18554
J. Phys. Chem. 1996, 100, 18554-18561
Stability of Chlorophyll a at the Gas-Water Interface in Pure and Mixed Monolayers. An Evaluation of Interfacial pH C. Mingotaud,*,† J.-P. Chauvet,‡ and L. K. Patterson§ Centre de Recherche Paul Pascal-C.N.R.S., AVenue Schweitzer, 33600 Pessac, France, Ste´ re´ ochimie et Interactions Mole´ culaires, Ecole Normale Supe´ rieure, 46 alle´ e d’Italie, 69364 Lyon Cedex 07, France, and Radiation Laboratory and Department of Chemistry, UniVersity of Notre Dame, Notre Dame, Indiana 46556 ReceiVed: February 29, 1996; In Final Form: July 8, 1996X
The stability of chlorophyll a, Chl a, in neat and mixed lipid monolayers at the nitrogen-water interface has been measured using Langmuir trough and HPLC techniques. In neat monolayers, at subphase pH less than 8, Chl a degrades rapidly to produce pheophytin a, Phe a. Above this pH, Chl a appears to hydrolyze, giving a new product, Chl a hyd, spectroscopically similar to the parent compound. This latter process appears to involve alteration at the pentanone ring peripheral to the porphyrin structure. For mixed monolayer studies, four oleoyl-bearing lipids with headgroups differing in charge have been used to explore the influence of such headgroups on these two pH dependent processes. Consistent with a mechanism by which a negatively charged headgroup may enhance H3O+ concentration in the monolayer region, it has been shown that L-Rdioleoylphosphatidyl-DL-glycerol markedly increases pheophytinization while eliminating the formation of Chl a hyd. By contrast, positively charged dioleoyl-N-(3-trimethylammoniopropyl)carbamoylglycerol serves to generate formation of Chl a hyd with no production of Phe a. In neutral but polar 1,2-dioleoyl-sn-glycerol monolayers, product yields were observed that are consistent with the larger acidity in the region of Chl a. Finally, L-R-dioleoylphosphatidylcholine, which bears a zwitterionic headgroup, significantly enhanced apparent Chl a hydrolysis. These results are interpreted in terms of alterations by the host lipid in pH local to Chl a.
Introduction The chemical stability of chlorophyll a, Chl a, has been widely studied, and a number of well-defined degradation processes have been reported, especially in homogeneous solution. However, with several reactive sites available on the molecule, the course of reaction may depend on specific interactions with surrounding media and yield results difficult to predict a priori. This can be especially true for anisotropic media in which all sites of activity do not experience the same microenvironment. It has been shown that the central magnesium ion may easily be displaced by even weak acid to produce pheophytin a,1 Phe a, a compound readily monitored by UV-visible spectroscopy2 or HPLC analysis.3 By contrast, hydrolysis processes can occur through reaction with a basic reagent in organic solvent.4,5 Scission can take place at the ester group connecting the phytyl chain to yield chlorophyllide, while a more complete hydrolysis can occur involving both ester functions and rupture of the cyclopentanone ring. This latter process leads to the formation of phyllin e6.6 Hydroxide anions or other efficient basic reagents can also abstract the hydrogen at C10, the site for initiation of the chlorophyll allomerization process, to create an enolate form of the ketone function located on the cyclopentanone ring. Since chlorophyll is found naturally in anisotropic and even organized environments, it is of interest to define the extent to which such media can influence the course of acid-base processes. A good model system in which to examine the effects of molecular organization on such reactions is the spread monolayer at the gas-water interface, which can bring to bear some of the same microenvironmental factors experienced by chlorophyll in complex biological media. Furthermore, it is a †
Centre de Recherche Paul Pascal-C.N.R.S. Ecole Normale Supe´rieure. § University of Notre Dame. X Abstract published in AdVance ACS Abstracts, October 1, 1996. ‡
S0022-3654(96)00650-8 CCC: $12.00
system in which the degree of molecular organization can be both monitored and controlled. Indeed, monolayer systems have been widely utilized for chlorophyll studies, and among such studies some work is to be found relating chlorophyll stability to acidity. Hughes, in 1936, reported an increase in the surface potential when the aqueous subphase pH was decreased from 7.5 to 6.7 This behavior was confirmed by A. E. Alexander8 and attributed to Phe a formation at pH lower than 6. Further confirmation of this transformation was provided by Colmano using UV-visible spectroscopy on material removed from the water surface.9 A kinetic study of pheophytinization was carried out by Rosoff and Aron10 through the monitoring of changes in chlorophyll surface pressure after spreading on an aqueous subphase. They reported a rate with a first-order dependence on subphase H3O+ and also a decrease in reaction rate with surface pressure. Most importantly, they reported a 13-fold increase in the rate of pheophytinization in a pure monolayer when compared to reaction in an acetone-water mixture and suggested some influence on the local proton concentration generated by the dipole orientation of chlorophyll in the interface. Though these several studies have been reported for pheophytinization in monolayers, no work has been reported concerning the effects of a basic subphase on the stability of chlorophyll in spread monolayers. In this present investigation, the stability of chlorophyll in neat monolayers has been examined over the pH range 3-11. In addition to the formation of Phe a at pH lower than 8, we have observed the formation of a new base dependent species at pH greater than 8. Both thermodynamic and HPLC measurements confirm the presence of this compound at the gas-water interface. Yields for production of this molecule are shown to be sensitive to conditions of pressure. Here, we have also examined lipid headgroup effects on the acid-base behavior of Chl a in spread monolayers of various © 1996 American Chemical Society
Stability of Chlorophyll a
J. Phys. Chem., Vol. 100, No. 47, 1996 18555
dioleoyl lipids. It is found that the extent of acid-base processes is highly influenced by the nature of the headgroup and that, even for neutral or zwitterionic headgroups, there is significant effect on the course of reaction. This behavior may be explained by the influence of the headgroup structure on interfacial pH.
