RESEARCH NOTE BLACK LIPID MEMBRANES (BLM) CONTAINING CRYSTALLINE CHLOROPHYLL I. CSORBA, J. SZABAD, L. ERDEIand Cs. FAJSZI Hungarian Academy of Sciences, Biological Research Center, Institute of Biophysics, H-6701 Szeged, P.O.B. 521, Hungary (Received 10 October 1914; accepted 10 January 1975)
Earlier many investigations were carried out to characterize the so-called crystalline chlorophyll because this highly ordered form of the main photosynthetic pigments (chlorophyll a and h) was believed to have some structural features in common with the in v i m pigment states (Ke, 1966). Recently an aggregated chlorophyll form absorbing at about 740 nm, where the crystalline chlorophyll has a characteristic absorption band, was observed in intact plants (Thomas et al., 1970; lnoue et ul., 1972). Using artificial thin lipid membranes containing chlorophyll Tien (1968) observed a band at about 745 nm in the photoelectric action spectrum of the membrane. Although the membrane forming conditions seem to meet the requirements needed for the formation of the socalled crystalline chlorophyll, investigations revealing the presence of crystalline chlorophyll in model membranes are lacking. The present work was designed to study the formation of the so-called crystalline chlorophyll in membranes of various compositions.
(Szabad and Csorba, 1973). For these measurements we used a special sample holder in a highly sensitive spcctrophotometer (UNICAM SP18OOB UV spectrophotometer equipped with a n AR 25 recorder-Pye Unicam, Cambridge). For screening the Plateau-Gibbs border, black platinum sheets wcre put into the sample holder and thus only the central bimolecular part of the membrane was examined. Thinning of the membrane was controlled by the extinction changes at 668 and 743 nm. Emulsions. In order to gain insight on the effect of hydration of chlorophyll, 10 p-4" aliquots of the membrane forming solutions were hand shaken in 8 m Y of 0.1 M KCI solution for 5min. Absorption spectra of the light scattering emulsions were taken at the sample position near the photomultiplier of a UNICAM SP1800B UV spectrophotometer immediately after shaking. Studying thc effect of the relative lipid content o n the formation of crystalline chlorophyll, emulsions of different chlorophyll a/ lecithin molar ratios were used. Chlorophyll a and cgg yolk lecithin were prepared chromatographically o n sugar column (Strain and Svec, 1966) and A1,03 column (Singleton et a/., 1965), respectively. All chemicals were analytical reagent grade and were used without further purification. Sugar for column chromatographic separation of chlorophyll a was purchased from commerical sources. For preparing the aq solutions water, twice distilled from an all glass still was used.
MATERIALS AND METHODS
E.xtracts of spinach leaves. For a comparative study the RESULTS AND DISCUSSION BLMs wcrc made from 2 different extracts of spinach leaves. One of the extracts was prepared by the centrifugaThe absorption spectra of the membrane forming tion method of Tien e t al. (1968), and had a low chlorophylljlipid molar ratio of about 0.2 (L-extract), while the solutions which can be characterized by low and high other, prepared by a slightly modified procedure of Strain chlorophyll/lipid molar ratios (L- and H-solutions) and Svec (1966), had a high molar ratio of about 1.8 (H- showed the two main bands in the Soret and red extract). The modification means that after combining the regions. The characteristic so-called crystallinc methanol-petrolcum ether extracts of the boiled leaves in a separatory funnel, 330 m Y additional petroleum ether chlorophyll absorption band, however, could not be (30-40') per 25g leaves was added to the material. The detected in these nonaqueous hydrocarbon solutions. chlorophyll and lipid contents of the extracts were anaThe aqueous emulsion of the L-solution showed the lysed by spectrophotometric and thin layer chroma- same 2 absorption bands as the anhydrous solutions tographic mcthods, respectively. U L M f o r i t i h g solutions and BLM fbrmation. The BLM's did, while in the case of the other emulsion prcpared wcre formed by placing a drop of the 5% (w/v) n-octane from the H-solution, the 743 nm band appeared. The s o h of the air-dry L- and H-extracts (L- and H-solutions) modified H-solution behaved similarly to the on a 2 mm hole of a Teflon septum (Tien, 1968). In order L-solution, i.e. the emulsion made from it did not show to reduce the 1.8 chlorophyll/lipid molar ratio of the H any absorption change at 743 nm, even after repeated solution to 0.2 (thc value of L-solution) phospholipid preshaking with the aqueous solution. In the emulsions pared from egg yolk (Singleton et al., 1965) was added in ii-octane (modified H-solution). The absorption spectra of mixed solutions of chlorophyll a and lecithin the of the membrane forming solutions were measured by a amount of the so-called crystalline chlorophyll inUNICAM SP1800B UV spectrophotometer using a creased very sharply between the molar ratios of I cuvette of variable path length. and 3 reaching a maximal value (Fig. 1). At the value Light ahsorption of' BLM's. Spectral absorbance of the central part of BLM's prepared from the 2 different mem- of I or below, crystalline chlorophyll could not be brane lorming solutions were taken as described elsewhere detected. 377 PAP21
5-
I
I. CSORBA,J. SLABAD, L. ERDEIand Cs. FAJSZI
378
Chlorophyll 0 /lecithin molar ratio
Figure I . Formation of crystalline chlorophyll as a function of chlorophyll ullecithin molar ratio. The amount of crystalline chlorophyll is characterized by the ratio of extinctions at 743 and 668 nm. r -
I
Figure 2. Absorption spectra of BLMs made from the LH- ( ) solutions. The H-solution modified by the addition of phospholipid showed a similar spectrum to that of the L-solution. The spectra were recorded after 1Omin thinning and the membranes had become fully black.
