f=l .0) measurements.'^^"^
3.2. Results The solvent effect on photoisomerization of MC 540 was investigated in n-alkyl alcohols and n-alkanenitriles. The study of MC 540 in polar solvents provides information about solute/solvent friction, and solute/solvent dielectric interactions in activated barrier crossing processes for cis-trans isomerization reactions. Photophysical parameters of MC 540 in n-alkyl alcohols and n-alkanenitriles are given in Table 3.1 at 25 °C. The fluorescence quantum yield is related to the radiative rate constant, kf, and to the nonradiative rate constant, knr, by the photophysical equation 4>f=kr /(knr + kr)
(3.La) 35
Table 3.1: Photophysical Parameters and Solvent Properties of MC 540 in n-Alkyl Alcohols and n-Alkanenitriles at 25 °C
Solvent
ET(30),akcalmol-i Ti,bcP
f)-l].
(3.2)
The measured values of f can be directly converted to values of knr, if the values of kf are known. The quantum yield measurements were carried out over a temperature range of 0-80°C to obtain knr values, which were then used to calculate the activation barrier energy, Ea, for each solvent. The quantum yield and knr values for MC 540 in each solvent at different temperatures are given in Tables 3.2-3.17. The Strickler-Berg (SB) equation (eq 2.9) was utilized in this work to obtain the values of kf."^^ Typical absorption and fluorescence spectra (corrected) of MC 540 that were used to calculate kr by the SB equation (eq 2.9) are given in Figure 3.1. As shown in the eq 2.9. kr is directly proportional to n^ and previous studies have shown that the measured values of kr increases as n^ increases. In Table 3.1, the n values are given. The values of n^ increases from methanol to n-octanol as the size of alkyl group increases. The index of refractive increases from 1.326 to 1.427 for n-alcohols from methanol to n-octanol and in the series of alkanenitriles, n increases from 1.342 to 1.426 for acetonitrile. According to eq 2.9 kr is expected to increase by 15 % in going from methanol to n-octanol and by 13% in going from acetonitrile to nonanenitrile. This was not observed for kr values, as seen in the Table 3.1, because the 20-25% accuracy of the SB equation masks the variation in kr due to n^. The average values of kr calculated from the SB equation is 0.48 ± 0.004 ns"' for the alcohols and 0.49 ± 0.003 ns'* for the nitriles. The values of kr that were obtained by using the SB equation are in good agreement with results obtained by
37
Table 3.2: The Quantum Yields and knr Values in Methanol Temperature ( °C),^
Of. ^
knr, ^ ns-'
15.0
0.17
2.34
20.0
0.15
2.72
25.0
0.13
3.21
35.0
0.11
3.88
50.0
0.08
5.52
60.0
0.06
7.52
Table 3.3: The Quantum Yields and knr Values in Ethanol Temperature ( °C),^
(Df b
knr, ^ns
15.0
0.27
1.22
20.0
0.24
1.43
25.0
0.20
1.80
35.0
0.17
2.80
50.0
0.12
3.30
60.0
0.10
4.05
3 Uncertainty of temperature ±1.0°C. b Fluorescence quantum yield obtained from eq 2.8; uncertainty ±10%. c Nonradiative rate constant calculated from eq 3.2; uncertainty ±30%.
38
Table 3.4: The Quantum Yields and knr Values in l-propanol Temperature (°C),^
Of, b
knr, "^ ns
15.0
0.29
1.05
20.0
0.26
1.22
25.0
0.23
1.44
35.0
0.18
1.96
50.0
0.13
2.88
60.0
0.10
3.87
Table 3.5: The Quantum Yields and knr Values in 1-butanol Temperature ( °C),3
f, b
knr, ^ ns '
15.0
0.36
0.80
20.0
0.32
0.96
25.0
0.28
1.20
35.0
0.23
1.51
50.0
0.16
2.36
60.0
0.13
3.01
3 Uncertainty of temperature ±1.0°C. ^ Fluorescence quantum yield obtained from eq 2.8; uncertainty ±10%. ^ Nonradiative rate constant calculated from eq 3.2: uncertainty ±30%.
39
Table 3.6: The Quantum Yields and knr Values in 1-pentanol Temperature (°C), ^
5.0
0.74
0.17
10.0
0.69
0.22
15.0
0.64
0.27
20.0
0.58
0.35
25.0
0.52
0.44
35.0
0.44
0.61
50.0
0.33
0.97
60.0
0.27
1.30
Table 3.17: The Quantum Yields and knr Values in Nonanenitrile Of, b
knr-^ns'
5.0
0.84
0.09
10.0
0.79
0.12
15.0
0.75
0.15
20.0
0.70
0.20
25.0
0.62
0.28
35.0
0.54
0.38
50.0
0.40
0.68
60.0
0.36
0.80
Temperature ( °C), ^
3 Uncertainty of temperature ±1.0°C. b Fluorescence quantum yield obtained from eq 2.8; uncertainty ±10%. c Nonradiative rate constant calculated from eq 3.2; uncertainty db30%.