Experimental Section All monolayers were prepared on a Teflon Langmuir trough previously described.11 The trough was enclosed in a thermostatically controlled chamber at 22 ( 0.5 °C under an atmosphere of nitrogen at 80% relative humidity. The spreading solvent throughout was chloroform (high purity, BurdickJackson). A Millipore system was utilized to purify the water for these experiments. Water resistivity in excess of 16 MΩ cm-1 was achieved in this fashion. The pH of this water was measured before each experiment. Buffer materials (phosphate, borate, and citrate) for control of pH were purchased from either Aldrich or Merck. Except for dioleoyl-N-(3-trimethylammoniopropyl)carbamoylglycerol (the chloride) (DOCA), the lipids employed heresL-R-dioleoylphosphatidylcholine (DOL), L-Rdioleoylphosphatidyl-DL-glycerol (the sodium salt) (DPG), and 1,2-dioleoyl-sn-glycerol (DOG)swere purchased from Avanti Polar Lipids (98 or 99% purity) and used without further purification. A chromatographically pure sample of DOCA was graciously supplied for this work by Dr. A.-F. Mingotaud. The details of its synthesis are described elsewhere.12 Chl a was obtained by extraction from spinach leaves and purified according to a literature procedure.13 The purity was monitored by UV-visible spectroscopy and by HPLC analysis described below. Scheme for Measurement of Chl a Product Yields from Monolayers. Measured volumes of millimolar Chl a or Chl a/ lipid mixtures in chloroform were spread on the subphase of the trough to produce the onset of positive surface pressure. The area per molecule was determined with an accuracy of 0.5%. The pH of the subphase in these experiments was controlled to within 0.05 units. Twenty minutes after spreading, the monolayer was rapidly compressed during a period of less than a minute. A Pasteur pipet was used to aspirate the film into a decanting vial. The aqueous phase in the vial was extracted 3 times with dichloromethane, which was subsequently evaporated. The residue was then dissolved in about 0.7 mL of benzene for analysis by HPLC. Control experiments (Chl a in dichloromethane solution aspirated, shaked with water, and extracted as described above) show no degradation of the pigment during such procedure. Analysis of Monolayer Materials by HPLC. Two different instrumental arrangements have been used for HPLC analysis. (a) One arrangement consisted of an LKB instrumental system equipped with a 2152 monitor and dual 2150 flow pumps. The column employed was an inverse phase LiChrosorb RP-18, (10 mm, 4 × 250 mm). The detector was a variable wavelength UV-visible detector (Model KRATOS Spectroflow 757). A wavelength of 380 nm was used to detect Chl a and monolayer products. (b) Another arrangement consisted of a Waters 510 pump connected to a type 4 µm Nova-Pak C-18 cartridge and to a 990 photodiode array. Pure methanol was used for the carrier phase, and flow rates of 1.0 mL/min were employed to separate peaks from Chl a, Phe a, and Chl a hyd.
Figure 1. Isotherms taken at the nitrogen-water interface for Chl a spread on buffered solution in which measurements were begun 20 min after spreading: (a) pH 8.0, (b) pH 3.4, (c) pH 11.0. The compression speed was ca. 5 Å2/molecule/min.
