]) were assumed: at pH 6.3 (O), dpH 0.3, at pH 7.3 (A),

ApH 0.5 and at pH 8.0 (D), ApH 0.6

taken up and the d p H were measured simultaneously a t varying light intensities and a t different external pH values. At low pH, the ApH (or proton-concen- tration ratio) underwent “saturation” much earlier than the proton uptake activity. Beyond saturation further proton uptake changed the internal pH only slightly. At higher pH where the buffering region is reached only after most of the protons have been pumped in, the proton uptake and proton-concen- tration ratio respond similarly to the varying light intensity. This is also represented in Fig. 8 where the absolute amount of protons pumped in is plotted against the internal pH of the chloroplasts (i.e. a titration curve). At the three pH values a buffering group a t an internal pH around 4.5 is clearly visible.

DISCUSSION A new technique for the measurement of ApH in

chloroplasts by means of fluorescent amines has been developed. It is based on the measurement of their uptake which can be easily followed by the disappearance of their fluorescence. This quenching has already been shown to be a consequence of uptake in the case of atebrin and its mechanism was discussed [el.

I n this report we show that the different amines tested distributed according to the proton-concentra- tion ratio, as expected from their different dissociation constants. The technique requires a determination of osmotic space, and one should be careful to ascertain that big changes in this volume do not occur during

the reaction. When osmotic-volume values are available, it provides a fast and elegant means of continuously monitoring the ApH of the system. It is important to realize that several assumptions are made in order to allow a quantitative treatment of the phenomenon: firstly that the uncharged species is freely permeable ; secondly that the charged species does not permeate to a significant extent, a t the concentration used; thirdly it is assumed that the amine is concentrated in the water phase and does not bind significantly to chloroplast constituents ; fourthly that the amine does not affect chloroplast processes a t the Concentration tested; and finally that the quenching is due to penetration of the amine into the chloroplast osmotic space and is complete when the amine is inside.

The first two assumptions are t’he general accepted assumptions in treating amine movements [5,6]. The third is suggested by the observations that the same ApH has been calculated from a large variety of amines, and using a wide range of concentrations of any of these. As regards the fourth, measurements should be done with concentrations far lower than those which uncouple. The relation between uptake and quenching has been treated in[4], and the assumption that quenching is complete can be inferred from the fact that in every case tested essentially complete light-induced quenching of the fluorescence was achieved provided enough chloroplasts were present.

The lowest values of ApH that we could measure with the technique were in the order of 1 pH unit; i.e., a quenching of the fluorescence of about 0.2 to 1.00/, under the normal conditions employed. The use of higher concentrations of chlorophyll in order to increase the sensitivity can lead to artifacts due to the complexity of the optical system. Optical quenching of fluorescence is already detected a t very low chlorophyll concentration and is due mostly to the absorbance ofthe exciting light. Thus, chloroplasts a t a concentration of IOpglml will absorb almost goo/ , of the incident light a t 430 nm [14].

For routine work it is simplest to employ an amine having a single ionisable species with a dissociation constant low enough so that it will be always lower than the external H+ concentration (pK , >> pHout). It is also advantageous, of course, that the quantum yield of fluorescence be as high as possible and as insensitive as possible to changes in environment (mostly polarity and viscosity of the medium). All of these requirements are f a l l e d satisfactorily by 9-aminoacridine [12] and it therefore seems to be the easiest probe for routine measurements.

As regards uncoupling, our results are in agree- ment with the proposal [6] that the uptake of an amine is an essential prerequisite for its acting as an uncoupler. Mechanistically it is also reasonable to assume that a high internal concentration of the

70 S. SCHULDINER, H. ROTTENBERG, and M. AVRON: Fluorescent Amines and d p H in Chloroplasts Eur. J. Biochem.

charged species is necessary so that its leakage out will be sufficient to produce a cyclic movement of the protons leading to an energetically useless pumping and therefore to uncoupling.

We wish to thank Dr Rf. Shinitski for many valuable discussions.

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S. Schuldiner, H. Rottenberg, and M. Avron Biochemistry Department, Weizmann Institute of Science P.O. Box 26, Rehovot, Israel