45
""•
1
]
1
1
1
r
2.5
2.0
— Methanol — - Acclonilrilc — Butanol — Heplanenilnle
E ^
1.5
•^
1.0
w
0.5
400
450
500
550
600
650
700
Wavelength (nm)
1.0 -
0.8 c
3
Urn
0.6
a
'—^ >-. 4—•
C/3
0.4
(U
0.2 -
500
550
600
Wavelength (nm)
Figure 3.1. Steady-state absorption (a, top) and corrected emission (b, bottom) spectra of merocyanine 540 in four solvents at 25°C.
46
others. For instance, the kr for methanol is 0.42 ns'^ by Davila et al.'^JS and the kr for ethanol is 0.40 ns"' by Aramendia et al.^'l^ The quantum yield of MC 540 in methanol and ethanol has been previously measured. Our experimental value of f for methanol agrees with the one reported by Davila et al.17,18 (Of=0.13±0.01), within experimental error, but differs from that (f=0.26±0.01) reported by Hoebeke et al.-l The values of f for ethanol agrees with the one reported by Aramendia et al.^ for ethanol (f=0.20±0.02) but differs from that (4>f=0.39) reported by Hoebeke et al.-^ Table 3.1 contains the quantum yields at 25 °C for comparison. The fluorescence lifetime, Xf, was calculated from the measured values of f and kr by using the equation Tf = I / (knr + kr) = (f/kr).
(3.3)
Tf values calculated by using this equation were compared with literature values for methanol and ethanol solvents. The calculated value of Tf is 0.27 ns (methanol) and 0.44 ns (ethanol), in good agreement with the literature values of 0.23 and 0.43 ns, respectively.^'^^'l^ The nonradiative rate constant of MC 540 depends strongly on the solvent as shown in the Table 3.1. The nonradiative rate decreases in a homologous series as the length of the alkyl chain increases, and it is faster in an alcohol solvent than in a nitrile solvent with the same alkyl chain. This correlation is illustrated in Figure 3.2. The main purpose of this study is to extract kiso from knr- This can be difficult because there are other nonradiative transitions such as internal conversion, kic, and intersystem crossing, kisc, which should be taken into account. The photophysical transitions for MC 540 are illustrated in Figure 1.4. The dominant process in the nonradiative decay in MC 540 is the isomerization pathway. Davila et al. and Aramendia et al. found that the triplet quantum yield of MC 540 is typically less than 0.05 in the
47
-I
r
~-i-
-1
r-
O Alcohols • Nitriles
3 -
c
r
2
c
0
2
4
6
Number of CH2 groups
Figure 3.2. Correlation of the nonradiative rate at 25°C with the number of CHo groups in the alkyl chain of solvent.
48
temperature range of our measurements."^''^'l"^ Therefore, kisc can be neglected relative to (kic + l^iso)Aramendia et al. and Hoebeke et al. reported that internal conversion (kic) does not take place in the N state (trans-excited ) given in Figure 1.4.9>19,20 Recently. 4>f and the photoisomerization quantum yield, Oiso, of MC 540 in glycerol/ethanol mixtures were measured by Hoebeke et al. and the values of Oiso in these mixtures were determined by directly detecting P state by flash photolysis.^^ To see the effect of viscosity on quantum yields, they varied the relative amounts of glycerol and ethanol in the mixtures. As the viscosity of the mixtures is increased, ^ increases, while 4>iso decreases. One can write *^f + ^iso"= 1 for all values of the viscosity used quantum yield measurements. In addition, the viscosity dependence of quantum yield is the same for both 4>f and Oiso within experimental error. Furthermore, Aramendia et al. showed that the photoisomer directly arises from the first excited singlet state.^'^^ These results imply that internal conversion does not occur or is not a major pathway for nonradiative decay in MC 540. Therefore, we can make the nonradiative rate equal to the photoisomerization rate so that knr = kic + kiso " kiso •
(3.4)
3.2.1. Activation Energies of Solvents The temperature dependent studies of fluorescence spectra showed that the intensity of the fluorescence decreased with increasing temperature but that the shape of the fluorescence spectrum did not change. Davila et al. have studied the temperature dependence of the absorption spectrum of MC 540 and reported that the absorption spectrum of MC 540 does not change with temperature. ^^ The temperature dependence of kr and knr must be known to obtain activation energies. As mentioned earlier in this chapter, the temperature dependence of kr comes from n^ and as mentioned Chapter II, n^ does not change very much in the temperature range of the experiment. Therefore, kr can 49
be assumed to be relatively independent over the temperature range of our measurements. It varies by at most 3%, which is within the accuracy of the SB equation. In the case of knr, the temperature dependence can be determined from the equation 3.2. This equation can be rearranged to gi\ c knrf) - 1| versus 1/T gives information on the temperature dependence of knr- Equating to the Arrhenius equation knr =Anrexp(-Ea/RT)
(3.6)
gives
l(l/f)- ll=A0exp(-Ea/RT)
(3.7)
where AQ = Anr^kp Figure 3.3 shows that plots of ln[( \/f) - 1] versus 1/T and they are given in Table 3.18. The rates of internal conversion are weakly dependent upon temperature. Note that the linearity of the plots of ln[( l/f) 1 ] versus 1/T is also consistent with knr =* kiso- This indicates that if internal conversion is not negligible for nonradiative decay, knr would approach a constant value at low temperatures, which corresponds to kic- Therefore, the plots of ln{( l / ^ ) - 1] versus l/T would display nonArrhenius behavior if kic were not negligible compared to kiso-
3.2.2. Discussion 3.2.2.1. Solvent Effects The rate of photoisomerization is affected by solvent properties such as solvent viscosity and solvent polarity. The photoisomerization rate of MC 540 decreases as solvent 50
-T
1
1 — I — • — ' — '
r
I
Methanol 1 Acetonitrile Butanol Heptanenitrile.;
4
O--..