Results and Discussion I. Neat Monolayers of Chl a. A. Thermodynamic Studies. Surface pressure-area isotherms for chlorophyll monolayers on buffer at pH values of 3.4, 8.0, and 11.0 are presented in Figure 1. In each case, compression was started 20 min after spreading. The subphase ionic strength was 1 × 10-3 mol L-1 for these measurements. It may be seen in accord with literature reports that at lower pH, the curve contracts toward a smaller area per molecule as pheophytinization occurs. By contrast, the isotherm taken at pH 11.0 shows an increased area per molecule at comparable surface pressures. Most interestingly, one may observe that the collapse pressures are quite different for the three curves. This may be taken as an indication of different species at the interface, each exhibiting a different hydrophilic character. A survey of the literature over the past 50 years concerning collapse pressures for monolayers of pheophytin and chlorophyll provides ranges of values for Chl a of 27-29 mN/m and for Phe a of 19-20 mN/m.8,10,14-18 There appears to be no collapse pressure reported for Chl a on a subphase of pH higher than 9. To obtain a more complete characterization of this parameter and its dependence on subphase pH, a number of isotherms have been measured on subphases with pH between 3 and 11. A plot of the collapse pressure as a function of subphase pH is presented in Figure 2. The results indicate several distinct regions that one may interpret in terms of different species resident on the surface. For pH less than 5, the monolayer contains only Phe a (as will be shown below) and the collapse pressure is constant (ca. 19 mN/m) over this region. The removal of the Mg2+ center and protonation of porphyrin nitrogen sites produce a less polar headgroup, corresponding to this collapse pressure. In the pH region of 5-8, there is a transition to a second plateau, which may be seen between pH 8 and 10. This is consistent with a region of chlorophyll stability. However, above pH 10 there is a second increase in collapse pressure, which suggests the formation of another product at the expense of chlorophyll. This collapse pressure suggests the appearance of a product with a significantly higher polarity than for the parent compound. Such a plot is useful not only for predicting that reactions of Chl a take place but also for marking out the regions of stability to be expected for both chlorophyll and products.
18556 J. Phys. Chem., Vol. 100, No. 47, 1996
Figure 2. Collapse pressures (Π) for surface pressure-area isotherms taken from monolayers of Chl a spread on buffered subphase as a function of pH. Experimental conditions are those for data in Figure 1.
B. Measurements by HPLC. To determine quantitatively the Chl a monolayer composition at various subphase pH values, HPLC analyses were carried out. Procedures for HPLC measurements of photosynthetic pigments are well established.3,19-21 The conditions used here have been described above. Three chromatograms characteristic of these measurements are given in Figure 3. The first, Figure 3a, provides a reference chromatogram for pure Chl a taken from solution. It exhibits a principal peak at 25.2 min under a liquid phase flow rate of 0.3 mL/min (at the flow rate of 1 mL/min used throughout this study, this peak moves to 8.4 min). Two small peaks, one on either side of the main band, may be seen that are consistent with the findings of Schaber et al.21 who assigned such bands to 10-hydroxychlorophyll a and chlorophyll a′. Attempts to further purify Chl a led to little improvement over the purity (>95%) seen here. Figure 3b is the chromatogram for material gathered from the aqueous surface when the subphase pH was adjusted to 4.4. The appearance of a peak at
(a)
(b)
Mingotaud et al. 32.4 min is consistent with the presence of a compound less polar than Chl a. The spectrum of this derivative in methanol has been recorded using the HPLC spectral detector in the range 350-550 nm. This spectrum is consistent with that of Phe a. One may further note that HPLC analysis of pure pheophytin gave the same retention time. Finally, material gathered from the surface of a subphase at a pH of 10.8 exhibits not only the parent Chl a peak but also a new one at 6.4 min, which is assigned to the new base dependent product Chl a hyd (see Figure 3c). This product exhibits an absorption spectrum almost identical with that of Chl a. This spectral behavior suggests that the metal center of the compound remains intact, while the position of the new band in the chromatogram indicates generation of a compound more polar than Chl a. HPLC studies were carried out on products of chlorophyll reactions in various solutions (pure methanol in contact with air, degassed THF mixed with pyridine, degassed THF with KOH or saturated with LiOH, etc.) according to literature procedures.26 No peak corresponding to Chl a hyd was obtained, suggesting that the compound produced here is unique to the interfacial environment. Preliminary experiments were carried out to identify the structure of Chl a hyd. Monolayers containing the product have been transferred onto solid substrate by the Langmuir-Blodgett technique and analyzed by FT-IR, NMR, and mass spectroscopy. Together, the results of these measurements point to a reactive site at the cyclopentanone ring of Chl a. However, the small quantities of material that can be gathered and the product stability with time require a substantial effort, currently underway, to obtain a definitive structure of Chl a hyd. Monolayers of pure Chl a were spread on buffers at various pHs and then analyzed according to the procedure described above. Measurements were carried out in the pH range 3.410.8 at a constant ionic strength of 1 × 10-3 mol L-1. The overall evolution of monolayer composition as a function of subphase pH is reported in Figure 4. The data shown have been obtained through the calculation of the relative areas of each of the three main peaks corresponding respectively to compounds Chl a hyd, Chl a, and Phe a. Figure 4 shows that the Chl a
(c)
Figure 3. HPLC chromatograms for Chl a and products of reaction on buffered subphase: (a) pure Chl a taken at a liquid phase flow rate of 0.3 mL/min; (b) pure Phe a resulting from complete Chl a transformation on a buffered subphase at pH 4.4 in which the liquid phase flow rate was 1.0 mL/min and the arrow indicates the retention time for Chl a under the same conditions; (c) mixture collected from a Chl a monolayer on a buffered subphase at pH 10.8 in which the liquid phase flow rate was 1.0 mL/min and the arrow indicates the retention time of the product Chl a hyd.
