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Refractive index determination of lecithin black films
E. Biol. (1967) 30, 551653
efractive Index Determination
of Lecithin
Black Films
Huang & Thompson (1965) used an optical method to determine the thickness of lecithin black films formed in an aqueous electrolyte solution The thickness was calculated from the reflectivity assuming that the film was a single homogeneous layer of refractive index r~. The value of m was determined from Brewster angle measurements and found to be l-66 f 0.03. The authors noted this as a surprisingly high value, as the calculated value for l-palmitoyl,2-oleoylphosphatidylcholiue is l-49. This phosphatide has a similar structure and molecular weight to the structure and average molecular weight of the egg phosphatidylcholine used in their experiments. ther workers (Tien, 1966; Babakov, Ermishkin & Liberman, 1966) have used similar optical methods to determine the thickness of lecithin films, but no values for the refractive index were given. Vreeman (1966) measured the reflectivity of lecithin films and, assuming a refractive index of 1.48 (the value for parafEn), calculated the thickness as 46 A. If he had used Huang & Thompson’s value of the refractive index, the film thickness would have been about 20 d. Such a value disagrees with both Huang & Thompson’s value of 72 A, and the thickness of the ydrocarbon part of the film, 48 b, as determined by electrical measurements (Hanai, Haydon & Taylor, 1964). We have made black films from egg lecithin dissolved in decane plus tetradecaue by a similar technique to that described by van den Berg (1965). The Brewster e of these films was determined at 6328 A using light from a helium-neon laser. refractive index of the film was calculated from the equation tan is = n/n,
(1) where n, = 1.334 is the refractive index of the aqueous phase, and iB is the Brewster angle. The refractive indices of some pure phospholipids were also determined. Values for single crystal platelets of dimyristoyl-nL-phosphatidylethanolamine were obtained by measuring the Brewster angle for light reflected from the face of the platelet. Values for this material in the liquid crystalline state (obtained by heating the lipid) and for dioleoyl-L-lecithin were determined by pressing the phospholipid onto a glass block and measuring the critical angle at the interface. The results of these various measurements are given in Table 1. While our value of the refractive index of lecithin black 6lms is clearly in disagreement with that of Huang & Thompson, it cannot be directly related to any single component, or homogeneous mixture of components, of t,he solution from which the membrane was formed (Table 2). Huang & Thompson suggested that their observed anomalous refractive index value for these Glms may be due to an optical anisotropy resulting from the ordering of the molecules in the membrane. Our measurements d.imyristoylphosphatidylethanolamine crystals do not support this explanation. e crystals are in the form of platelets with the hydrocarbon chains of the phospholipid molecules lying approximately perpendicular to the face of the platelet (Chapman, Byrne & Shipley, 1966). This is a similar arrangement to the proposed molecular structure of the black films. We also find that the refractive index of the 551
852
R.
J.
CHERRY
AND
D.
TABLE
CHAPMAN
I
Refractive index values Material
Refractive
index
Lecithin
black
film
1.37
Dimyristoyl-DL-phosphatidylethanolamine
(crystals)
Dimyristoylphosphatidylethanolamine
(liquid
& o-02
1.46 *
crystalline)
0.02
1~49 & 0.01
Dioleoyl-r&&thin
1.48 -& 0.01
Dioleoyl-L-lecithin All measurements
+ 24%
water
(w/w)
were
made
at 20°C using
P-44 f the helium-neon
Q-01
laser.
TABLET Refractive indices of wmpomnh
of membrane solution R&active
Material
Decane
1*415=f
Tetradecane 1-Pahnitoyl, T From $ From
index
1.446-f 2-oleoylphosphatidylcholine
International Critical Hung & Thompson
Tables, (1965).
1*493$ vol.
