ARCHIVES OF RIOCHEMISTRY AND RIOPHYSICS 166, 134-142 (1973)

Cation-Dependent Quenching of the Fluorescence of Chlorophyll a

in Viva by Nitroaromatic Compounds

GEORGE PAPAGEORGIOU AND CHRIS ARGOUDELIS’

Nuclear Research Center (‘Democritus,” Department of Biology, Athens, Greece

Received October 3, 1972

The quenching of the Chl a2 fluorescence from spinach chloroplasts and chloroplast fragments by nitroaromatic compounds and the effect of added met,al cations on the quenching rate is investigated. The extent of the quenching with nitrobenzene and 1,3-dinitrobenzene was found to be independent of whether Chl a is excited directly, or through Chl b by means of electronic energy transfer. On the basis of this, the contribution from a purely static mechanism is considered as unlikely.

Nitroaromatics substituted with ionizable groups are almost equally effective quenchers for the fluorescence of Chl a in vivo and in methanol. On the other hand, nitroaromatics which are slightly soluble, or nearly insoluble, in water quench more strongly the fluorescence of Chl a in vivo. The overriding factor that determines the relation between the apparent and the true quenching constant appears to be the partition of the quencher in the lipid and the aqueous phases of the membrane sus- pension.

Divalent metal cations enhance the quenching by nitrobenzene dramatically, most likely by increasing the hydrophobic character of the chloroplast membranes. This enhancement occurs at cation concentrations higher than those corresponding to the maximal turbidity increase of the membrane suspension; hence, it is attributed to ultrastructural changes of the membrane rather than to volume changes of the thyla- koid. These changes may affect the extent of the quenching both by an increase in the local concentration of the nitroaromatir, and by an enhanced rate of excitation exchange among the chlorophylls.

Electrolytes are known to influence the morphology and the functions of chloro- plasts. The electrolyte content affects the configuration of the thylakoids (l-3), their spatial disposition and association into grana (4-7), as well as the ultrastructure of the thylakoid membrane (2, 3). The electrolytes control, also, the distribution of electronic excitation in the pigment populations of photosystems II and I (g-12), the accessibil- ity of the in situ chlorophylls to excitation quenchers (13-15), and they are essential

1 On sabbatical leave of absence from the Uni- versity of Illinois, Department of Food Technol- ogy, Urbana, IL 61801

2 Abbreviations used: Chl, chlorophyll; Tris, tris-hydroxymethylaminomethane cation; DCMU, dichlorophenyl-dimethylurea; ANS, l,%anilino- naphthalene-sulfonate.

for energy-coupled photosynthetic electron transport (16-20). In these phenomena, the divalent metal cations are, in general, 20-50 times more effective than the monovalents, while t’he kind of the associated anion is usually of secondary importance.

Diffusion-dependent fluorescence quench- ing is an appropriate technique to ascertain cation-induced ultrastructural changes in the neighborhood of Chl a in vivo. In solu- tions, according to the Stern-Volmer (21) formulation, the inverse of the fluorescence intensity (proportional to the fluorescence yield) is a linear function of the concentra- tion C of the quencher.

Fo - = 1 + KQC. F

Here, Fo and F are the fluorescence intensi-

134 Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

FLUORESCENCE QUENCHING BY NITR.OAROMATICH 1%

ties in t’he absence and in the presence of the quencher, and K, is the quenching con- stant, which is equal to the product of the collision rate constant (k), the probability of quenching upon collision (y), and the life- time of the unquenched fluorescence (7). An equation, formally similar to the above, applicable to a model in which excitation- exchanging fluorochromes are held on a supporting matrix, and hence they are not free to move, has been derived by Teale (Ref. 22, Eq. 6).

FO -7 E

=l+fKT,

where q is the average number of excitation transfer steps of sequences terminating at energy-trapping centers, K the relative prob- ability of excitation transfer from a fluo- rester to a trap, and T the fraction of the fluorescer molecules, that have become trap- ping centers because of the proximity of a quencher molecule.

