39
PHEOPHYTIN a AND PHEOPHYTIN b MONOLAYERS
A Comparative Study of Pheophytin a and Pheophytin b Monolayers by W. E. Ditmars, Jr., and Q. Van Winkle Department of Chemistry, The Ohw State University, Columbus, Ohio
@.??lo
(Received February 17,1067)
Monomolecular films of pheophytin a and pheophytin b at air-water interfaces have been prepared and studied. If the pH of the aqueous subphase is less than about 5.0 and if low-intensity diffuse green lighting is used, the films are chemically stable. At higher pH's, the pheophytins undergo a chemical change that appears to be essentially allomerization. Surface pressure and surface potential of the pheophytins as a function of molecular area at 21" have been studied at pH 4.0 and 3.0. Decrease of pH results in an increase of molecular area and of apparent surface moment at given values of surface pressure. The phenomena, and the chemical stabilization, are evidence of salt formation by entry of one or two protons at the center of the chlorin ring. The results are discussed in terms of a simple model. A method is described for analyzing the composition of dilute benzene or ether solutions containing mixtures of chlorophyll a, allomerized chlorophyll a, pheophytin a , and allomerized pheophytin a.
Introduction There is considerable evidence that the chlorophylls, in vioo, exist in the form of oriented noncrystalline
monolayers,' lying at what is essentially an oil-water interface. Monolayer studies of the chlorophylls and their closely related derivatives in vitro provide useful information about their intermolecular interaction properties. In particular, insight can be gained into conditions necessary for chemical stability and the variations of interfacial dipolar characteristics with change of molecular composition and superficial environment. As a surface chemistry problem, study of the chlorophyll and derivative monolayers presents unusual difficulties not only because of their chemical and photochemical instability, but also because of problems of obtaining homogeneously spread films. These problems may, in part, account for the lack of agreement in recent work.$J Qualitative comparison shows differences in surface pressure ( n ) by as much as a factor of 2 for given values of area per molecule ( u ) . Exploratory work in this laboratory on the chemical stability of chlorophyll and pheophytin monolayers is in general agreement with observations made by C ~ l m a n oRosoff ,~ and Aron,2 and Bellamy, et aL3 Our findings differ from those of the latter authors in several respects, however. We find that at air-buffer (pH 8.0-9.0) interfaces, in dim, diffuse green light, homogeneously spread monolayers of chlorophyll a, pheophytin a, and pheophytin b undergo a chemical change that appears to be essentially allomerization (Le., oxidation of the cyclopentanone ring). Allomerization of the pheophytins occurs to the extent of about 75% in 4.5 hr. In the same period, chloro-
phyll a undergoes about 11% allomerization. We found that the pheophytins were stable ( 55% decomposition) for periods of at least 6 hr if the pH of the aqueous phase was equal to or less than 4.5. A comparative study was made of their surface pressures (r) and surface potentials (AV) as a function of molecular area ( u ) . Film pressure (r) and surface potential (AV) measurements were made simultaneously at 21 f 0.5". The only published work found, for the pheophytins under similar experimental conditions, is that of Rosoff and Aron.2 These authors apparently studied only pheophytin a and indicate that the properties are the same at pH 8.0 as at pH 4.0. This is in variance with our observations on chemical stability as a function of pH and with our findings that, at pH 4.0,areas per molecule at given values of .rr are at least 20y0 greater than those reported at pH 8.0.
Experimental Section A . Materials. All solvents and reagents were CP analytical grade. The solvents used in the spectroscopic analyses with determinants were saturated with water and contained dissolved oxygen and carbon dioxide in amounts probably 210-6 M . The distilled water used had a specific resistance of 8 X 106 ohms/cm. Benzene and ether were evaluated as spreading solvent. (1) For example, see: Brookhaven Symposia in Biology, No. 11, "The Photochemical Apparatus-Its Structure and Function," Brookhaven National Laboratory, Upton, N. Y. (Available from the Office of Technical Services, Department of Commerce, Washington 25,D. C.), 1958,J. J. Wolken, p 87; J. A. Bergeron, p 118; R. Sager, p 101. (2) M.Rosoff and C. Aron, J . Phye. Chem., 69,21 (1965). (3) W. D. Bellamy, G. L. Gaines, Jr., and A. G. Tweet, J . Chem. Phys., 39, 2528 (1963). (4) G.Colmano, Biochim. Bwphys. Acta, 47, 454 (1961).