( _ _ . _ _ _ _and )
talline chlorophyll form, provided the chlorophyll/ lipid molar ratio of the membrane forming material is sufficiently high. Such a structural model is in agreement with the results of Jacobs et ul. (1953; 1954) who found that the chlorophyll micrwrystals are built up from bimolecular units, in the formation of which hydration plays an important role. Later, Ballschmiter and Katz (1969) published details on the nature of interaction between chlorophyll and water and found that 2 adjacent porphyrin groups are connected to each other via a water bridge so that the phytyl tails become parallel. Present investigations show that in a composite system containing chlorophylls and lipids the special bonding betwcen chlorophylls, which is necessary for the formation of the crystalline form, can be hindered by the interposition of lipid molecules. This is thought to be a steric effect of the lipid molecules instead of a competition between the lipid and water molecules for the coordination sites in porphyrin ring. The amount of crystalline chlorophyll reaches a saturation value when the chlorophyll/lipid molar ratio is about 3. According to combinatoridl considerations, this value corresponds to a sudden increase in the probability of hexagonal arrangement of the bulk constituent of a two component system, suggesting a similar pattern for the organization of crystalline chlorophyll. Recently, a two-dimensional structure of crystalline ethyl chlorophyllide a was described by Strouse (1974), which is in a good agreement with the former suggestion as well. It may be concluded that the artificial membranes present the possibility of creating appropriate systems for studying the highly organized aggregates of the main photosynthetic pigments, such as the so-called crystalline chlorophyll.
Figure 2 illustrates that only the membranes made from the H-solution showed the crystalline chlorophyll absorption peak a t 743 nm. The spectra of the membranes prepared from the L-solution and modified H-solution d o not show any absorption around 743nm, suggesting that these BLMs do not contain crystalline chlorophyll. The results suggest that the BLM's, which are Acknowledyemerzts~Wcwish to thank Dr. B. Karvaly and accepted as bimolecular layers, may contain the crys- Dr. Agnes Faludi-Daniel for valuable critical discussions.
REFERENCES
Ballschmiter, K. and J. J. Katz (1969) J . Am. Chem. Soc. 91, 2661-2677. Inoue, Y., T. Ogawa and K. Shibata (1972) Plant Cell Physiol. 13, 385--389. Jacobs. E. E., A. S. Holt and E. Rabinowitch (1953) 1.Chem. Phys. 22, 142-143. Jacobs, E. E., A. E. Vatter and A. S. Holt (1954) Arch. Biochrm. Biophys. 53, 228-238. Ke, B. (1966) In The Chlorophylls (Edited by L. P. Vernon and G. R. Seely), pp. 253-279. Academic Press, New York. Singleton, W . S., M. S. Gray, M. L. Brown and J. L. White (1965) J . Am. Oil Chem. Soc. 42, 53-56. Strain, H. H. and W. A. Svcc (1966) In The Chlorophylls (Edited by L. P. Vernon and G. R. Seely), pp. 22-66. Academic Press, New York. Strouse, C. E. (1974) Proc. Natl. Acad. Sci. U.S. 71, 325-328. Szabad, J. and I. Csorba (1973) Scan-Pye Unicam 2, 15-16. Thomas, J., G. P. Phondke, V. G. Tatake and A. R. Gopal-Ayengar (1970) Photochem. Photohiol. 11, 85-92. Tien, H. T. (1968) J. Phys. Chem. 72, 4512-4519. Tien, H. T., W . A. Huemoeller and H. P. Ting (1968) Biochem. Biophys. Res. Commun. 33,207-212.