We may assume, plausibly, that in a sus- pension of chloropla& the density of the quencher-introduced trapping centers is a function of the concentration of the quencher in the microenvironment of the chlorophylls. This, in turn, depends on the concentration of the continuous aqueous phase, and on the ability of the quencher to penetrate to the pigment bed through the nonchlorophyllous elements of the lamella. In such systems, the apparent, quenching constant K,, which is determined on the basis of the macroscopic concentration of the quencher in the sus- pension, measures the ability of the quencher molecules to penetrate to the pigment bed. In principle, then, conditions that modify the conformation of the lamella will be re- flected in the extent of the quenching, since they may influence both the density of the extrinsic excitation energy traps, and the energy-transfer rate among the chlorophylls.

The present investigation was undertaken as an exploration of the capabilities of the fluorescence-quenching technique for the de- tection of cation-induced ultrastructural changes in photosynthetic membranes. In our experiments, we studied the Chl a fluorescence of isolated chloroplasts and of chloroplast fragments in the absence and in

the presence of added cloctrolytes. To avoid complications from the electron transport- linked quenching of Chl a fluorescence, the quenchers employed are nonreducible nitro- aromatic compounds. Some of these nitro- aromatics, notably nitrobenzenc, behave as hydrophobicity probes, becoming more ef- fective quenchers n-hen the hydrophobic character of the membrane is raised by the addition of metal cations. Apparent quench- ing constants are calculated on the basis of the macroscopic quencher concentration of the suspension. Depending on the partition of the quencher in the aqueous and lipid phases of the suspension, an apparent quenching constant can be larger, equal, or smaller than the true quenching constant.

MATERIALS AND METHODS

Chloroplasts were isolated from fresh market spinach. Thoroughly washed and deveined leaves were blended in cold buffer (400 mM sucrose, 50 mM Tris-HCl, 10 mM NaCl, pH 8.0) and the frac- tion precipitating between 5009 for 1 min and 50009 for 5 min was collected. Sonicated chloro- plasts were prepared from this fraction by a lo- min (total) treatment in an MSE ultrasonic disin- tegrator. To avoid heating, the sonication was carried out intermittently and in ice bath, and the suspension was stirred during the rest periods. This preparation, designated as “chloroplast, sonicate,” was used without further fractionation. In some experiments chloroplasts isolated in 50 rnM Tris-HCl, pH 8.0, were sonicated intermit- tently for 10 min while in ice bath, and then cen- trifuged at 3000g for 10 min; the supernatant fraction, diluted to a final 10 mM Tris-HCl, was used with the designation “chloroplast frag- ments.” The Chl a to Chl b ratio of the crude sonicate and of the chloroplast fragments ranged between 2.05 and 2.20, indicating a pigment com- position of the fragments which is typical of that of the whole chloroplasts.

Chl a was extracted from the unicellular cyano- phyte Anacystis nidulans by methanol, and the concentration of the methanolic solution was determined according to Ozerol and Titus (23).

Fluorescence was excited and measured with an assembled recording spectrofluorometer. The main components of this instrument are a 250.mm Bausch and Lomb monochromator as the ana- lyzing element and a High Intensity Bausch and Lomb monochromator equipped with a 150 W Xe arc as the excitation source. Fluorescence is de- tected by an EMI 9558B photomultiplier tube, whose signal is amplified by a Keithley 150B

136 PAPAGEORGIOU AND ARGOUDELIS

Microvolt Ammeter and recorded by a Varian G-1000 strip chart recorder. Appropriate wide- band Corning glass filters eliminated higher order spectral bands from the excitation light, and red sharp cut-off filters prevented stray excitation from entering the analyzing monochromator. Fluorescence was collected from the same side of the cuvette on which the excitation impinged.