Volume 7.9, Number I
January 1968
40
W. E. DITMARS, JR.,AND Q. VANWINKLE ~~
Table I : Visible Absorption Parameters of the Pheophytins Red molar absorption coeff, Ref
a
fR
x
XR(max),
XE(max),
mfi
mfi
Riiz,
BIR
R(min)
This research Zscheile and Comar Smith and Benitez Holt and Jacobs
5.30 5.14 5.55 5.10
Pheophytin a (mol wt 871), Ethyl Ether 667.0 408.5 2.03 18.1 665.0 401 2.14 18 667 408.5 2.07 666 408.5 2.10 -23
This research Bellamy, Gaines, and Tweet
5.42 4.88
670.2 669
This research Zscheile and Comar Smith and Benitez Holt and Jacobs
3.18 3.27 3.73 3.30
Pheophytin 654.3 653 655 654.8
Pheophytin a, Benzene 414.3 2.03 414 2.06
19.2 16.3
h (mol wt 885.2), Ethyl Ether 433.2 4.89 7.33 433 5.32 8.0 434 5.15 432 5.26 -10
R/505"
mfi
4.52 4.38 4.37 4.44
16.5
4.97 4.65
18.4 19.0
47.9 50
2.97 2.92 2.96 3.0
16.0 16.0
16.2 22.0
17.0
18.0
The ratio is that of the optical densities of the peaks of the main red band and the second green band.
16.6 17.0
But, mfi
51.8
The latter for pheophytin
b occurs a t 522.1 mp.
The latter was adopted since it was found to give more homogeneously spread films. Surface-active impurities derived from the materials used could have given the following probable positive errors in n : 0.05 dyne/cm for 210 A2 2 u 2 155 A2, 0.1 dyne/cm for 150 A2 2 a 2 130 A2, and 0.2 dyne/cm for 125 A2 1 u 2 100 A2. Double recrystallization of the Baker reagent grade phthalate salt, used to prepare the 0.025 M buffer solutions, had no detectable influence on experimental accuracy (see below). Within experimental error, increase or decrease of buffer concentration by a factor of 2, use of a citrate buffer, and use of unbuffered HC1 substrate gave the same results. Crystalline samples of chlorophyll a and chlorophyll b were prepared by a method similar to that of Jacobs, Vatter, and H ~ l t . The ~ pheophytins were prepared by treating acetone solutions of the chlorophylls with degassed 10% (by volume) HC1 solution for 1 min with agitation and then were transferred by washing to ethyl ether or benzene. All operations were carried out in dim, diffuse green light or in total darkness. The purity of the pheophytjns was estimated to be 90% or better, the main impurity being 5 7 % allomerized pheophytin. A comparison of spectral parameters of this work and those reported in the literatureg is given in Table I. The values of the molar extinction coefficients of this work were calculated from the measured optical densities of the pheophytin solutions of known concentration. The accuracies of the concentrations depended on those of the parent chlorophyll solutions. The crystalline chlorophylls were thoroughly desiccated to remove water before solution preparation. Samples were taken from different prepThe Journal of Physical Chemistry
arations of crystals whose spectroscopic parameters agreed to within better than 1%. A detailed comparison (unpublished) of molar extinction coefficients of this work and those available in the literature indicated an absolute purity of 97% or better for the chlorophylls used in this research. B. Monolayer Stability Test Procedure. The monolayer was spread a t an area per molecule (2240 A2) corresponding to pressures less than 0.1 dyne/cm. At the end of a test period, about 57% of the monolayer was compressed onto a large glass plate, collected in a known volume of reagent grade solvent, and analyzed spectroscopically. All of the apparatus (glassware and nickel tongs) used in transferring the monolayer were thoroughly cleaned and wetted with the aqueous substrate used for testing. It was essential that the plate remain completely wetted during the whole time of transfer (5 min); partial drying resulted in the occurrence of allomerization on the plate. C . Correlation of Spectroscopic Data. Spectroscopic measurements, a t 25", were made with a Cary 14 recording spectrophotometer run on automatic slit control. Pheophytinization and allomerization of the chlorophylls are the simplest decomposition processes that can occur under many usual laboratory conditions when water, carbon dioxide, and oxygen are present to (5) E. E. Jacobs, A. E. Vatter, and A. S. Holt, Arch. Biochem. Biophys., 53, 228 (1954). (6) F. P. Zscheile and C. L. Comar, Botan. Caz., 102, 463 (1941); J. H. C. Smith and A. Benitez in "Modern Methods of Plant Analysis," K. Paech and M. V. Traoey, Ed., Vol. 4, Springer-Verlag, Berlin, 1955, p 142; A. S. Holt and E. E. Jacobs, Am. J . Botany, 41, 710 (1954).