-O-
•G"
--o •'©'—.
L
B-. B-^
O—. < ^ . . . "]i-^
-o-
-O
-
--D
0
^S J
3.0
^ ^
u
3.2
3.4
3.6
1000/T(K-1)
Figure 3.3. Typical Arrhenius plots of (l/f) - 1 for merocyanine 540 in four solvents. Lines are linear least-squares fits of data. Arrhenius parameters are listed in Table 3.18.
51
Table 3.18: Arrhenius Parameters for Solvent Viscosity and Nonradiative Rates in n-Alkyl Alcohols and n-Alkanenitriles Solvent
Til) 10-^ cP
Er|,^ kcal mol '
ICHAo,*'
Ea,^''=kcal mol 1
methanol
7.70
2.53 ±0.10
1.5
4.57±0.94
ethanol
3.60
3.37±0.10
1.5
4.89±0.22
l-propanol
1.40
4.29±0.10
2.5
5.27±0.24
1-butanol
1.10
4.60±0.10
2.3
5.40±0.29
1-pentanol
0.53
5.22±0.24
1.6
5.24±0.80
1 -hexanol
0.64
5.25±0.12
1.5
5.33±0.38
I-heptanol
0.41
5.66±0.41
1.5
5.53 ±0.49
I -octanol
0.25
6.09±0.20
3.0
6.02±0.60
acetonitrile
19.70
1.70±0.16
1.02
4.70±0.20
propionitrile
18.00
1.86±0.10
1.08
4.89±0.26
butanenitrile
10.60
2.33±0.20
1.07
5.03 ±0.3 2
pentanenitrile
9.95
2.50±0.10
1.5
5.35±0.39
hexanenitrile
10.80
2.63 ±0.20
2.4
5.80±0.83
heptanenitrile
7.23
3.02±0.20
3.5
6.18±0.74
octanenitrile
4.74
3.43±0.32
8.3
6.81 ±0.97
nonanenitrile
3.40
3.76±0.44
8.0
6.82±0.95
3 The viscosity prefactor, i^o, and viscosity activation energy, E^, obtained from refs 52 and 53 or by fitting viscosities at different temperatures to eq 3.19. ^^ Arrhenius parameters obtained by linear least-squares fit of semilogarithmic plots of K1/^) - 1] over temperature range 0-80°C. '^ Calculation of error is described in the appendix.
52
shear viscosity increases. 1 •7,20,55 j ^ addition, the rate is also affected by the solvent polarity as shown in Table 3.1. The rate is faster in alcohols than in nitriles. If one compares the rate in I-propanol to that in nonanenitrile at 25°C, it is five times faster in 1propanol than in nonanenitrile, although the solvent shear viscosities are the same for these solvents and equal to 1.95 cP. Solute/solvent friction and dielectric interactions must be separated to understand the nature of this dependence on the solvent. The activated barrier crossing formula is utilized to accomplish this separation:5-'53,56,57 kiso = F(U exp(-Eo/RT)
(3.8)
The prefactor F(u) is a dynamical quantity that is dependent on the solute/solvent friction, t: E
r~~i—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—j-
T—m—r
0.8 1/T m 1/T.
0.6
0.4
0.2 -
0.0
•
U_L_1_L
10
•
'
'
I
'
20
l
l
I
•
'
'
•
I
I
30 40 Temperature (°C)
I
I
I
I
I
50
I
I
I
L
60
Figure 4.6. Plot of 1/xrot versus temperature used to obtain actual rotational relaxation times of merocyanine 540 in CTAB micellar solution.
95
^—1—r 1
0.2
1
1
1
a
\/T^
O
1/Tp
1 r
1
1
1
1
1
1
1
1
1
—I
1 1 1 1 T 1 I
1O .
—1—1—r
0.3
1
o
—
-
—
O
o o
O.l
o -
o
_0
D "
0.0
D 1
I
1
1
10
• 1
1
° 1
1
1
20
D 1
.
D
D .
.
. . . .
1 .
30 40 Temperature (°C)
1
1
1
1
50
1
1
.
.
1"
60
Figure 4.7. Plot of 1/xrot versus temperature used to obtain actual rotational relaxation times of merocyanine 540 in Triton X-lOO micellar solution.