Spectroscopic measurements were carried out with samples poisoned with 10 PM DCMU, and whose 67%nm absorbance was adjusted to 0.2. The poison as well as the high intensity of excitation eliminated all complications that would arise from the induction phenomena. Accordingly, the time-invariant Chl a fluorescence measured was the sum of the “constant yield” and “variable yield” components. Photodestruction of the chlorophylls was prevented by turning the excita- tion beam on the sample for short times (2-3 set) only. New Xe lamps, free from the aperiodic intensity changes of the Xe arc, were used in these experiments, and each sample was measured al- ways relative to a blank minus the quencher. Both elements, then, of the Fe/F ratio corre- spond to the same excitation intensity.

The samples were incubated with the added electrolyte for at least 15 min at room temperature in dim light. This was found necessary for the full expression of cation-induced effect. The nitro- compounds used were either reagent grade, or they were recrystallized by standard procedures. The concentrations of their ethanolic stock solu- tions were corrected spectrophotometrically on the basis of absorptivities given in the literature (24), except for 2,4,6-trinitrobenzoic acid which was corrected titrimetrically. Microliter quanti- ties of the stocks were added directly to the sam- ples whose final ethanol content never exceeded 2yc. Incubation with the added nitroaromatic was found to be unnecessary.

All measurement,s were carried out at room temperature.

RESULTS

Nitrobenzene has been shown to quench the fluorescence of Chl a in solution with Fe/F deviating from linearity in the positive sense as the concentration of the quencher is raised (25). Nonlinear Stern-Volmer plots for nitrobenzene as a quencher are typical also of fragmented lamellae from Chlorella (14), as well as of isolated spinach chloro- plasts and of the spinach chloroplast soni- cate (Fig. 1, curve A). In the presence of added Mg salts, the quenching ability of nitrobenzene for the fluorescence of Chl a in viva is further intensified (Fig. 1, curves B and C). MgCL, Mg(NO&, and MgS04 gave identical results, indicating that the enhancement of the quencher-fluorescer in- teractions is a property of the cation. Mono- valent metal cations, as we have shown before (14), have little or no effect on the nitrobenzene quenching of the fluorescence of Chl a in vivo.

The nonlinear Stern-Volmer plots of Fig. 1 suggest that more than one process con- tribute t’o the extent of the quenching, as the concentration of nitrobenzene in the sus- pension is raised. Although on the strength of the evidence presented here these proc- esses cannot be resolved, we may, neverthe- less, rule out the interference from a purely static mechanism, since the nitroaromatics used in this investigation, even at the high- est concentration employed, had no effect

1.0 0 2 4 6 8 0 2 4 6 8

NITROBENZENE, mM NITROBENZENE, mM

p FIG. 1. Stern-Volmer plots of the quenching of the Chl a fluorescence of spinach chloroplasts and of the chloroplast sonicate by nitrobenzene. The green material was suspended in 400 mM sucrose, 50 mM Tris-HCI, 10 mM NaCI, 10 p~ DCMU, pH 8.0, to a final 676nm absorbance of 0.2. A, without added MgCls ; B, with 0.5 Y MgCls ; C, with 1 M MgClz . Fluorescence excitation, 436 nm. Fluorescence ob- servation, 685 nm; half-band width 13.2 nm.

FLUORESCENCE QUENCHING BY NITROAROMATICS 137

on the absorption spectrum of the spinach chloroplast fragments. Livingston and Be (25) have attributed similarly curved Stern- Volmer plots, obtained with solutions of Chl a, to the superposition of diffusional and of quasi-static quenching. The latt,er case refers to the interaction of proximal Chl u-quencher pairs immediately after the exci- tation, and at times much shorter than allowed by diffusion. The quasi-static mech- anism is a dcfinitc possibility for Chl a in vim, as well, especially since the calculated radius of the sphere within which quasi- static quenching occurs for Chl a and nitro- aroqatics ill, organic solvents ranges from 23 A to 7 A (25). These magnitudes are within the thickness of the chlorophyll- lipid phase of the lamella (26).