PHEOPHYTIN a AND PHEOPHYTIN b MONOLAYERS
41
interact.7 Study of the spectral changes of dilute solutions in instances where decompositionhad occurred suggested that mixtures of pheophytinized and allomerized products can be formed. Using the optical densities of pure solutions with known concentrations of chlorophyll a, pheophytin a, allomerized chlorophyll a, and allomerized pheophytin a, the spectra of solutions in which decornposition had occurred were analyzed by the standard method of determinants. The allomerized chlorophyll a was prepared by the method which Halts used to prepare fraction 2 (the lactone with the expanded isocyclic ring structure). This compound is predominantly formed when water is present. The visible absorption spectrum is the same whether a hydroxyl group or a methoxy group is present on the C-10 atom. Allomerized pheophytin a was obtained by the procedure used to prepare the pheophytins, but using a solution of allomerized chlorophyll a as the starting material. The optical densities and wavelengths used for the four components and values of the denominator determinants are given in Table 11. The optical densities are arrayed in the order which gives the value of the determinant. Wavelengths in the regions of the maxima of the primary red and blue bands were chosen to minimize the effect of small errors. By calculating
Table 11: Optical Density ( d ) Values and Wavelengths for 0.99 X 10-6 M Solutions at 25” and Values of the Denominator Determinants Used for Component Correlation Wavelength ,
w
Chl a
Optical density Allom Allom chl a pheo a
7
Pheo a
A. For Benzene (HzO,Oa, CO,) Solutions 667.5 405.0 427.5 415.0
405.0 662.5 427.5 415.0
0.723 0.569 0.840 0.633
0.226 0.652 0.646 0.907
Determinant A = -96.8686 0.569 0.652 0.696 0.343 0.840 0.646 0.633 0.907
0.282 0.989 0.283 0.720
0.501 0.902 0.465 1 ,088
X 10-8 0.989 0.182 0.283 0.720
0.902 0.329 0.465 1.088
Determinant B = f40.1329 X B. 420 665 415 405 405 415 667.5 420
For Ethyl Ether (HaO, 02,GO?) Solutions 0.796 0.659 0.690 0.688
Determinant C 0.688 0.690 0.477 0.796
0.955 0.221 0.983 0,725
0.347 0,300 0.521 0.902
0.609 0.505 0.882 1 ,022
= +46.11707 X 10-8
0.725 0.983 0.121 0.955
0.902 0.521 0.344 0.347
Determinant D = +39.917798 X 10-3
1.022 0.882 0.525 0.609
the unknown concentrations from two different sets of simultaneous equations, the possibility of spurious correlations is reduced. Use of the method to analyze the changes in pigment samples from numerous tests on decomposition during pigment preparation, storage in solutions, and in monolayers showed that, in the absence of extensive photobleaching, pheophytinization and/or allomerization were the predominant decomposition reactions. An uncertainty of f5% or less was found in the concentrations of the four components, if extensive allomerization had not occurred. Where extensive allomerization was indicated, concentration uncertainties as large as f10% were found, indicating that more than one type of allomerized product had been formed. The percentage decomposition of pheophytin b in the monolayer stability tests was estimated by determining the change in the ratio of the peak optical densities of the main blue band and the blue satellite band. The percentage recovery was estimated from knowledge of the amount spread and comparison of the peak optical densities of the red, blue, and blue satellite bands with those from a solution of known concentration of pure pigment. On the basis of the similarities in the type spectroscopic changes to those caused by transformation of pheophytin a into allomerized pheophytin a, it seems likely that the primary intact-ring product was the allomerized form of pheophytin b. D. Surface Measurement Apparatus and Procedures. The Wilhelmy plate methodg was used to measure surface pressure (precision f0.02 dyne/cm). Microscope cover glasses, slightly etched in hot Haemosol (Meinecke and Co., Inc.) and trisodium phosphate preserved a zero contact angle, whereas tantalum and platinum did not. Etching caused adsorption of the pheophytins on the wet glass at a < 0.1 dyne/cm. To obtain zero contact angle and reproducible adsorption the slide was inserted after the monolayer was spread. The correction for surface concentration, due to adsorption, was determined for each run. Surface potentials (precision i8 mv or better) were measured with both the vibrating disk and the ionizing electrode methods.’O