96
Table 4.8: Actual Rotational Relaxation Times of MC 540 in Triton X-100, CTAB, and SDS Micellar Solutions
Xp, ns
Xp, ns
Xp, ns
in SDS
in CTAB
in Triton X-100
5.0
5.88±0.17
11.11±1.02
16.67±0.46
10.0
5.26±0.10
9.10±0.67
15.38±0.38
15.0
4.00±0.16
7.69±0.50
11.76±0.24
20.0
3.70±0.16
5.56±0.32
9.43±0.18
25.0
3.33±0.16
4.55±0.15
8.13±0.14
35.0
3.13±0.19
3.33±0.15
5.49±0.11
50.0
2.85±0.20
2.04±0.07
3.88±0.10
60.0
2.70±0.18
1.45±0.03
2.85±0.05
Temperature (°C)
97
is localized further into the hydrophobic sites of the interi'ace in CTAB and in Triton X100. An important parameter in determining the microviscosity is the hydrodynamic volume of the probe, Vhyd- Vhyd was determined in n-alkyl alcohols at room temperature by using the steady-state anisotropy and fluorescence lifetime data. Xrot was obtained for each alcohol solvent by using the Perrin's equation (eq 4.2) and then the hydrodynamic volume of MC 540 was calculated from the slope of the plot of Xrot versus r]. Data are given in Table 4.9 and the plot is illustrated in Figure 4.8. Vhyd is found to be 1569±134 A-\ Quitevis and Horng have reported that Vhyd is equal to 990±20 A^ by utilizing the picosecond polarized transient bleaching technique.-^ This technique gives the reorientational d> namics of the probe in the ground state. The fluorescence depolarization measurements indicate that the hydrodynamic volume of the excited state is greater than the ground state. This difference could be caused by a change in conformation when the probe undergoes photoisomerization, hydrogen bonding between dye molecule and solvent molecule resulting in a size increase, or by dielectric friction. Dielectric friction is a type of solute-solvent interaction arising from the long-range interaction of the surrounding dielectric medium with the dipole of the solute molecule.^ The medium is polarized by the electric field associated with the dipole of the probe molecule. The change in polarization of the medium lags as the dipole rotates because the response of the medium is not instantaneous. This causes a deviation from DSE behavior which can accounted for by an additional term in the expression for the rotational diffusion time. Quitevis and Homg have given a detailed discussion on this effect for MC 540.-^ They found that its effect was not important compared to the contribution of the hydrodynamic friction.
98
Table 4.9: Rotational Relaxation Times and of MC 540 in n-Alkyl Alcohols at 25°C
Solvent
Ti,McP)
Xrot,^(ns)
Ethanol
1.14
0.54±0.04
l-propanol
1.95
0.88±0.05
1-butanol
2.60
1.17±0.06
1-pentanol
3.57
1.45±0.76
1 -hexanol
4.54
2.02±0.15
1-heptanol
5.81
2.28±0.10
1-octanol
7.32
2.82±0.13
^ Solvent shear viscosity, r\, obtained from refs 52 and 53. ^ Rotational relaxation times were calculated from eq 2.15.
99
—I
1
r-
"T
~~-r~
V3
c
£
c o
d
2 -
c o o
0 - n 0
2
4 Viscosity (cP)
6
8
Figure 4.8. Rotational relaxation times of merocyanine 540 in n-alkyl alcohols versus the shear viscosity of the each n-alkyl alcohol from ethanol to 1-octanol.
100
4.1.4.2. Micro\ iscosity of Micelles To understand the dynamics of MC 540 in the micelles the microviscosities of the micelle systems will be examined. The hydrodynamic volume value which was determined by Quitevis and Horng-^ will be used to calculate the microviscosities or apparent viscosities of each micellar system at the temperatures reported for the measurements. In particular, the temperature dependence of microviscosity for each system will be studied. The micro\ iscosity \ alues of micelle systems were calculated by utilizing the DSE equation (eq 2.16), the Xp values of MC 540 in micelles, and Vhyd=990.0±20 A^. The variation of microviscosity with temperature for each micelle system is shown in Figure 4.9 and data are given in Table 4.10. The microviscosity of the micelles increases in the order SDS \
4
1.5^ 3.0
3.1
3.2
3.3
3.4
3.5
\
! J
3.6
1000/T (K-^
Figure 4.13. Photoisomerization plot of kiso for merocyanine 540 in CTAB micellar solution. Line through points is linear least-square fit of data.
109
"T
1
1—!
I
.
r-
"T
r
I
1
1
1
1
(—-T
(
1
1
J—1
1
1
1
1
1
1
1
r—t
1
r-
-0.5 o on
1.0 -
-1.5 .
3.0
'
'
•
'
—
'
3.1
'
'
•
3.2
•
'
3.3
•
•
•
•
3.4
, I , , . , I ,
3.5
3.6
lOOOAF (K-1)
Figure 4.14. Photoisomerization plot of kiso for merocyanine 540 in Triton X-100 micellar solution. Line through points is linear least-square fit of data.
110
are 5.65±0.19 kcal mol-> in CTAB, 4.90±0.10 kcal mol' in Triton X-100, and 0.95±0.07 kcal moL' at higher temperatures and 4.20±0.29 kcal moL' at low temperatures in SDS. The value of isomerization activation energy. Ea, obtained for micelle systems and value of the Eo obtained from the eadier studies in pure liquid can be used to obtain Emic values for each system.