Stability of Chlorophyll a
Figure 4. Composition of Chl a and products collected from Chl a monolayers on buffered subphases (ionic strength ) 1 × 10-3 mol L-1) as functions of subphase pH. The area per molecule was that required for the onset of positive surface pressure, and surface materials were gathered 20 min after spreading.
J. Phys. Chem., Vol. 100, No. 47, 1996 18557
Figure 6. Molecular structures of the lipids used in this study.
Figure 7. Surface pressure-area isotherms for pure lipid monolayers on pure water. The compression speed was ca. 5 Å2/molecule/min. Figure 5. Effect of compression on generation of Chl a hyd in spread monolayers of pure Chl a over a subphase of pH 10.8 at an ionic strength of 2.6 × 10-3 mol L-1 adjusted by addition of NaCl or NaNO3. Area per molecule is expressed in Å2/molecule.
stability is maximum at pH 7.5-7.8. Already at pH 8, a small amount of Chl a hyd is generated. Although Phe a formation appears to be almost complete at pH lower than 4, a yield of only 50% Chl a hyd is found at the highest pH investigated. Such yield describes the composition of the monolayer 20 min after spreading. Complementary experiments show a larger amount of Chl a hyd when the delay between spreading and analysis of the monolayer is increased. Finally, the dependence of chlorophyll stability on subphase pH, both for basic as well as for acid media, would suggest its potential utility to monitor pH in the vicinity of Chl a reactive sites. A study of chlorophyll stability in lipid monolayers and the correlation of this stability to local pH has been carried out and is reported below. C. Effect of Monolayer Compression on the Reaction Chl a f Chl a hyd. The yield of Chl a hyd as a function of surface pressure is shown in Figure 5. Only small changes in Chl a hyd yield are observed at molecular areas exceeding 200 Å2/ molecule. Because of this, most experiments throughout this study were conducted in this area range. At smaller areas, there is a 50% increase until a surface pressure of about 3 mN/m (or an area of 125 Å2/ molecule) is reached. With further increase in surface pressure, the yield drops markedly. Although a decrease in reactivity with pressure is observed in various
systems,22,23 including pheophytinization,10,24 the enhanced reactivity about a pressure of 3 mN/m is unique in monomolecular films at the gas-water interface. Since the mean planar area of the chlorophyll ring is about 225 Å2/molecule, compression to smaller areas requires an orientation of the porphyrin ring structure that is not parallel to the water surface. Such reorientation, which can change interactions of the polar Chl a groups with the surface, may increase the local pH over a very narrow range of molecular area before further compression restricts accessibility of OH- to reactive sites. II. Mixed Monolayers of Chl a and Lipid. To examine the role of headgroup structure in altering the stability of Chl a, four lipids were selected, each of which exhibits different charge character. The structures of these compounds are given in Figure 6. Additionally, each of these lipids includes oleoyl ester moieties in the 1- and 2-glycerol positions. As may be seen from their surface pressure-area isotherms, each exhibits a liquid-compressed phase without transition to a solid-like packing (see Figure 7). This aspect of the lipid structure ensures that the monolayer will not undergo reorganization to form isolated Chl a domains.27 The Chl a stability in each of the mixed systems was measured on a pure water subphase as a function of chlorophyll/lipid mole percent, and the results are given below. A. Mixed Spread Monolayers of DOG/Chl a on Pure Water. Yields of Chl a, Phe a, and Chl a hyd (expressed in area percent from chromatograms) are presented as functions of DOG/Chl a composition in the monolayer spread on pure water. These
18558 J. Phys. Chem., Vol. 100, No. 47, 1996
(a)
Mingotaud et al.
