KINETICS OF MONOMOLECULAR FILMS OF CHLOROPHYLL a
Acknowledgment. Acknowledgment is made to the donors of the Petroleum Research Fund, administered
21
by the American Chemical Society, for support of this research.
Reaction Kinetics of Monomolecular Films of Chlorophyll a on Aqueous Substrates
by Morton Rosoff and Carl Aron I B M Watson Laboratory, Columbia University, New York, NEW York
(Received February 8 , 1964)
The kinetics at constant area of the monolayer reaction of chlorophyll a to pheophytin a on an acidic substrate were studied. The rate constants were found to depend upon pH, the initial surface pressure, the presence of O2at the interface, and divalent metal ions such as Ca+*and YIg+2in the subphase. The half-life of chlorophyll a at 23' on a pH 4 substrate and at an initial surface pressure of 6 dynes/cm. was about 6 min. Evidence was obtained for the participation of H 2 0 in the reaction and the probable existence of a charged intermediate.
Introduction of chlorophyll spread a t an Film balance aqueous interface afford a unique way of approximating a molecular state of organization and environment which is closer to that present in vivo than the one which exists in simple solutions of photosynthetic substances in organic solvents. In the course of investigating the stability, reproducibility, and physical properties of chlorophyll monol a y e r ~it, ~was found that pheophytin was the primary product of chlorophyll decomposition a t the surface. This work reports on the utilization of the techniques of monolayer reactions to study the kinetics of the conversion of chlorophyll a to pheophytin a in the presence of H + ion. Since the relationship between surface properties and time depends upon the interaction between reactant and product, the properties of mixed monolayers of these substances were also examined.
Experimental The preparation of pigments, the apparatus, arid spreading techniques used in these experiments have
been described elsewhere. Briefly, the chlorophyll a was prepared from fresh spinach leaves by the method of Jacobs, et aL6 Pheophytin was formed from chlorophyll by the addition of HC1 and rechromatographing. The surface balance was of a semiautomatic Wilhelmy type employing a Teflon trough and was sensitive to 0.1 dyne/cm. Surface potentials were measured by means of a Ra226ionizing source and a Keithley 6lOH electrometer. Concentrations of solutions of pigments were obtained from measurements of optical density and previously determined extinction coefficients. Solute was delivered to the substrate surface by means of a Hamilton microliter syringe using benzene as the carrier solvent. Phosphate buffer, M , was used (1) A. E.Alexander, J. Chem. Soc., 1813 (1937). (2) H. J. Trurnit and G. Colmano, Biochem. B w p h y s . Acta, 36, 447 (1958). (3) W. 0.Bellamy, J. L. Gaines. Jr.. and A. G. Tweet, J . Chem. Phys., 39, 2528 (1963). (4) M.Rosoff and C. Aron, hrature, to be published. ( 5 ) E. E. Jacobs, A. E. Vatter, and A. S. Holt, Arch. Biochem. Biophys., 53, 278 (1954).
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MORTONROSOFFAND CARLARON
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for measurements a t pH 8. Conversion to pheophytin was carried out by the addition of about 1 ml. of HC1 to M. the substrate to obtain a concentration of The initial effect of spreading chlorophyll a t constant area on a pH 4 substrate was to elevate both the pressure (-3 dynedcm.) and surface potential (-100 mv.) above that found a t pH 8. The isotherms and surface potentials of pheophytin, however, were unaffected a t pH 4. Since the voltages for pheophytin are almost the same as the initial values for chlorophyll at pH 4, the change in surface pressure was chosen as the more sensitive parameter to follow the conversion rate.
Results Monomolecular Films of Mixture. An equimolar mixture of pheophytin and chlorophyll spread a t the air-water interface a t pH 8 gave n-a isotherms which agree within experimental error with the calculated average curve, based on simple additivity of the two individual monolayers. The individual pressure-area relations as well as that for a mixture are given in Figure 1. If the molecules of the mixed film have no effect on each other, the applicable equation U M * = fiQl*
+
1
AREA PER MOLECULE LJ-
(A')
Figure 1. Surface isotherms of chlorophyll a and pheophytin a. Points are experimental d a t a for a 1:1 mixture; solid line calculated from eq. 1 for a 1: 1 mixture.