The values of Emic obtained from the rotational diffusion and
photoisomerization measurements can then be compared. The value of EQIO be used will be one obtained from a solvent system that possesses the same micropolarity features as the micelle system. Because the polarity of the microenvironment determines the absorption and fluorescence maxima,-^^ this will be determined by comparing the spectral properties of MC 540 in micelle systems with those in the pure solvents. 1-octanol has the same spectral features, particularly the emission spectra, as found in the three micelle system. Therefore, Eo was taken to be equal to that of MC 540 in 1-octanol. For 1-octanol Eo « 0.0. This implies that photoisomerization process in micelle systems is barrierless and that the activation energy is due entirely to the microviscosity, i.e., Ea « Emic- If the Smoluchowski limit is valid and Eo = 0, Emic values obtained from rotational diffusion should match the values obtained from photoisomerization. When we compare the Em,e values, one obtained from rotational diffusion is slightly greater than the one obtained from photoisomerization except in the case of SDS at high temperatures. When we take ratio of Emic from photoisomerization to Emic from rotational diffusion, we obtain 0.91±0.05 for CTAB, 0.90±0.04 for Triton X-lOO, 0.87±0.07 for SDS at low temperatures, and 1.58±0.13 for SDS at higher temperatures. Cleariy, SDS system does not behave in the same way as the other micelle systems. These ratios indicate that the Smoluchowski limit is not completely invalid in these micelle systems. If the ratios were equal to 1.0, we would admit that the Smoluchowski limit of photoisomerization applies for the dye molecules in micellar systems.
Ill
As calculated for n-alkyl alcohols and n-alkanenitriles, the difference between Ea and Emic was calculated to obtain the intrinsic barrier height, Eo. For micelles the Emic values were taken to be equal the values obtained from rotational diffusion. This result can be called either the aviscous activation energy or the intrinsic barrier height of the system. The values of Eo are -0.56±0.34 kcal mol"' in CTAB, -0.54±0.24 kcal mol"' in Triton X-lOO, and 0.35±0.10 kcal mol"' at higher temperatures and -0.62±0.34 kcal mol"' at low temperatures in SDS. Although some of these values are negative, within experimental error they are close to zero. This implies that the isomerization dynamics in micelles is controlled primarily by frictional effects associated with collisions between the surfactant molecules and the probe molecule. The barrier in this case is due to energy needed to displace the surfactant molecules during the isomerization.
4.1.5. Conclusion This study has shown that MC 540 binds to micelles regardless of the nature of the micelle (anionic, cationic, or nonionic) but that different micelle-dye types of interactions occur. These different interactions causes Xp and rimic in these micelles to be different. MC 540 reorients more freely in SDS micelles than in CTAB and in Triton X-100 micelles. MC 540 is predominantly in the hydrophobic region of water-micelle interface of CTAB and Triton X-100. This leads to a greater restriction of the movement of the dye molecule in the micelle. The higher values of Xp and rimic for MC 540 in CTAB and Triton X-lOO are consistent with this interpretation. In the case of SDS, electrostatic repulsion prevents MC 540 from being drawn into the hydrophobic region of the micelle.
The
photoisomerization of the probe in micelle systems is similar to that of in pure 1-octanol and is close to the Smoluchowski limit.
112
4.2. MC 540 in Vesicles 4.2.1. Introduction The rotational dynamics in the hydrocarbon region of lipid bilayer membranes have extensively been investigated by measuring the fluorescence polarization of hydrophobic probes such as perylene and l,6-diphenyl-l,3,5-hexatriene.27,28,36,9l Jhese probes are sensitive indicators of the rotational mobility of the hydrocarbon chains, as exemplified by the finding that their fluorescence polarization changes abruptly at the phase-transition temperature of the membrane. The temperature-dependence of the steady-state fluorescence polarization of perylene and 1,6-diphenyl-l,3,5-hexatriene in membranes has been interpreted in terms of the microviscosity of the membrane interior. This interpretation assumes that the rotational motion of the probe is isotropic. It was also found that the addition of cholesterol to the phospholipid membranes increased the magnitude of their steady-state fluorescence polarization.^^ Hence, it was inferred that cholesterol increases the microviscosity of the membrane interior. The interactions between cholesterol and phospholipids have been extensively studied. Most of the work has used phosphatidylcholines, but other lipids such as phosphatidylethanolamine or sphingomyeline have also been studied.-^ There is reasonable agreement on the phenomenological description of cholesterol-phospholipid mixtures, but there is no consensus on the interpretation of data in terms of specific structural models. X-ray and neutron scattering show that cholesterol inserts normal to the plane of the bilayer with the -OH group near the ester carbonyl of the lipid. However, Raman spectroscopy indicates that no actual hydrogen bond is formed with these carbonyls. The presence of cholesterol has a substantial effect on the order parameters measured along the lipid hydrocarbon chain by ^H-NMR and on the phase transition of the phospholipid.