(b)
(c)
(d)
Figure 8. Analysis of the composition of Chl a/lipid mixed monolayers 20 min after spreading on pure water at the onset of surface pressure as a function of lipid mole percent: (a) DOG; (b) DOL; (c) DOCA; (d) DPG. The subphase pH was 5.5 except for DOL where pH was 7.
measurements were carried out under the conditions used for neat monolayers. The results are presented in Figure 8a. At lipid mole percent less than 50, little effect of the lipid is observed. Above this point, however, the composition of the layer moves strongly toward production of Phe a at the expense of Chl a and Chl a hyd until one may, at very low Chl a content, extrapolate to a 95+% pheophytin yield. Such behavior strongly suggests that though the lipid is neutral, the polar character of the headgroup exerts considerable influence on the structure of aqueous domains local to the monolayer and/or on the organization of the Chl a itself. The data are consistent with an enhancement of acidity by DOG in the microenvironment of Chl a at the lipid interface. B. Mixed Spread Monolayers of DOL/Chl a on Pure Water. Similar measurements have been carried out with DOL as the host lipid. This zwitterionic compound, though also exhibiting a net neutral charge, acts in a fashion opposite to that of DOG. At low lipid mole percent, there is a slow net increase in the yield of Chl a hyd with a corresponding loss of Chl a and Phe a (see Figure 8b). At 70 lipid mol % and above, Chl a disappears quite rapidly with a corresponding sharp rise in the yield of Chl a hyd. Such behavior is consistent with the alignment of headgroups in such a fashion that the ammonium groups face the aqueous subphase and tend to localize OH- in the vicinity of the chlorophyll reactive site. C. Mixed Spread Monolayers of DOCA Chl a and DPG/ Chl a on Pure Water. With the data from DOG and DOL in hand, one would expect a significant enhancement of Chl a hyd yield in the presence of positively charged DOCA, and indeed,
this is the case (see Figure 8c). In mixed monolayers on a pure water subphase at a pH of 5.5, the apparent hydrolysis reaction increases almost monotonically with the addition of DOCA to the layer, while pheophytinization practically disappears at lipid contents above 20%. At high lipid content, the generation of Chl a hyd considerably exceeds that observed for pure chlorophyll monolayer on buffer above pH 11. The most striking behavior, however, appears for DPG layers in which all apparent hydrolysis is lost with 1 mol % lipid and complete pheophytinization occurs at and above 5 mol % (see Figure 8d). Qualitatively, such behavior is to be expected, since the negatively charged headgroup should be capable of markedly altering the balance of OH- and H3O+ toward much lower pH in the microenvironment of Chl a. However, the behavior at low lipid content compared to the other cases considered is quite striking. D. Effect of Lipid Headgroup on Local pH. To provide some quantitative perspective for the effects on Chl a acid-base reactions produced by the lipids in the layer, one may compare the Chl a reaction yields obtained in various lipid/Chl a mixtures to the corresponding yields obtained in pure Chl a layers over buffer. The most direct approach consists of identifying the pH in Figure 4 at which the Chl a/Phe a/Chl a hyd composition corresponds to that from product analysis in lipid/Chl a mixtures (Figure 8) and assigning an equivalent or apparent pH to the effect produced by the lipid. One may then extrapolate to 100% lipid to obtain a value for the maximum lipid effect on Chl a reaction. Such extrapolation is not feasible with either the case of DOCA or that of DPG where, within the constraints of the
Stability of Chlorophyll a
J. Phys. Chem., Vol. 100, No. 47, 1996 18559 a pure Chl a film (at 6 mN/m) than in solution. They suggested that this observed difference could be ascribed to a change in H3O+ concentration local to the chlorophyll layer. The kinetics of pheophytinization in that work were expressed in terms of a first-order dependence on H3O+. If one assumes that the difference in reaction rates between the two media is due principally to an alteration in local pH, one may calculate ∆pH°chlorophyll. The rate of reaction in solution becomes
νo ) ko(H3O+)o
(5)
where (H3O+)o is the concentration of protons in solution (equivalent to the aqueous subphase utilized here). For the monolayer, one may write Figure 9. Evaluation of local pH in mixed monolayers of Chl a and DOG. The ordinates of the data points are determined by plotting each Chl a composition (after product analysis) as the pH of the subphase for a pure Chl a layer giving the same percent of Chl a from product analysis.