The latter method provided for scanning over the entire surface. To prevent irreversible changes in the film with use of the polonium electrode,“*l2it was held at a distance of 1 cm from the surface during the brief period of automatic recording of (7) For example, E. I. Rabinowitch, “Photosynthesis,” Interscience Publishers, Inc., New York, N. Y., 1945-1956, pp 400, 452, 462, 1773-1774. (8) A. S. Holt, Can. J . Bwchem. Physwl., 36, 439 (1958). (9) Fcy example, W. D. Harkins, “Physical Chemistry of Surface Films, Reinhold Publishing Corp., New York, N. Y., 1952, pp 121-124. (10) For example, A. W. Adamson, “Physical Chemistry of Surfaces,” Interscience Publishers, Inc., New York, N . Y., 1960,p 119. (11) Similar observations have been made for protein monolayers.la (12) J. F. Chambers, Australian J. Chem., 16, 180 (1963).
Volume 71, Number 1 January 1068
42 meaaurements and removed from the surface at other times. The following procedure gave satisfactory results in M obtaining homogeneously spread films. A solution of the pigment was added dropwise in increments of -0.005 cc/lO sec. The drops were distributed evenly over the surface such that the initial total surface area of the film was from 40 to 80 cm2greater than that corresponding to the start of measurable changes of surface pressure. The initial area per molecule was from 230 to 245 A2. A well-spread monolayer showed variations in AT' (surface potential) of h 2 0 mv or less at a < 1.0 dyne/cm and f2 mv at higher compression. These variations were reproducible on decompression and recompression provided that the collapse pressure had not been greatly exceeded. More rapid spreading led to large variations in AT' (A50 to f 150 mv) over the surface that persisted on high compression. With improper spreading, smaller values of a at given u's were observed, which indicated three-dimensional aggregation that was not reversible in the time of measurement (5 hr). Use of a spreading peg (wire, slide, etc.)13 did not, in itself, lessen heterogeneity. Compression and decompression of the films were completely reversible if the area per molecule was not reduced to less than about 85 A2 and if the rate of area change was 9 A2/molecule min or less. Equilibration times of 5-10 min were allowed between area changes corresponding to about 4% of the total area per molecule.
W. E. DITMARS, JR.,AND Q. VAN WINKLE pheophylin a
\
120-
I
0
I
120
100
60
-p H -
4.0
----pH-
3.0
,.--,---,
140
160
I
MO
180
IS
220
Area per moleoule, u,A*.
Figure 1. Film pressure ( ?r, dynes/cm) and surface moment ( p / D , D.) us. area per molecule ( u, A2 per molecule) for pheophytin a monolayers a t air-water (0.025 M phthalate buffer) interfaces; pH 4.0 and 3.0; temperature 21'.
r
1
8.0
1
PheODhYlin b
--- p H = 3 0 -pH =
- 1.4 ,-------
4.0
6.0
-1.3
\ 2.0
0 80
I 100
-1.2
\
\
\
I 120
140
d d 2
-
-----
160
180
0
200
Area per moleoule, u,Aa.
Results The surface pressure and surface potential data are summarized in Figures 1 and 2 and Table 111. The experimental accuracy in area per molecule, for a given value of a, is estimated to be h3%. The average deviation in surface potential was found to be A 8 mv over the whole range of a. Film collapse, as evidenced by decrease of surface pressure and surface potential with time, was observed at compressions several dynes per centimeter above the highest values recorded in the table. The surface potentials are positive in sign. This suggests that the ester groups are anchored in the water phase, with the phytol group and the chlorin head oriented in the air phase. The smaller over-all surface moments of the pheophytin b systems are probably due primarily to the presence of the carbonyl group at the 3 position on the chlorin ring. The average difference at both pH's is about 0.5 Debye unit. The compressibilities, at compressions greater than about 1.0 dyne/cm, are characteristic of liquid expanded films, ranging from 0.08 cm/dyne for pheophytin b at low compressions and pH 3.0 to 0.022 cm/dyne for pheophytin a at high compressions and pH 4.0. Decrease of pH results in an increase of compressibility. The pheophytin b monolayers show greater compresThe Journal of Physical Chemistry
Figure 2. Film pressure ( ?r, dynes/cm) and surface moment ( p / D , D.) us. area per molecule (a, A2 per molecule) for pheophytin b monolayers at air-water (0.025 M phthalate buffer) interfaces; pH 4.0 and 3.0; temperature 21'.