Earlier many investigations were carried out to characterize the so-called crystalline chlorophyll because this highly ordered form of the main photosynthetic pigments (chlorophyll a and h) was believed to have some structural features in common with the in v i m pigment states (Ke, 1966). Recently an aggregated chlorophyll form absorbing at about 740 nm, where the crystalline chlorophyll has a characteristic absorption band, was observed in intact plants (Thomas et al., 1970; lnoue et ul., 1972). Using artificial thin lipid membranes containing chlorophyll Tien (1968) observed a band at about 745 nm in the photoelectric action spectrum of the membrane. Although the membrane forming conditions seem to meet the requirements needed for the formation of the socalled crystalline chlorophyll, investigations revealing the presence of crystalline chlorophyll in model membranes are lacking. The present work was designed to study the formation of the so-called crystalline chlorophyll in membranes of various compositions.
(Szabad and Csorba, 1973). For these measurements we used a special sample holder in a highly sensitive spcctrophotometer (UNICAM SP18OOB UV spectrophotometer equipped with a n AR 25 recorder-Pye Unicam, Cambridge). For screening the Plateau-Gibbs border, black platinum sheets wcre put into the sample holder and thus only the central bimolecular part of the membrane was examined. Thinning of the membrane was controlled by the extinction changes at 668 and 743 nm. Emulsions. In order to gain insight on the effect of hydration of chlorophyll, 10 p-4" aliquots of the membrane forming solutions were hand shaken in 8 m Y of 0.1 M KCI solution for 5min. Absorption spectra of the light scattering emulsions were taken at the sample position near the photomultiplier of a UNICAM SP1800B UV spectrophotometer immediately after shaking. Studying thc effect of the relative lipid content o n the formation of crystalline chlorophyll, emulsions of different chlorophyll a/ lecithin molar ratios were used. Chlorophyll a and cgg yolk lecithin were prepared chromatographically o n sugar column (Strain and Svec, 1966) and A1,03 column (Singleton et a/., 1965), respectively. All chemicals were analytical reagent grade and were used without further purification. Sugar for column chromatographic separation of chlorophyll a was purchased from commerical sources. For preparing the aq solutions water, twice distilled from an all glass still was used.
MATERIALS AND METHODS
E.xtracts of spinach leaves. For a comparative study the RESULTS AND DISCUSSION BLMs wcrc made from 2 different extracts of spinach leaves. One of the extracts was prepared by the centrifugaThe absorption spectra of the membrane forming tion method of Tien e t al. (1968), and had a low chlorophylljlipid molar ratio of about 0.2 (L-extract), while the solutions which can be characterized by low and high other, prepared by a slightly modified procedure of Strain chlorophyll/lipid molar ratios (L- and H-solutions) and Svec (1966), had a high molar ratio of about 1.8 (H- showed the two main bands in the Soret and red extract). The modification means that after combining the regions. The characteristic so-called crystallinc methanol-petrolcum ether extracts of the boiled leaves in a separatory funnel, 330 m Y additional petroleum ether chlorophyll absorption band, however, could not be (30-40') per 25g leaves was added to the material. The detected in these nonaqueous hydrocarbon solutions. chlorophyll and lipid contents of the extracts were anaThe aqueous emulsion of the L-solution showed the lysed by spectrophotometric and thin layer chroma- same 2 absorption bands as the anhydrous solutions tographic mcthods, respectively. U L M f o r i t i h g solutions and BLM fbrmation. The BLM's did, while in the case of the other emulsion prcpared wcre formed by placing a drop of the 5% (w/v) n-octane from the H-solution, the 743 nm band appeared. The s o h of the air-dry L- and H-extracts (L- and H-solutions) modified H-solution behaved similarly to the on a 2 mm hole of a Teflon septum (Tien, 1968). In order L-solution, i.e. the emulsion made from it did not show to reduce the 1.8 chlorophyll/lipid molar ratio of the H any absorption change at 743 nm, even after repeated solution to 0.2 (thc value of L-solution) phospholipid preshaking with the aqueous solution. In the emulsions pared from egg yolk (Singleton et al., 1965) was added in ii-octane (modified H-solution). The absorption spectra of mixed solutions of chlorophyll a and lecithin the of the membrane forming solutions were measured by a amount of the so-called crystalline chlorophyll inUNICAM SP1800B UV spectrophotometer using a creased very sharply between the molar ratios of I cuvette of variable path length. and 3 reaching a maximal value (Fig. 1). At the value Light ahsorption of' BLM's. Spectral absorbance of the central part of BLM's prepared from the 2 different mem- of I or below, crystalline chlorophyll could not be brane lorming solutions were taken as described elsewhere detected. 377 PAP21
5-
I
I. CSORBA,J. SLABAD, L. ERDEIand Cs. FAJSZI
378
Chlorophyll 0 /lecithin molar ratio
Figure I . Formation of crystalline chlorophyll as a function of chlorophyll ullecithin molar ratio. The amount of crystalline chlorophyll is characterized by the ratio of extinctions at 743 and 668 nm. r -
I
Figure 2. Absorption spectra of BLMs made from the LH- ( ) solutions. The H-solution modified by the addition of phospholipid showed a similar spectrum to that of the L-solution. The spectra were recorded after 1Omin thinning and the membranes had become fully black.