ν ) ko(H3O+)m
(6)
with (H3O+)m as the effective concentration of protons. Comparison of the two rates gives, by definition,
∆pH°chlorophyll ) -log((H3O+)/(H3O+)o) ) -log(ν/νo)
(7)
From the difference of 13 in the rates, one may approximate a value of
∆pH°chlorophyll ≈ -1.1
Figure 10. Definition of ∆pH as evaluated from mixed and pure Chl a monolayer data.
experimental procedures adopted, the chlorophyll has completely disappeared at a lipid mole percent much less than unity. However, with DOG, this type of treatment is possible and is presented in Figure 9. In the region between pure chlorophyll and equimolar mixtures of lipid with Chl a, the apparent pH remains at 5.45 but then decreases almost linearly to 4.25 at 100% of DOG, an apparent pH change (∆pH) of -1.2 units. The definitions of parameters in this approach are provided in Figure 10. The value of ∆pH calculated above represents only the difference between pH local to the pure lipid layer, pHlocal (lipid), and that local to pure Chl a, pHlocal(Chl a):
∆pH ) pHapp (lipid) - pHlocal(Chl a)
(1)
One may define the difference, ∆pH°chlorophyll, between the local pH, pHlocal(Chl a), experienced by the chlorophyll in a pure Chl a layer and the pH of the aqueous subphase, pHsubphase:
∆pH°chlorophyll ) pHlocal(Chl a) - pHsubphase
(2)
Furthermore, introducing ∆pH°lipid as the difference in pH between the lipid interface and subphase,
∆pH°lipid ) pHlocal(lipid) - pHsubphase
(3)
One may then write
∆pH°lipid ) ∆pH + ∆pH°chlorophyll
(4)
Determination of ∆pH°lipid from eq 4 requires a value for ∆pH°chlorophyll, which one may not assume to be zero. However, one may approximate that number from literature data. In the study of pheophytinization by Rosoff and Aron,10 it was shown that the reaction rate at pH 4 was about 13 times more rapid in
(at 6 mN/m)
(8)
However, taking into account the results of Rosoff and Aron at surface pressures of 6 and 16 mN/m and assuming a linear relationship between the rate constant and surface pressure, one may obtain a value of approximately -1.5 for ∆pH°chlorophyll at 0 mN/m. Equation 4, then, gives an apparent pH change along the DOG monolayer relative to the subphase:
∆pH°DOG ) - 2.7
(9)
The sign of this parameter indicates an increased concentration of H3O+ ions local to the DOG monolayer compared to pure water. This is, of course, consistent with the enhanced pheophytinization occurring in presence of DOG. The value of ∆pH°DOG calculated here is in close agreement with that of -2.9 obtained in DOG monolayers using a fluorescent coumarin derivative as a probe of interfacial pH.25 A similar treatment of Chl a/DOL data from experiments with pure water as the subphase leads to a value for ∆pH of +7.2. Consequently, ∆pH°DOL is equal to +5.7. The positive value obtained in this case is consistent with an increased concentration of OH- near Chl a in DOL monolayers. A measure of ∆pH°DOL was also made using the fluorescent coumarin derivative mentioned above.25 By contrast with the results found in DOG monolayers, ∆pH°DOL values obtained by the two methods are completely different, with a value of -4.0 obtained by coumarin probe compared to +5.7 found here. The two molecules, Chl a and coumarin derivative, may be situated differently in the interface, with their reactive sites sampling different ionic environments. Then the difference between ∆pH°DOL values may well reflect the heterogeneity in ionic concentration at the interface, due to the presence of zwitterionic headgroups composed of two opposite charges. Such heterogeneity is expected to be large because of the very low ionic strength of the subphase (i.e., pure water). This approach to the evaluation of local pH assumes that the mechanisms of reaction in a mixed monolayer closely approximate those in pure layers. For example, it is necessary to suppose that generation of products in pure layers does not cause the reaction to deviate from a single first-order process. Additionally, one must assume that the change in rate is due to
18560 J. Phys. Chem., Vol. 100, No. 47, 1996
(a)
Mingotaud et al.
(b)
Figure 11. (a) Stability of Chl a in mixed monolayers with DOL when spread on buffered subphase at various pHs, where the ionic strength was maintained constant at 1 × 10-3 mol L-1: (-) pure Chl a; (b) 40 mol % lipid; (+) 65 mol % lipid; (×) 80 mol % lipid; (O) 90 mol % lipid. Each monolayer was removed for analysis 20 min after spreading. (b) Variation in δpH for these mixtures as a function of subphase pH. Symbols are as in part a.