The possibility of very fast quencher- fluoresccr encounters raises the prospect of quenching interactions between nitrobcn- zene and Chl b, before the latter has a chance to transfer its excitation to Chl a. If, in addition to the trapping centers in the Chl a population, the quencher can intro- duce trapping centers in the Chl b popula- tion, as well, then photons absorbed by Chl b molecules will have a smaller chance to be reemitted as Chl a fluorescence, than those directly absorbed by Chl a molecules. This is inferred from the fact that, in vim, Chl b transfers all its excit,ation to Chl a, in a unidirectional manner.

The macroscopic manifestation of this mechanism would be a larger apparent

10

a Exc:435nm

6- I3 FI :

quenching constant for Chl b-sensitized Chl a fluorescence, than for directly excited Chl a fluorescence. As Fig. 2 illustrates, however, the nitrobenzene quenching of Chl a fluorescence obevs identical Stern- Volmer plots, both for &rectly excited Chl a (435 nm) and for Chl a sensitized through excitation of Chl 6 (480 nm). The apparent quenching constants obtained from the limit- ing slopes of this particular case are K4d5 =

480 KQ = 180 M-’ without added Mg, and

435 KQ zz K4,8O = 230 ;M+ in the presence of 1 M MgCI,. For clarity, Fig. 2 does not re- produce all the experimental points in the region of low quencher concentrations. Simi- lar apparent quenching constants were ob- tained in scvcral experiments.

This result rules out both ground state and excited state interactions between Chl b and nitrobenzene, that would prevent the former from transferring its electronic exci- tation to Chl a. By reference to the similar chemical structure of these chlorophylls, it further suggests that ground state Chl a is not likely to be affected either. It is note- worthy that Mg2+ raises the Fe/F values throughout the nitrobenzene concentration range, evidencing an increased accessibility of the pigment bed to the quencher, irre- spective of the quenching mechanism by which it becomes apparent. In general, this accessibility is influenced both by the extent of fragmentation of the thylakoids and the salt composition of the suspension medium. Fragmented lamellac in a low ionic-strength

10

8

r-7

Exe: UOnm

6 I3

O-O “OU 0 4 a NITROEENZENE, mM NITROBENZENE. mM

FIG. 2. Stern-Volmer plots of the nitrobenzene quenching of directly excited (435 nm), and of Chl b sensitized (480 nm), Chl a fluorescence of spinach chloroplast fragments. The green material was sus- pended in 10 mM Tris-HCl, 10 PM DCMU, pH 8.0, to a final 676.nm absorbance of 0.2. $, without added MgC12 ; B, with I M MgClt . Other experimental detail as in Fig. 1.

138 PAPAGEORGIOU AND ARGOUDELIS

TABLE I

QUENCHINGCONSTANTSIN M-I FORNITRORENZENE QUENCHING OF THE Chla FLUORESCENCE FROM ISOLATED SPINACH CHLOROPLASTS, CHLORO-

PLAST SONICATE, AND MEMBRANE FRAGMENTS WITHOUTANDWITHADDED MgC12a

Preparation MgClz

0 0.5M 1 Id

Chloroplasts 116 168 180 Chloroplast sonicate 145 175 234 Chloroplast fragments 165 195 230

a Experimental conditions as in Materials and Methods.

‘OOOW 1000

MgC12, mM

FIG. 3. The limiting (C+O) quenching con- stants of the nitrobenzene quenching of Chl a fluorescence as a function of the concentration of the added MgC12. Experimental detail as in Fig. 1.

medium (chloroplast fragments, cf.,Materials and Methods) are more susceptible to quenching than those in a high ionic-strength medium (chloroplast sonicate), and these more susceptible than the unsonicated chlo- roplasts (Table I).

tions of Mg salts and declines in the range of higher concentrations. The cation-induced turbidity rise, like the light-induced one (3), must reflect mostly volume changes of the thylakoids (configurational effect). The en- suing reversal of the turbidity rise at higher salt concentrations must, however, reflect a direct structural change of the membrane (conformational effect), since at those con- centrations the penetration of the quencher to the pigment bed is facilitated, as the en- hanced quenching suggests. Similar effects on the turbidity of chloroplast fragments were observed with SrClz and BaC12, while Ca salts caused t’he membranes to aggre- gate. Contrary to the alkaline earth salt,s, KC1 up to 110 mM had no effect on the 550-nm absorbance of the chloroplast frag- ments.