1 (1926).
ordered crystalline state only differs by a small amount from that of the liquid crystalline state, in which there is less or no over-all orientation of the molecules. Recent studies in our laboratory of lecithin-water mixtures have shown that several water molecules may be strongly bound to the polar head of these phospholipids (Chapman, Williams i% Ladbrooke, 1967). Measurements of the refractive index of dioleoyl-rAecithin plus 24% water (the maximum amount of water which can be bound by the lecithin) indicate that the presence of this water lowers the refractive index of the lecithin, but not sufficiently to account for the value we obtain for the black hhn. We believe that the d.ifEoulty in accounting for the observed Brewster angles of lecithin black fihns arises &from the assumption Dhat the Elm is a single layer characterized by a single refractive index. The film may be more correctly represented by a structure consisting of several layers with different refractive indices. The diEerent layers might include the hydrocarbon chain region and the polar head groups with bound water. It is known that the “Brewster angle” of multi-layer films may take on a wide range of values depending on the optical parameters of the individual layers (Duyvis, 1962). Solvent remaking in the fihn could also affect the refractive index of the inner layer and may account for different Brewster jangles being observed with phospholipid films prepared from different solvents.
LETTERS
TO
THE
EDITOR
563
We are, at present, calculating the optical properties of various multi-layer structures to see how far they can account for the experimental results. Until the interpretation of the optical measurements is more ftiy understood, estimates of mensions and structural properties of these black films must remain uncertain. Molecular Biophysics Unit Unilever Research Laboratory The Frythe, Welwyn, He&s, Received
21 July
R. D.
5. kE2RRY CXIAP~KA~X
England
1967 REFERENCES
Babakov, A. V., Ermishkin, L. N. & Liberman, E. A. (1966). Nature, 210, 953. van den Berg, Ip. J. (1965). J. Mol. BioZ. 12, 290. Chapman, D., Byrne, I?. St Shipley, G. G. (1966). Proc. Roy. Sot. A, 290, 115. Chapman, D., Williams, R. M. & Ladbrooke, B. D. (1967). Chem. Phys. Lipids, press. Duyvis, E. M. (1962). Thesis, University of Utreeht. Wanai, T., Haydon, D. A. & Taylor, J. (1964). Proc. Roy. Xoc. A, 281, 377. Huang, C. C%Thompson, T. E. (1965). J. Mol. Biol. 13, 183. International Critical Tables (1926). New York: McGraw-Hill Book Company. Tien, H. T. (1966). J. Mol. Biol. 16, 577. Freeman, B[. J. (1966). Thesis, University of Amsterdam.
in tlhe
efractive Index Determination
of Lecithin
Black Films
Huang & Thompson (1965) used an optical method to determine the thickness of lecithin black films formed in an aqueous electrolyte solution The thickness was calculated from the reflectivity assuming that the film was a single homogeneous layer of refractive index r~. The value of m was determined from Brewster angle measurements and found to be l-66 f 0.03. The authors noted this as a surprisingly high value, as the calculated value for l-palmitoyl,2-oleoylphosphatidylcholiue is l-49. This phosphatide has a similar structure and molecular weight to the structure and average molecular weight of the egg phosphatidylcholine used in their experiments. ther workers (Tien, 1966; Babakov, Ermishkin & Liberman, 1966) have used similar optical methods to determine the thickness of lecithin films, but no values for the refractive index were given. Vreeman (1966) measured the reflectivity of lecithin films and, assuming a refractive index of 1.48 (the value for parafEn), calculated the thickness as 46 A. If he had used Huang & Thompson’s value of the refractive index, the film thickness would have been about 20 d. Such a value disagrees with both Huang & Thompson’s value of 72 A, and the thickness of the ydrocarbon part of the film, 48 b, as determined by electrical measurements (Hanai, Haydon & Taylor, 1964). We have made black films from egg lecithin dissolved in decane plus tetradecaue by a similar technique to that described by van den Berg (1965). The Brewster e of these films was determined at 6328 A using light from a helium-neon laser. refractive index of the film was calculated from the equation tan is = n/n,
(1) where n, = 1.334 is the refractive index of the aqueous phase, and iB is the Brewster angle. The refractive indices of some pure phospholipids were also determined. Values for single crystal platelets of dimyristoyl-nL-phosphatidylethanolamine were obtained by measuring the Brewster angle for light reflected from the face of the platelet. Values for this material in the liquid crystalline state (obtained by heating the lipid) and for dioleoyl-L-lecithin were determined by pressing the phospholipid onto a glass block and measuring the critical angle at the interface. The results of these various measurements are given in Table 1. While our value of the refractive index of lecithin black 6lms is clearly in disagreement with that of Huang & Thompson, it cannot be directly related to any single component, or homogeneous mixture of components, of t,he solution from which the membrane was formed (Table 2). Huang & Thompson suggested that their observed anomalous refractive index value for these Glms may be due to an optical anisotropy resulting from the ordering of the molecules in the membrane. Our measurements d.imyristoylphosphatidylethanolamine crystals do not support this explanation. e crystals are in the form of platelets with the hydrocarbon chains of the phospholipid molecules lying approximately perpendicular to the face of the platelet (Chapman, Byrne & Shipley, 1966). This is a similar arrangement to the proposed molecular structure of the black films. We also find that the refractive index of the 551
852
R.