the presence of the lipid headgroup and not primarily due to a change in interactions among Chl a molecules, which occurs as their concentration is lowered. The behavior of the Chl a/ DOG system in which little change in rate is observed with increasing DOG to 50%, even though the Chl a at the surface is being largely diluted, is consistent with this approximation. Moreover, the interpretation above is based on results taken from neat Chl a on buffered subphases and those obtained from mixed Chl a/lipid monolayers on pure water. To show the validity of this approach, it is also necessary to compare neat chlorophyll and chlorophyll/lipid systems on the same subphase. Again, because of the extremes in their behavior, DOCA and DPG are not good candidates for this type of comparison. On the other hand, Chl a stability in DOL systems is accessible over a rather wide pH range, and this system was chosen for more detailed measurements on buffered subphases. Such measurements provide a basis for comparisons not only between pure Chl a and Chl a/DOL monolayers on a buffered subphase but also between DOL/Chl a layers on buffer and on water. E. Stability of Chl a in DOL/Chl a Mixed Monolayers on Buffered Subphases. In Figure 11a, the stabilities of Chl a for various mixtures with DOL in spread monolayers are given as functions of buffered subphase pH with a constant ionic strength of 1 × 10-3 mol L-1. These curves exhibit some similarities in shape compared with that of Chl a on a pure water subphase. However, the maxima of the curves may be seen to be displaced toward lower pH in a regular fashion with increasing lipid content. At pH values higher than that for maximum Chl a stability, the curves are parallel for all the mixtures considered. At pH values lower than the maximum, all curves tend to converge on the value found in neat Chl a systems at pH 4. This suggests that the pheophytinization mechanism in the presence of DOL obeys a different type of dependence on subphase pH than does the apparent hydrolysis reaction. The consequences of the parallelism observed in the baserelated reaction from the curves as presented in Figure 11b can best be demonstrated by a consideration of the data in terms of a δpH, which may be defined as the difference between the subphase buffer pH for a given Chl a stability in a mixed film and the pH of the subphase buffer for an equivalent Chl a stability in the pure Chl a monolayer. It may be seen in Figure
11b that δpH is constant at pH values above that for maximum Chl a stability. To interpret this behavior, one may consider the two equilibria involving H3O+ in the subphase and at the interface,
H3O+subphase T H3O+mixed film H3O+subphase T H3O+pure Chl film
(10)
from which one may write the equilibrium constants as
Kmixed film ) (H3O+)mixed film/(H3O+)subphase
(11)
Kpure Chl film ) (H3O+)pure Chl film/(H3O+)subphase
(12)
and
to give
δpH ) pHmixed film - pHpure Chl film ) pKmixed film pKpure Chl film (13) Accordingly, the values of pK and, hence, δpH should remain constant over the range of subphase pH. Although the activity coefficients involved are not available, the data as presented in Figure 11b appear to support this analysis in the region of Chl a hyd formation. Once again, utilization of product yields to determine an apparent pH by comparison between pure Chl a and mixed films suggests that the Chl a reactions are similar in both media and that the absolute concentration of products, much higher in the former case than the latter, do not significantly alter the course of reaction. The parallelism in behavior between pure Chl a layers and highly diluted layers tends to confirm this assumption. Using the values taken from Figure 11b, one may plot plateau values of δpH vs DOL mole percent to obtain the curve given in Figure 12. The extrapolated value obtained for Chl a stability at 100% DOL is equivalent to ∆pH defined above for the treatment of Chl a/DOG systems, but on a buffered subphase. Utilization of eq 4 gives a value for ∆pH°DOL of +1.7. This is much less than the value of +5.7 on pure water and indicates
Stability of Chlorophyll a
J. Phys. Chem., Vol. 100, No. 47, 1996 18561 of chlorophyll at very low surface pressure. The results obtained all correlate with the charge character of the headgroups or, in the case of DOG, its dipole character. In the cases of the charged lipids, DPG and DOCA, comparison with pure Chl a layers suggests reaction rates normally associated with extremely high values of H3O+ and OH- local to Chl a. By use of the data from pH dependent behavior of pure Chl a layers, it is possible to suggest some quantitative description of the effects produced by DOG and DOL. The magnitudes of the changes observed on pure water are rather higher than those found on buffer. This reflects the effect of ionic strength on the equilibria between ions in the bulk phase and those at the interface.