The Journal of Physical Chemistry
~ M A= V 12n(fip1 f
fw2)
(2)
where pl and p2 are the apparent surface moments of species 1 and 2 a t U M , and AV is in mv. Outside of experimental error, there were no systematic exceptions to eq. 1 and 2. However, near the collapse points the slopes of the AV-a curves were similar to that of pheophytin alone and may be related to the mechanism of mixed film collapse. From the mixture data it is evident that chlorophyll and pheophytin form a two-dimensional solution in which there is little interference or cooperation between the two molecules in the monolayer, i.e., the excess free energy of mixing is zero. n-a plots for both chlorophyll and pheophytin are nearly linear and can be represented over most of the range by an equation of the form
a=a-ba (3) It follows from eq. 1 that the surface pressure for a mixture is a linear function of composition given by
(1)
flU2"
where UM = the molecular area of the mixture a t surface pressure n,ulTand aZu = the molecular areas of species 1 and 2 a t surface pressure n,andfl andf2 are mole fractions of species 1 and 2 in the mixture, was found to hold over the composition range 5 : 1 to 1:5 for chlorophyll :pheophytin. Similarly, the surface potential,
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AV, of the mixed film assuming no interaction is given by
(4)
where the averages are taken with respect to mole fractions. If the variation of the slopes of the mixture isotherms is not too great, then TM will be a linear fraction of composition. This will be particularly so a t areas close to collapse, where the slopes of the pheophytin and chlorophyll isotherms are nearly identical! I t was found empirically that the collapse pressure of the mixture was a linear function of the collapse pressures of the pure constituents weighted by their respective mole fractions. Using this relationship, 2 or 3% pheophytin was detectable from the experimental collapse pressure of mixed films, and this technique afforded a more sensitive method of estimating pheophytin than spectrometric determinations of films lifted off the subphase. Kinetics of Pheophytinization. Measurements a t constant area of the rate of change of surface pressure with time were carried out for a chlorophyll film spread on an acidic substrate. At pH 4, but not at higher pH values, the rate of pheophytinization was conveniently measurable. Since molecular areas of mixtures of starting material and reaction product were found to be additive, the data were treated using the expression for a pseudounimolecular reaction In
(n
- n,)
=
-k[H+]t
+ constant
where nm is the surface pressure when the reaction proceeds to completion, [H+]is the substrate hydrogen ion
KIKETICS OF
h1030MOLECULAR
FILMS OF
23
CHLOROPHYLL U
concentration, and IC is the specific rate 'constant in min. -l M . -' Rate constants were calculated from the initial slopes of the linear plots. The reliability of the linear relationship found was tested by making up mixed films of 1 : 1 pheophytin-chlorophyll and showing that the slope so obtained agreed closely with that given by pure chlorophyll. The rate constant a t the air-water interface was found to be 1.36 x lo3 min.-' M-' at an initial pressure of about 6 dynes/cm.; i e . , u = 120 i.2/molecule. It was found that increasing the initial pressure to about 16 dynes, cm. produced a marked decrease in the rate constant to a value of 1.43 X lo2 min.-' hi-' as shown in Figure 2 . These results suggest that the change in orientation of the molecules in the interfacial film is responsible for these different rates and may be related to the availability of the ring magnesium for reaction. The possibility that oxidation of the isocyclic ring (V) may accelerate the rate of removal of magnesium was z environchecked by carrying out the kinetics in an S ment after saturating the substrate and solution with purified Y2. A rate constant of 4.46 X lo2min-' M-' was found; this is about three times slower than that in air. Addition of 0.5% hydroquinone to the substrate in air produced no change in the initial rate, but the extent of the reaction was markedly reduced. When GO2 was bubbled through the substrate and the pH adjusted to 4.0, the rate constant, 9.60 X lo2, was close to the value found in air. The slightly slower rate may be due to O2having been displaced. A series of experiments was carried out to determine the effect of various ions on the rate of pheophytinization. For the addition of C a f 2at a concentration level in the substrate OF J I , there was a 20% decrease in the velocity of the reaction; a t lop2M , the rate decreased by about 35%. Mg+2 ion, however, at a concentration of lop2A', slowed down the reaction rate by somewhat more than one-half.