J.
CHERRY
AND
D.
TABLE
CHAPMAN
I
Refractive index values Material
Refractive
index
Lecithin
black
film
1.37
Dimyristoyl-DL-phosphatidylethanolamine
(crystals)
Dimyristoylphosphatidylethanolamine
(liquid
& o-02
1.46 *
crystalline)
0.02
1~49 & 0.01
Dioleoyl-r&&thin
1.48 -& 0.01
Dioleoyl-L-lecithin All measurements
+ 24%
water
(w/w)
were
made
at 20°C using
P-44 f the helium-neon
Q-01
laser.
TABLET Refractive indices of wmpomnh
of membrane solution R&active
Material
Decane
1*415=f
Tetradecane 1-Pahnitoyl, T From $ From
index
1.446-f 2-oleoylphosphatidylcholine
International Critical Hung & Thompson
Tables, (1965).
1*493$ vol.
1 (1926).
ordered crystalline state only differs by a small amount from that of the liquid crystalline state, in which there is less or no over-all orientation of the molecules. Recent studies in our laboratory of lecithin-water mixtures have shown that several water molecules may be strongly bound to the polar head of these phospholipids (Chapman, Williams i% Ladbrooke, 1967). Measurements of the refractive index of dioleoyl-rAecithin plus 24% water (the maximum amount of water which can be bound by the lecithin) indicate that the presence of this water lowers the refractive index of the lecithin, but not sufficiently to account for the value we obtain for the black hhn. We believe that the d.ifEoulty in accounting for the observed Brewster angles of lecithin black fihns arises &from the assumption Dhat the Elm is a single layer characterized by a single refractive index. The film may be more correctly represented by a structure consisting of several layers with different refractive indices. The diEerent layers might include the hydrocarbon chain region and the polar head groups with bound water. It is known that the “Brewster angle” of multi-layer films may take on a wide range of values depending on the optical parameters of the individual layers (Duyvis, 1962). Solvent remaking in the fihn could also affect the refractive index of the inner layer and may account for different Brewster jangles being observed with phospholipid films prepared from different solvents.
LETTERS
TO
THE
EDITOR
563
We are, at present, calculating the optical properties of various multi-layer structures to see how far they can account for the experimental results. Until the interpretation of the optical measurements is more ftiy understood, estimates of mensions and structural properties of these black films must remain uncertain. Molecular Biophysics Unit Unilever Research Laboratory The Frythe, Welwyn, He&s, Received
21 July
R. D.
5. kE2RRY CXIAP~KA~X
England
1967 REFERENCES
Babakov, A. V., Ermishkin, L. N. & Liberman, E. A. (1966). Nature, 210, 953. van den Berg, Ip. J. (1965). J. Mol. BioZ. 12, 290. Chapman, D., Byrne, I?. St Shipley, G. G. (1966). Proc. Roy. Sot. A, 290, 115. Chapman, D., Williams, R. M. & Ladbrooke, B. D. (1967). Chem. Phys. Lipids, press. Duyvis, E. M. (1962). Thesis, University of Utreeht. Wanai, T., Haydon, D. A. & Taylor, J. (1964). Proc. Roy. Xoc. A, 281, 377. Huang, C. C%Thompson, T. E. (1965). J. Mol. Biol. 13, 183. International Critical Tables (1926). New York: McGraw-Hill Book Company. Tien, H. T. (1966). J. Mol. Biol. 16, 577. Freeman, B[. J. (1966). Thesis, University of Amsterdam.
in tlhe