Figure 12. Plateau values of δpH taken from Figure 11b and plotted as a function of DOL mole percent with an extrapolation to behavior in a pure DOL layer.
the extent to which increasing ionic strength may diminish the influence of the headgroups. That the value of ∆pH°DOL is positive in both cases is consistent with a dominant role of the ammonium moiety in influencing apparent pH local to the reactive group in Chl a. In the case of DOL, the behavior of pheophytinization follows no such regular pattern. Rather, all pH data including that for pure Chl a converge in Figure 11 at a pH of 4. The underlying reason for this is not wholly clear but may well be related to the geometry of the chlorophyll in relation to its contact with the aqueous phase. One may suggest that the chlorophyll is oriented to the water surface by the pentanone cycle with the macrocycle plane inclined at an angle to the surface.6 Under such conditions, the Mg2+ in the porphyrin ring experiences a different average interaction with the lipid and subphase than does the site of Chl a hyd formation. Summary This work has been directed toward a quantitative measure of Chl a stability in spread monolayers under a carefully controlled set of conditions and toward correlating that stability with pH of the subphase. As anticipated, pheophytinization is observed in the pH range below 8. Above pH 8, it is clear from HPLC measurements that a second product is generated. This behavior has not been reported previously, and the product generated appears to be unique to spread monolayers. The spectral similarity between Chl a and this base product, Chl a hyd, shows that the base-related reaction does not involve rupture of the porphyrin ring. Furthermore, the monolayer behavior of Chl a hyd and the HPLC measurements demonstrate that the phytyl chain is not removed. It would appear that the cyclopentanone ring presents the most likely site for reaction. By use of the two acid-base dependent reactions of Chl a at the nitrogen-water interface, an effort has been made to relate the headgroup structure of four lipids to acid-base reactivity
Acknowledgment. The research described herein was supported by the Office of Basic Energy Sciences of the U.S. Department of Energy. This is Document No. 3478 from the Notre Dame Radiation Laboratory. The sponsorship and support of Rhone-Poulenc S.A. for the work of C.M. in the Radiation Laboratory is gratefully acknowledged. References and Notes (1) Willsta¨tter, R.; Hocheder, F. Ann. Chem. 1907, 354, 205. (2) Goedheer, J. C. In The Chlorophylls; Vernon, L. P., Seely, G. R., Eds.; Academic Press: New York, 1966; p 147. (3) Falkowski, P. G.; Sucher, J. J. Chromatogr. 1981, 213, 349. (4) Brown, S. B.; Houghton, J. D.; Hendry, G. A. F. In Chlorophylls; Scheer, H., Ed.; CRC: Boca Raton, FL, 1991; p 466. (5) Hynninen, P. H. In Chlorophylls; Scheer, H., Ed.; CRC: Boca Raton, FL, 1991; p 945. (6) Seely, G. R. In The Chlorophylls; Vernon, L. P., Seely, G. R., Eds.; Academic Press: New York, 1966; p 21. (7) Hughes, A. Proc. R. Soc. London 1936, A155, 710. (8) Alexander, A. E. J. Chem. Soc. 1937, 1813. (9) Colmano, G. Biochim. Biophys. Acta 1961, 47, 454. (10) Rosoff, M.; Aron, C. J. Phys. Chem. 1965, 69, 21. (11) Vaidyanathan, S.; Patterson, L. K.; Mobius, D. J. Phys. Chem. 1985, 89, 491. (12) Mingotaud, A. F.; Patterson, L. K. J. Colloid Interface Sci. 1993, 157, 135. (13) Strain, H. H.; Svec, W. A. In The Chlorophylls; Vernon, L. P.; Seely, G. R., Eds.; Academic Press: New York, 1966; p 21. (14) Bellamy, W. D.; Gaines, G. L. J.; Tweet, A. G. J. Chem. Phys. 1963, 39, 2528. (15) Heithier, H.; Ballschmiter, K.; Mo¨hwald, H. Photochem. Photobiol. 1983, 37, 201. (16) Turnit, J.; Colmano, G. Biochim. Biophys. Acta 1959, 31, 434. (17) Heithier, H.; Galla, H. J.; Mo¨hwald, H. Z. Naturforsch. 1978, 33, 382. (18) Chapados, C.; Leblanc, R. M. Biophys. Chem. 1983, 17, 211. (19) Yoshiura, M.; Iriyama, K.; Shiraki, M. Chem. Lett. 1978, 103. (20) Iriyama, K.; Yoshiura, M.; Shiraki, M. J. Chromatogr. 1978, 154, 302. (21) Schaber, P. M.; Hunt, J. E.; Dries, R.; Katz, J. J. J. Chromatogr. 1984, 316, 25. (22) Ahmad, J.; Brian Astin, K. Langmuir 1990, 6, 1098. (23) Gaines, G. L. Insoluble monolayers at liquid-gas interfaces; Intersciences Publishers: New York, 1966; p 314. (24) Wang, Y.-M.; Patterson, L. K. J. Phys. Chem., submitted. (25) Mingotaud, C.; Chauvet, J.-P.; Patterson, L. K. Thin Solid Films 1994, 242, 243. (26) Svec, W. A. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978; Vol V, pp 375-383. (27) Agrawal, M. L.; Chauvet, J. P.; Patterson, L. K. J. Phys. Chem. 1985, 89, 2979.
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