( _ _ . _ _ _ _and )
talline chlorophyll form, provided the chlorophyll/ lipid molar ratio of the membrane forming material is sufficiently high. Such a structural model is in agreement with the results of Jacobs et ul. (1953; 1954) who found that the chlorophyll micrwrystals are built up from bimolecular units, in the formation of which hydration plays an important role. Later, Ballschmiter and Katz (1969) published details on the nature of interaction between chlorophyll and water and found that 2 adjacent porphyrin groups are connected to each other via a water bridge so that the phytyl tails become parallel. Present investigations show that in a composite system containing chlorophylls and lipids the special bonding betwcen chlorophylls, which is necessary for the formation of the crystalline form, can be hindered by the interposition of lipid molecules. This is thought to be a steric effect of the lipid molecules instead of a competition between the lipid and water molecules for the coordination sites in porphyrin ring. The amount of crystalline chlorophyll reaches a saturation value when the chlorophyll/lipid molar ratio is about 3. According to combinatoridl considerations, this value corresponds to a sudden increase in the probability of hexagonal arrangement of the bulk constituent of a two component system, suggesting a similar pattern for the organization of crystalline chlorophyll. Recently, a two-dimensional structure of crystalline ethyl chlorophyllide a was described by Strouse (1974), which is in a good agreement with the former suggestion as well. It may be concluded that the artificial membranes present the possibility of creating appropriate systems for studying the highly organized aggregates of the main photosynthetic pigments, such as the so-called crystalline chlorophyll.
Figure 2 illustrates that only the membranes made from the H-solution showed the crystalline chlorophyll absorption peak a t 743 nm. The spectra of the membranes prepared from the L-solution and modified H-solution d o not show any absorption around 743nm, suggesting that these BLMs do not contain crystalline chlorophyll. The results suggest that the BLM's, which are Acknowledyemerzts~Wcwish to thank Dr. B. Karvaly and accepted as bimolecular layers, may contain the crys- Dr. Agnes Faludi-Daniel for valuable critical discussions.
REFERENCES
Ballschmiter, K. and J. J. Katz (1969) J . Am. Chem. Soc. 91, 2661-2677. Inoue, Y., T. Ogawa and K. Shibata (1972) Plant Cell Physiol. 13, 385--389. Jacobs. E. E., A. S. Holt and E. Rabinowitch (1953) 1.Chem. Phys. 22, 142-143. Jacobs, E. E., A. E. Vatter and A. S. Holt (1954) Arch. Biochrm. Biophys. 53, 228-238. Ke, B. (1966) In The Chlorophylls (Edited by L. P. Vernon and G. R. Seely), pp. 253-279. Academic Press, New York. Singleton, W . S., M. S. Gray, M. L. Brown and J. L. White (1965) J . Am. Oil Chem. Soc. 42, 53-56. Strain, H. H. and W. A. Svcc (1966) In The Chlorophylls (Edited by L. P. Vernon and G. R. Seely), pp. 22-66. Academic Press, New York. Strouse, C. E. (1974) Proc. Natl. Acad. Sci. U.S. 71, 325-328. Szabad, J. and I. Csorba (1973) Scan-Pye Unicam 2, 15-16. Thomas, J., G. P. Phondke, V. G. Tatake and A. R. Gopal-Ayengar (1970) Photochem. Photohiol. 11, 85-92. Tien, H. T. (1968) J. Phys. Chem. 72, 4512-4519. Tien, H. T., W . A. Huemoeller and H. P. Ting (1968) Biochem. Biophys. Res. Commun. 33,207-212.