Figure 3 depicts the dependence of the Livingston and Kc (25) have shown 1,3- apparent nitrobenzene quenching constants dinitrobenzene to quench the fluorescence (calculated from the limiting slopes) on the of Chl a in solution in accordance to a non- concentration of the added Mg salt. Both in linear Stern-Volmer plot, and Teale (22) the unsonicated chloroplasts and in the demonstrated the high efficiency of 1,3-di- chloroplast sonicate these constants begin nitrobenzena quenching of Chl a fluores- to increase when the added salt concentra- cence in the green alga Chlorella pyrenoidosa. tion exceeds 10 rnx. On the other hand, the Very efficient quenching by this compound turbidity of the chloroplast fragments (meas- is demonstrable, also, with isolated spinach ured as the 550-nm absorbance; Fig. 4) rises chloroplasts and with the spinach chloro- rapidly to a maximum at low conccntra- plast sonicate (Fig. 5). On the basis of the

E c

=: z 0.10 Q

0” 0.0%

z fj 0.06

k

0 O.OL

tl f 0.02 2 2 0 -u-v.- Q

0 20 LO 60 80 100 120

ADDED SALT,mM

FIG. 4. Cation-induced turbidity changes of chloroplast fragments suspended in 10 mM Tris- HCl, 10 PM DCMU, pH 8.0. Turbidity was meas- ured with a Bausch and Lomb Spect,ronic 505 spectrophotometer as the difference in 550-nm absorbance between the sample and a blank minus the added electrolyte.

FLUORESCENCE QUESCHIN’G BY NITROAROJIATICS 139

3.0 .- CHLOROPLASTS

0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8

m-DINITROEENZENE, mM m-DINITROBENZENE ,mM

FIG. 5. Stern-Volmer plots of the quenching of the Chl a fluorescence of spinach chloroplasts and of the chloroplast sonicate by 1,3-dinitrobenzene. A (circles), without added MgC$ ; B (squares), with 0.5 M MgClz . Other experimental detail as in Fig. 1.

macroscopic quencher concentration of the suspension, we determined in several experi- ments apparent quenching constants ranging from 2500 M-l to 3100 M-l.

These values are unusually high compared to the quenching constant for methanolic Chl a and 1,3-dinitrobenzene, which we found to be 74 M-‘. Two causes can account for this. First, the random motion of the exciton among the in vivo chlorophylls, because of which its chance to be captured at a trapping center increases; and second, the preferential migration of the nitroaro- matic from the continuous aqueous phase to the lipid phase of the lamella, which raises the local concentration of the quencher. As in the case of nitrobenzene, we were unable with 1,3-dinitrobenzene, also, to establish a clear difference in the apparent quenching constants obtained by excitation at 435 nm (Chl a), and at 450 nm (Chl b). Salts of Mg did not produce the dramatic enhancement in the extent of the quenching by 1,3-di- nitrobenzene, and in some experiments smaller quenching constants were calculated in the presence of high Mg salt.

Several other nitroaromatic compounds were tested and found to quench the Chl a fluorescence of unsonicated and sonicated chloroplasts, and of a methanolic (10 pg/ml) solution giving linear Stern-Volmer plots. Their quenching constants, listed in Table II, indicate that nitroaromatics without ionizable subst’ituents (nitrobenzene, 1,3- dinitrobenzene) are more effective for Chl