sibility than the pheophytin a films at a given pH, a t compressions above about 1.0 dyne/cm. In the same range, the areas per molecule of the b systems are smaller than those of the a by an average of about 7% at pH 4.0 and 9% at pH 3.0 for the same value of a. Alexande1-1~and later Trurnit and Colmano14 made comparative studies of chlorophyll a and chlorophyll b monolayers. While their results cannot be compared directly with those of this research, there are certain features which are in common. Their work showed that the chlorophyll b films had greater compressibilities than the a films, and, at given values of a, showed smaller values of molecular area. Alexander's work showed that the surface moment of chlorophyll b films was about 0.27 D. less than that of the a films. As shown in Figures 1 and 2 the pheophytins are characterized by a decrease of over-all surface moment (13) A. E. Alexander, J. Chem. Soc., 1813 (1937). (14) For example, H. J. Trurnit, and G. Colmano, Biochim. Bwphys. Acta, 31, 434 (1959).
43
PHEOPHYTIN a AND PHENOPHYTIN b MONOLAYERS Table I11 : Film Pressure (r)and Surface Potential (AV) Values for Pheophytin a and b a t pH 4.0 and 3.0 Pheophytin 5---------p-H --,
100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210
r----pH
3,0-----1
r,
AV,
dynes/cm'
mv
11.04 9.50 8.00 6.56 5.30 4.10 3.05 2.25 1.50 1.05 0.80 0.63 0.50 0.42 0.37 0.30 0.27 0.23 0.20 0.17 0.15
561 553 544 536 527 517 508 497 486 476 464 454 442 431 420 409 398 388 379 369 361
Pheophytin b,-------pH
r^-1 1 -
_ _ _ _ I _ _
Ares/ molecule, A=
--pH
4.0-----
3.0---
4.0------
AV,
r,
AV,
dynes/om
mv
dynes/om
mv
dynes/cm
mv
12.85 10.46 8.25 6.24 4.35 2.80 1.60 1.04 0.68 0.50 0.41 0.36 0.31 0.27 0.24 0.21 0.18 0.16 0.14 0.12 0.10
597 586 573 561 548 535 521 508 492 480 463 450 436 424 412 401 387 377 367 358 349
8.03 6.92 5.83 4.90 4.02 3.30 2.63 2.10 1.64 1.29 1.01 0.83 0.70 0.60 0.50 0.45 0.35 0.30 0.25 0.20
423 415 407 398 390 381 372 364 355 347 339 330 322 314 306 298 290 283 275 269
8.25 6.35 4.90 3.65 2.62 1.84 1.27 0.98 0.75 0.62 0.50 0.40 0.35 0.30 0.29 0.25 0.23 0.18 0.15 0.12 0.10
434 422 410 399 387 377 364 354 342 333 322 313 303 295 286 279 271 263 257 249 244
r,
( p / D ) with decrease of u at compressions above about 1.0 dyne/cm, at which point the compressibility has greatly decreased. Above about 1.0 dyne/cm, p / D decreases linearly with increase of ?r up to the collapse pressure. The pheophytins show an expansion in area per molecule, at a given value of ?r, with decrease of pH. Decrease of pH from 4.0 to 3.0 results in an average increase of 8% in u for ?r > 1.0 dyne/cm. The expansion below 1.0 dyne/cm averages about 13%. Comparison of the data of Bellamy, et u Z . , ~ on pheophytin a at pH 8.0 with the present data at pH 4.0 shows an average expansion of 20% in u at the lower pH for ?r > 1.0 dyne/cm. The average compressibility, in the range of 1.0-11.0 dynes/cm, increases from 0.018 cm/dyne at pl3 8.0 to 0.024 cm/dyne at pH 4.0. Table IV shows the effect of pH on surface moment. While a detailed investigation of the factors affecting monolayer stability has not been made, certain features can be outlined. At pH's less than 5.0, the allomeriaation of the pheophytins is retarded to about 0.7%/hr in periods up to 20 hr. There is indication that other, slower reactions also occur. At pH 8.0, the rate of allomerization of the pheophytins is about lla/o/hr. The corresponding allomeriaation rate for chlorophyll a is about 2.5%/hr. No pheophytin or allomeriaed pheophytin appears to be formed. When the monolayer and a t is under an inert atmosphere (-100 ppm of 02), high pH, the rate of allomerization of chlorophyll decreases to about O.S%/hr. The effect on the pheo-
r,
AV
I
Table IV: Effect of pH on p / D for Pheophytin a *,
P/D,
Ref
PH
dynes/cm
Debye units
Bellamy, et al. Bellamy, et al. This research This research This research This research
8.0 8.0 4.0 4.0 3.0 3.0
1.0 10.0 1.0 10.0 1.o 10.0
1.52 1.41 1.82 1.64 1.96 1.67
phytins has not yet been evaluated. Additives such as p-carotene, lecithin, or phytol in chlorophyll a monolayers appear to have little effect on the rate of decomposition.