In the liquid crystalline state (liquid phase) the sterol results in
conformational constraints on the phospholipid chain, whereas in the gel state the sterol 113
inhibits optimal packing of the all-trans chain configuration. The result is that lipidcholesterol mixtures behave in some ways (e.g., disorder) as intermediate between the gel and liquid crystalline states of the pure phospholipid. Basically, cholesterol acts as a "spacer" and reduces the attractive forces between the lipid hydrocarbon chains and disrupts the compact spacing of the headgroups by interacting with the acyl chain of the lipid bilayer.^ We have carried out steady-state anisotropy and fluorescence lifetime studies for MC 540 in vesicle systems to understand the reorientation dynamics of the probe. MC 540 differs in structure than previous probes used to study the reorientational dynamics at the surface of membrane systems. First, we will investigate MC 540 in pure PC and in mixtures of PC and CH to understand the packing effect of the vesicle system. Second, we will discuss the results in the DSPC, DOPC, and mixtures of DSPC and DOPC in terms of chain effects.
4.2.2. Vesicle Preparation The vesicles were prepared by using L-a-egg lecithin phosphatidylcholine (PC), cholesterol (CH). distearoylphosphatidylcholine (DSPC; chain length: 18, unsaturation: 0). and dioleoylphosphatidylcholine (DOPC: chain length: 18, unsaturation: 1 (9-cis) ). The unilamellar vesicles were prepared in phosphate buffer saline, pH = 7.4, by employing the sonication technique. The original lipid samples in chloroform solvent were dried by nitrogen and further kept under vacuum for 2 hours to ensure complete removal of the solvent. Samples were sonicated for 2 hours to obtain unilamellar vesicles after the buffer solution was added to the dried lipid samples. The sonication was carried out under nitrogen atmosphere to prevent any oxidation of the lipids.9- Vesicles were prepared below the phase transition temperatures of the lipids. PC, PC:CH, and DOPC vesicles were prepared at 4°C whereas DSPC and DSPC:DOPC systems were sonicated at 50°C. 114
The lipid-to-probe ratio in solution was 300:1.33 Different ratios of PC and CH mixtures were employed to understand the effect of the compact and disordered-vesicle systems on the orientation of the MC 540. The ratios of PC to CH were 1.0:0.0, 0.9:0.1, 0.8:0.2, and 0.7:0.3. The alkyl chain effect was studied in vesicles consisting of DSPC, DOPC, and a 1.0:1.0 mixture of DSPC to DOPC.
4.2.3. Results 4.2.3.1. Interactions of MC 540 in PC and Mixtures of PC and CH The excitation and emission spectra (corrected) of MC 540 in vesicle were recorded on the SLM 4800C fluorometer as a function of temperature. These spectra are illustrated in Figures 4.15.a and b. The spectra do not depend on temperature and amount of CH in the vesicle.
As discussed earlier for MC 540 in micelle, the lack of any temperature
dependence in the shape of the spectra indicate that the probe is mainly localized in a single type of environment. The spectra of dye in these vesicles are red-shifted from the spectrum in water. The absorption spectrum of MC 540 peaks at 567.0±l.O nm in 100% PC. at 567.0±1.0 nm in 90% PC, at 568.0±l.O in 80% PC, and at 569.0±1.0 in 70% PC system while absorption spectrum of MC 540 peaks at 533.0±1.0 nm in water. The emission maxima of the dye molecules are located at 591.0± 1.0 nm in 100% PC, 593.0± 1.0 nm in 90% PC, 594.0±1.0 nm in 80% PC, and 594.0±1.0 nm in 70% PC. In aqueous phase, the fluorescence spectrum of MC 540 peaks at 572.0±1.0 nm. The effect of CH is shown in Figure 4.15.b, and it is clearly seen that the composition change does not affect the region selectivity of the probe. It has been already established that MC 540 is preferably bound to surface of the membrane systems. The driving force is that there are two hydrophobic tetramethylenic tails in the structure which interact with the hydrophobic parts of the lipids in the membrane structure. The charge on the dye keeps the dye localized at
115
500
400
600
700
Wavelength (nm) T
!