TABLE II QUENCHING CONSTANTS Ix A~M-1 FOR THE QUENCH-

ING OF THE FLUOXJWXXE OF Chl a iv T’izlo AND

iu vi’ilro BY VLRIOUS NITKOAROMATICS

Nitroaromatic Chloro- plasts

Nitrobenzene 1,3-Dinitrobenzene 2-Nitrophenol 3-Nitrophenol 4Nitrophenol 2.Nitrobenzoic acid 3-Nitrobenzoic acid 4.Nitrobenzoic acid 4-Nitrobenzene sulfonic

acid 2,4-l)init,robenzoic arid 3,5-IXnit,robenzoic arid

116

3100 25 24

33 15 84

9; 10 I

“1 33 31 4s

2,4,6-Trinitrobenzoic acid; 280 1 276

Chloro- plast soni- cate

chl a in

neth- an01

33 74 61 24 16 35 38

r

1:

a Chl a concentration: 10 pg ml. Other condi- tions as in Materials and Methods.

a in vivo than in solution. Ionizable nitro- aromatics, on the other hand, are almost equally effective in either system. A special case is 4-nitrobenzoic acid, which is excep- tionally insoluble in water. This is a very weak quencher for Chl a in methanol, and a relatively strong quencher for Chl a in vivo.

DISCUSSION

The experiment’s reported here do not provide evidence concerning the topology of

140 PAPAGEORGIOU AND ARGOUDELIS

the photosynthetic units in the lamella; consequently, they must be interpreted in terms of a preexisting model. Several inves- tigators have concluded, recently, that exci- tation is exchanged across interunit bound- aries in a more or less unrestricted manner (27, 28). For the sake of simplicity, then, and since at our experimental conditions all the intrinsic trapping centers (reaction cen- ters of photosystem II) are closed, we adopt here the so-called lake or statistical model, in which the exciton moves freely in a two- dimensional array of chlorophylls.

It is likely that the morphological basis for the statistical model is the layer of por- phyrin rings, which according to Kreutz (26) borders a lipid layer on the inside, and a protein layer on the outside. The latter controls the passage of molecules from the continuous aqueous phase to the lipid- chlorophyll phase of the lamella. The ques- tion now arises whether the extrinsic trap- ping centers are held in fixed positions dur- ing the lifetime of Chl a fluorescence, as Lavorel and Joliot (29) have recently as- sumed, or whether the nitroaromatic can c$ffuse to appreciable distances within the 34 A thick lipid-chlorophyll phase. Taking 1.74 nsec as the lifetime of Chl a fluorescence in vivo (30), and assuming a diffusion con- stant as low as 0.2 X 10e5 cm2 see-‘, we can calculate an average displacement, inoa three-dimensional space, of P = 14 A. Actually, the diffusion constant of nitro- benzene can be larger (D = 1.8 X 1O-5 cm2 see-’ in benzene; Ref. 31) and this would lead to larger displacements of the trapping centers during the lifetime of Chl a fluorescence. These considerations lend support to the hypothesis of a diffusion- dependent quenching, at low concentrations of the quencher, and of an additional quasi- static process, at the range of higher quencher concentrations.

Two causes can account for the large ap- parent quenching constants observed with the chloroplastic material. First, the random walk of the exciton, which enhances its probability to be captured at a trapping center, and second, the preferential migra- tion of the nitroaromatics to the lipid- chlorophyll phase of the membranes. The last process is born out by the fact that

nitroaromatics that are slightly soluble in water are stronger quenchers of the fluores- cence of Chl a in viva, than water-soluble nitroaromatics. In general, nitroaromatics substituted with ionizable groups are about equally good quenchers for Chl a in vitro and in viva, but 4-nitrobenzoic acid, which is unusually insoluble in water compared to its isomers, is a strong quencher in vivo and a weak quencher in vitro (cf. Table II).