Discussion The evident uniform mixing and distribution of pheophytin and chlorophyll molecules in the mixed films shed some light on the possible state of association in the surface phase of chlorophyll itself. If it is assumed that chlorophyll exists in the surface as aggregates as well as single molecules, then for eq. 1 to hold, each species must be considered to form a perfect solution. In going from pure chlorophyll to a pheophytin-chlorophyll mixture in the ratio 5 : 1, .it would be expected that a one-sixth dilution of chlorophyll would affect the distribution of the various assumed single and multiplet species and result in an exception to eq. 1. Further, in
'-1
I
0
2
4
6
8
IO 12 14 16 18 2 0 22 24 26 28 TIME (MINUTES)
3
Figure 2. Rate of pheophytin formation on surface a t constant film area: temp. 23"; substrat: p H 4; 0 , u = 120 A.2, initial A = 6 dynes/cm.; 0, u = 99 A.2, initial A = 16 dynes/cm.
view of the random nature of the attack of H + during the conversion of chlorophyll to pheophytin on an acidic substrate, it is difficult to see why the assumed distribution should not be altered and consequently render the application of eq. 1 invalid. Since the mixture rule was found to apply even under the conditions of the kinetic experiments, it would seem that any appreciable association (to within the experimental uncertainty of about 5%) of chlorophyll molecules in the surface into discrete pairs or n-mers is unlikely. It is of interest to compare the rate of pheophytinization found in the two-dimensional state with the values obtained in bulk.6 At a comparable hydrogen ion conAI, and at 25", the specific rate concentration, stant in an acetone-water solution was 1.03 X lo2 min.-' k-'.The rate of conversion to pheophytin in the surface is approximately thirteen times faster. For most reactions carried out at constant surface area there is little deviation between the energies of activation and rate constants in the surface and the values of these for similar reactions in bulk phases.' The deviation that does occur in this case may be attributed to the high concentration of [ H + ] in the subphase resulting from the presence of a negative dipole end of the chlorophyll molecules oriented toward the substrate. The iondipole interaction can also be of importance in lowering the activation energy. (6) S. H. Schanderl, C. 0. Chichester, and G. L. Marsh. J . Org. Chem., 27, 3771 (1962).
( 7 ) J. T . Davies, Adban. Catalysis, 6 , 1 (1954).
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J a n u a r y 1965
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The observed variation of rate constant with surface pressure caused by the reduced accessibility of the reactive group supports the suggestions8 that high pressures below the collapse point exert a protective action on the decompositiori of chlorophyll. This contradicts the findings of Bellamy, et aL3 The possibility of a primary oxidized intermediate is indicated by the results in an oxygen-free atmosphere and also by the results when hydroquinone, a known inhibitor of autoxidation, is added. On this view the rate constant for the unoxidized form at the lower compression is about one-third less than the corresponding value in air. The absence of an effect of COZon the rate of pheophytin formation 111an acidic substrate appears to rule out this variable except insofar as it contributes to the hydrogen ion concentration. Some preliminary measurements have revealed that, even on a substrate buffered at pH 8, elevated temperatures, ie.! 37”, led to rapid pheophytinization. The possibility that water participates in the reaction is suggested, and the kinetic scheme should then include a specific rate constant due to the reaction of the surface chlorophyll molecules with water. An estimate of the velocity of this simultaneous hydrolysis shows that it is slower by about one-third than the reaction
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MORTON ROSOFFAND CARLARON
with hydrogen ion. It is interesting to note in comparison the stability of chlorophyll in bulk solutions of organic solvents such as benzene and carbon tetrachloride, where heating for 25 min. a t 55” produced no change in the visible spectra. These results indicate that care must be taken in preparative procedures to eliminate long exposures of chlorophyll solutions in contact with water, as in heterogeneous two-phase crystallizations. The apparent protection against pheophytinization exhibited particularly by Mg+2may indicate a specific ion effect, as well as competition with H + for sites in the surface. The actual mechanism of replacement of the Mg in the phorbin nucleus by H is still not quite clear. The evidence obtained from initial values that chlorophyll, when placed on a pH 4 substrate, gives an expanded film and more positive surface potentials suggests that the two hydrogen ions enter at different rates, and the first step may be the rapid substitution of one H + to give a singly charged intermediatee8
(8) G. Colmano, Bwchim. Bwphys. Acta, 47, 454 (1961) (9) E.Rabinowitch, “Photosynthesis,” Vol. I, Interscience Publishers, New York, N. Y.,1945,pp. 493,494.