1
r—
-I
— Pure PC - 70% PC & 30% CH
1.0 -
(
r-
(b)
>> 0.8 Urn
C3
0.6
"^ c
0.4
0.2
400
500
600
700
Wavelength (nm) Figure 4.15. Temperature dependence of excitation and emission (corrected) spectra of merocyanine 540 in pure PC vesicles (a) and the effect of cholesterol addition on excitation and emission (corrected) spectra of merocyanine 540 (b). 116
the surface and prevents the dye from being pulled into the interior of the membrane. The lack of any change in the spectra indicates, as in the case of MC 540 in micelles, that the dye remains bound to this site regardless of temperature. The data of steady-state anisotropics and fluorescence lifetimes for MC 540 in vesicles are given in Tables 4.11-4.14. The steady-state anisotropy values of MC 540 in 100% PC are greater than in the other vesicle systems. Addition of cholesterol to the vesicle systems causes the steady-state anisotropy to decrease. Other effects on steady-state anisotropy can be seen by varying the temperature. As the temperature increases, the steady-state anisotropy increases. To see these trends in data, the data are plotted in Figure 4.16. The fluorescence lifetime values show an opposite trend in these systems. The fluorescence lifetime of the probe is greater in 100% PC system at low temperatures than that of other vesicle systems. Addition of cholesterol reduces the lifetime but at higher temperatures the values of fluorescence lifetime are greater in 70% PC than the lifetime times of the probe in other vesicle systems. The temperature dependence of fluorescence lifetime is illustrated in Figure 4.17. The steady-state anisotropy data indicate that in pure PC vesicles, the structural configuration of the vesicle is more rigid, and tightly spaced at the head groups region (i.e., surface of the membrane) than in vesicles which contain cholesterol. Some other workers reported that pure PC systems are compactly spaced at the head group region.^^ These findings are consistent with our observed higher values of steady-state anisotropy in 100% PC system than in the mixed vesicle system. The data indicate that MC 540 does not intercalate completely into the hydrophobic regions of the lipid bilayers of the vesicles. If this were not true, the steady-state anisotropy values should have been higher for the probe in membranes containing cholesterol because cholesterol increases the order or rigidity in the hydrocarbon region of the bilayer. As seen in Figure 4.16, the steady-state anisotropy decreases, as the concentration of cholesterol increases. This trend is more pronounced at 117
Table 4.11: Steady-State Anisotropy and Fluorescence Lifetime of MC 540 in Pure PC Vesicles
Temperature (°C), ^
Xf, ^^ (ns)
r, '^
5.0
2.37±0.03
0.145
10.0
2.33±0.03
0.153
15.0
2.26±0.04
0.153
20.0
2.19±0.02
0.152
25.0
1.93±0.03
0.152
35.0
1.69±0.02
0.155
50.0
1.42±0.03
0.157
60.0
1.17±0.03
0.166
70.0
1.09±0.03
0.181
2 The uncertainty in temperature is ±1.0°C. t'The fluorescence lifetime was measured as described in Chapter II with SLM 48(X)C fluorometer. c The uncertainty in the steady-state anisotropy is ±3%.
118
Table 4.12: Steady-State Anisotropy and Ruorescence Lifetime of MC 540 in 90% PC and 10% CH Vesicles
Temperature (°C), ^
Xf, ^ (ns)
r. c
^'0
2.18±0.02
0.108
10.0
2.15±0.02
0.109
15.0
2.08±0.02
0.109
20.0
2.05±0.04
0.109
25.0
l.79±0.03
0.113
35.0
1.64±0.03
0.120
50.0
1.31 ±0.04
0.130
60.0
1.20±0.02
0.144
70.0
1.15±0.03
0.166
^ The uncertainty in temperature is ±1.0°C. ^The fluorescence lifetime was measured as described in Chapter II with SLM 4800C fluorometer. ^ The uncertainty in the steady-state anisotropy is ±3%.
119
Table 4.13: Steady-State Anisotropy and Fluorescence Lifetime of MC 540 in 80% PC and 20% CH Vesicles
Temperature (°C), ^
Xf, ^ (ns)
r, ^
5.0
2.15±0.02
0.108
10.0
2.16±0.02
0.108
15.0
2.07±0.02
0.107
20.0
2.01 ±0.03
0.107
25.0
1.86±0.02
0.110
35.0
1.63±0.03
0.112
50.0
1.32±0.03
0.122
60.0
1.17±0.04
0.135
70.0
1.07±0.02
0.150
3 The uncertainty in temperature is ±1.0°C. ^The fluorescence lifetime was measured as described in Chapter II with SLM 4800C fluorometer. c The uncertainty in the steady-state anisotropy is ±3%.
120
Table 4.14: Steady-State Anisotropy and Fluorescence Lifetime of MC 540 in 70% PC and 30% CH Vesicles
Temperature (°C), ^
T| , ^^ (ns)
r,
50
2.10±0.02
0.122
10.0
2.11 ±0.02
0.114
15.0
2.07±0.03
0.113
20.0
1.97±0.03
0.113
25.0
l.87±0.03
0.114
35.0
1.65±0.04
0.118
50.0
1.43 ±0.03
0.127
60.0
1.28±0.04
70.0
1.21 ±0.03
'
0.134 0.150
^ The uncertainty in temperature is ±1.0°C. ^The fluorescence lifetime was measured as described in Chapter II with SLM 4800C fluorometer. ^ The uncertainty in the steady-state anisotropy is ±3%.
121
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0.18
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Temperature (°C) 1—
^SA
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1
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60
Temperature (°C) Figure 4.20. Temperature dependent decrease of rotational relaxation times and fluorescence lifetimes of merocyanine 540 in pure PC vesicles, top plot, and in 90% PC vesicles, bottom plot. 132
20
40
60
80
Temperature (°C) 2.2
_
1
1
1 1
1
.
1
1
1
1
1
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.
40
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60
.
J
i
1
80
Temperature (°C) Figure 4.21. Temperature dependent decrease of rotational relaxation times and fluorescence lifetimes of merocyanine 540 in pure 80% PC vesicles, top plot, and in 70% PC vesicles, bottom plot. 133
increases. The rate of change of Xf with temperature is 2 times , 30 times, 5.3 times, and 4.4 times faster than the rate of change of Xrot with temperature in pure PC vesicles, in 90% PC vesicles, in 80% PC vesicles, and in 70% PC vesicles, respectively.