Discussion A plausible interpretation of the stabilization of pheophytin monolayers by low pH and the concurrent expansion of the films and increase of p / D with decrease of pH from 8.0 to 4.0 is that salt formation occurs with entry of a least one proton into the center of the ring. On this basis, the further expansion when the pH is decreased from 4.0to 3.0 can be viewed as an increase in the degree of protonation.16 The strong perturbing effect of acid on the visible (15) A. Neuberger and J. Scott [Proc. Roy. SOC.(London), ,4213, 307 (1952) ] present evidence that the closely related porphyrin derivatives can exist in mono- and diprotonated forms.
Volume 72, Number 1 January 1068
44
W. E. DITMARS, JR.,AND Q. VANWINKLE I
1
6 A'
H
-=
pheophytin o in ethyl ether = pheophytin o in ethyl ether sat'd with dry HCI go9
C =cyclopentonone X 370
410
450
490
530
570
610
650
!:.(
590
0%p h y l o l
mlr.
Figure 3. Visible absorption spectra of pheophytin a (10-6 M ) in ethyl ether and in ethyl ether saturated with dry HC1 gas.
absorption spectrum of pheophytin a, shown in Figure 3,16 can also be interpreted as arising from entry of protons into the center of the ring. The effect is reversed upon removal of acid. Addition of protons to the center of the chlorin ring of pheophytin a would tend to produce the same symmetry as that of chlorophyll a. The changes effected by the acid make the pheophytin a spectrum resemble that of chlorophyll a. Strong perturbing effects of acid on the spectrum of allomerized pheophytin a are also observed. Certain features of the pheophytin monolayers can be interpreted on the basis of a simple model. From a scale molecular model based on known atomic radii and bond angles, it was determined that the chlorin head with the ester groups can be approximated by a rigid rectangular parallelepiped, 13.1 X 13.1 X 4.5 A, and the phytol tail by a flexible cylinder with a length of 20 A and a diameter of 5.8 A. With the head and tail folded together and the plane of the head perpendicular to the air-water interface, the smallest excluded area is about 90 A2 (this includes the unoccupiable interstitial area between close-packed molecules). With the ester groups oriented in the water, this would be the smallest observable area per molecule for a monomolecular film of the pure pigment. If the angle of the folded unit, with respect to a normal to the interface, is increased to 42", the smallest excluded area is about 110 A2. The collapse areas in the range 100-110 A2, observed in this work, are consistent with the tilted, folded configuration. If the chlorin head were inverted into the water and both head and tail made perpendicular to the interface, molecular areas of 75 A2 or less would be observed. The sign of the surface potential would probably be reversed. Figure 4 shows an idealized cross-sectional view, in the plane of the interface, of a probable local arrangement of monolayer molecules and their counterions a t an average area per molecule of 130 A2. The rectangles The Journal of Physical Chemistry
1cx
ring
= e s t e r group = b u f f e r ion toil
;:;-I= p h e o p h y t i n
0
molecule
Figure 4. Arrangement of pheophytin molecules at an air-water interface at an average area of 130 A2 per molecule. A plan view of the plane of the interface. Rectangles repiresent the cross section of the chlorin head oriented at 42' to the normal to the interface.