Metal cations have been shown to aug- ment the hydrophobicity of the chloroplas- tic membranes and to promote their associa- tions by hydrophobic interactions (7). Thus, they are essential in cementing the indi- vidual thylakoids into grana formations, and they enhance the fluorescence of the membrane polarity probe ANS. The dra- matic increase in the quenching efficiency of nitrobenzene when Mg2+ is added to the membrane suspension must also be attrib- uted to an increase of hydrophobicity, prob- ably as a result of a lowering of the mem- brane bound negative charge. On the other hand, 1,3-dinitrobenzene is selectively con- centrated in the lipid phases of the mem- brane to such an extent that Mg2+ has little or no effect on its quenching rate. As in respect to several other variables (cf. Intro- duction), the divalent alkaline earth cations are more effective in enhancing the quench- ing interactions between Chl a in vivo and nitrobenzene than the monovalent metal cations.

Characteristically, the divalent metal ca- tions promote the fluorescence quenching by nitrobenzene only at elevated concentra- tions, i.e., in the range that corresponds to the descending portion of the turbidity curve (Figs. 3 and 4). Since it is known that the turbidity increase reflects mostly a decrease in the osmotic volume (3), it appears that this is not important for the enhancement of the quenching rate. On the other hand, the ultrastructural changes brought about at higher metal cation concentrations make the membrane suspension less turbid and enhance the rate of quencher-fluorescer encounters.

These changes may enhance the quench- ing by nitroaromatics not only because of the increased membrane hydrophobicity, but also because of an orientation-depend-

FLUORESCENCE QUENCHING BY ?JITROAROMATICS 141

ent increase in the lifetime of Chl a fluores- cence. It is apparent that the second mecha- nism should manifest itself irrespective of the nature of the quencher used. Since, however, this is not justified in the case of 1,3-dinitrobenzene (cf. Fig. 5), we consider the cation-enhanced hydrophobicity of the membrane as the predominant cause for the increased quenching. Murakami and Packer (7) have shown that the hydrophobicity of the chloroplastic membranes increases when t’he membrane-bound negative charge is lowered by acidification. We believe that the metal divalent cations behave in a similar fashion. The larger concentrations required can be attribut’ed to the higher dissociat’ion constants of the corresponding carboxylic acid salts.

Livingston and co-workers (25, 32) found that compounds with electron-poor aro- matic rings and oxidizing agents (nitroaro- matics, quinones) are strong quenchers of excited chlorophylls and related porphyrins. On the other hand, reducing agents and aro- matics substituted with electron-donating groups were found to be poor quenchers. Fork and Amesz (32) also reported a direct, electron transport-independent, quenching of the fluorescence of Chl a i?L vivo by various quinones. The oxidizing nature of these quenchers suggests that the quenching of excited Chl a may have a charge transfer character. Gout’erman and Stevenson (34) observed charge-transfer bands in the spcc- tra of chlorophylls dissolved in nitrobcnzene n-hm 1,3,5-trinitrobenzenc was added, and Tealr (22) invoked ground-state nonfluo- resting complexes of 1,3-dinitrobenzene and chlorophylls in viuo, on the basis of the negative temperature coefficient of t’he quenching process. On the other hand, Whitt#en et al. (35) found 110 evidence for such complexes between metal etioporphy- rins and mononitroaromatic compounds.

Ground-state complexes between nit’ro- aromatics and the chlorophylls in uivo, that dissipate their excitation by a nonradiative route, arc unlikely in the light of our results. On t#he basis of their molecular structure, wc would expect a very similar response of either Chl a or Chl b toward a given nitro- aromatic. If ground-st,at’c complexes existed, ~vc would expect a stronger quenching for

the Chl b-sensitized Chl a fluorescence, than for directly excited Chl a fluorescence. This, however, is not borne out by our results (cf. Fig. 2). In addition, the absorption spectrum of chloroplast fragments does not change in the presence of the nit8roaromatics of Table II. We consider, therefore, the quenching imeraction of these nitroaro- matics with the i?z vivo Chl a to be strictly of the dynamic type, in which the quencher recognizes only the excited population of the fluorcscer.

ACKNOWLEDGMENTS The authors thank Miss Joan Isaakidou for

technical assistance in some of the experiments. Thanks are also due to Drs. G. Ako-unoglou and Govindjee for a critical reading of the manuscript.

1.

2.

3.

4. 5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

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