represent cross-sectional projections of the heads, with the angle of tilt taken to be 42" from the normal. The closed circles are projections of the tails, and the dashed circles inside the head areas are projections of the phthalate counterions, considered to lie beneath the heads in the aqueous phase. The units are drawn to scale of the approximate molecular dimensions. The letter C's designate the position of the cyclopentanone ring, and the X's show the position of the ester groups. In addition to the horizontal displacement of alternate rows of molecules as shown in Figure 4, it is visualized that a relative vertical displacement between alternate rows occurs. This would further reduce the net lateral coulombic repulsion interactions. Vertical displacement of a molecule into the surface of the water, due to nearest neighbor repulsions, will be opposed by the work required for hydrophobic inmersion of the hydrocarbon parts of the molecule. Dispersion interactions, which make the main contribution to the effective cohesive pressure, will be large for head-tail and headhead interactions and small for tail-tail interactions. In this closely packed array, kinetic motions of the molecular units about their equilibrium positions and axial rotational motions of the phytol tails will contribute to the thermal pressure. Random wagging motions of the tails will be largely frozen out. The observed small increase of surface moment with increase of area per molecule can be simply interpreted as resulting from a decrease in the degree of relative vertical displacement between neighboring molecules, (16) Similar observations were made for a,P,7,6-tetraphenylchlorin: G. D. Dorough and F. M. Neunnekens, J. Am. Chem. Soc., 74, 3974 (1952).
PHEOPHYTIN a AND PHEOPHYTIN b MONOLAYERS accompanied by a slight increase in separation of some of the heads and their counterions. At film pressures less than about 1.0 dynelcm, pheophytin monolayers are characterized by compressibilities larger than those for cohering liquid films and by small fluctuations in the surface potential over the surface. The consistency and reproducibility of the phenomena make it unlikely that surface contaminants are the cause. A reasonable interpretation is that the films are characterized by coexisting regions of different states of molecular ordering. One type of region would consist, of molecules in the ordered local array suggested in Figure 4, while the other would consist of a more or less random interdispersion of heads and tails, characterized by a significantly increased kinetic motion of the tails. In the more disordered region, intermolecular coulombic repulsions would be less and could thereby result in a further decrease in the degree of relative vertical displacement and an increase in the separation of some of the heads and their counterions. This would account for the variations in surface moment (about *0.06 D. from the average value shown in Figures 1 and 2) over the surface. The areas per molecule where collapse begins to occur are similar for the pheophytin a and b systems. Collapse pressures for the b systems, however, are considerably smaller. From the viewpoint of the proposed model, these phenomena can be related rather simply to the presence of the 3-position carbonyl groups on the pheophytin b head units. As the film is compressed, the molecules approach a close-packed ordered array with increasing degree of relative vertical displacement between alternate rows of molecules. In the vertical staggering arrangement, intermolecular configurations
45
are possible where there are ion-dipole attractions between neighboring positively charged head centers and 3-position carbonyl groups. Increase of degree of relative vertical staggering by compression can increase ion-dipole attractions. These interactions, not present in the pheophytin a monolayers, will contribute to the cohesive pressure and tend to promote relative vertical displacement. Consequently, less compressional work is required to reduce film area and reach the collapse configuration. Increase of angle of tilt of the chlorin heads (with respect to a normal to the interface) with decrease of pH may account in part for both the increase of area where film collapse begins and the relatively small increase of apparent surface moment. At the area of start of film collapse the monolayer molecules are probably in the closest packed state. By the present model, angles of tilt of 31" at pH 4.0 for pheophytin a and b, 37" at pH 3.0 for pheophytin b, and 42" at pH 3.0 for pheophytin a are calculated. If stabilization of the pheophytin monolayers against allomerization results as a consequence of protonation at low pH, as seems likely, the following can be visualized. Protons, in the center of the chlorin ring, can act as strong electron acceptors. Such attraction transmitted to the cyclopentanone ring would decrease its electron density. This would increase the potential barrier in the first step of the allomerization process. Acknowledgments. We take pleasure in acknowledging the assistance of H. F. Blanck, Jr., in development of the determinantal method of analysis. The research work has been generously supported by grants from the National Science Foundation (GB2254) and the National Institute of Health (5R01 GM10856-06).
Volume 7.2,Number 1 January 1868