4.2.3.3. Interactions of MC 540 in DOPC, DSPC and Mixture of DSPC and DOPC «
The static organization of membrane lipids in a bilayer depends on temperature in a conspicuous way. Its mesomorphism finds general expression in the existence of two distinct lamellar phases, the L^ form at temperatures above the phase transition point, the Lp form at temperatures below it. In the Lp phase, the hydrophobic tails of the lipid are well-spaced and almost parallel-spaced each other while in L^ phase, hydrophobic tails of the lipid are kinked in configuration and disorganized. The phase transition temperature depends on the nature of the acyl chains of the lipids. The phase transition temperature for a single-lipid membrane is a well-deflned quantity which, due to the cooperative nature of the melting, may be narrowed down to less than a degree. In biological membranes, however, the passage from one phase to the other may extend over several degrees because of the heterogeneity of membrane composition.^ The L«form corresponds to a solid crystalline phase in which there is very little lateral movement of lipid molecules and where the acyl chains are in the trans configuration. In the La form the liquid-crystalline organization prevails and permits free lateral diffusion, as well as rotation around the axis normal to the membrane plane. This results in a frequent formation of rotational isomers of the acyl chains, based on a 120-degree turn of a C-C bond in either one or the other direction, the resulting configuration being called gauche.93 DOPC and DSPC differ only at one position where there is a double bond in the structure of DOPC, locating on C^ in cis configuration.^^ The structures of the lipids are illustrated in Figure 4.22. These two lipid systems were chosen to study the effects of acyl 134
CH^,
CH-
CH2-CH2-N^ — C H
CH2-CH2-N^ —CH2
O
O
CH^
O'
O
o-
O
CH-
P = 0 O
CH.
CH
CH-
O
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o=c
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c=o
0 =
CH
CH-
O
O
C
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(CHo)^
(CH2)7
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CH
CH
CH2
CH.
II
II
CH
CH
CH2
CH2
(CH2)7
(CH2)7
(CH2)-
(CH2)7
CH3
CH3
CH3
CH3
Dioleoylphosphatidylcholine
Distearoylphosphatidylcholine
Figure 4.22. Chemical structures of a saturated, distearoylphosphatidylcholine, and an unsaturated, dioleoylphosphatidylcholine, lipids. 135
chains on the molecular reorientation of MC 540. The stead\-state anisotropy and fluorescence lifetime data of MC 540 in DOPC. DSPC and mixture of DSPC and DOPC b\ the ratio o( 1.0:1.0 are given in Tables 4.19-4.21. In contrast to MC 540 in PC and PC:CH mixture vesicles, the data for MC 540 in synthetic lipid vesicles does not lend itself to a clean interpretation. To see the trends in the data, the steady-state anisotropy and fluorescence lifetime data uere plotted against the temperature. Figure 4.23 and Figure 4.24 illustrate the variation of r and Xf with temperature, respectively. As seen in Figure 4.23, MC 540 in DSPC shows different behavior compared to MC 540 in the other \ esicle systems. The steady-state anisotrops in DSPC is higher and almost constant at the temperatures between 20°C and 40°C. DSPC contains saturated acyl chains in trans configurations, and hence the spacing of the acyl chains is more compact compared to lipid s\stem which contains unsaturated acyl chains. Therefore. MC 540 in DSPC is in the gel phase of the vesicle system at the temperatures between 20°C and 40°C. The steady-state anisotropy starts to decrease at 45°C, indicating that MC 540 experiences phase transition from gel phase to liquid crystalline phase at higher temperatures. It is reported that DSPC shows onl\ one phase transition temperature at 51.0°C.^^'^^ In the liquid cr\stalline phase the probe has more space to execute and can rotate freely. As a result, the steady-state anisotropy goes down at those temperatures. DOPC contains one double bond and this causes a drop on phase transition temperature. The phase transition temperature of DOPC is reported as -22°C.^^^^ DOPC shows gel state below -22°C and liquid cr>stalline phase above it. In our studs, we also observed a transition point at 10°C in the case of DSPC system. This 10°C transition point obviously cannot be associated with the gel-liquid crystal transition that occurs at 5 r C . There must be another effect uhich at this time we are unable to explain. In the temperature range of our measurements, a pronounced phase transition was not observed for both DOPC system and mixture of DOPC and DSPC
136
Table 4.19: Steady-State Anisotropy and Fluorescence Lifetime of MC 540 in DOPC Vesicles
Temperature (°C), -'
J^,b (ns)
r, ^
0.0
2.60±0.02
0.121
5.0
2.52±0.06
0.120
10.0
2.39±0.04
0.121
15.0
2.36±0.04
0.117
20.0
2.18±0.04
0.125
25.0
2.11 ±0.02
0.127
30.0
l.97±0.02
0.137
35.0
1.88±0.001
40.0
1.73±0.02
0.121
45.0
1.64±0.03
0.136
50.0
l.46±0.00l
0.146
55.0
1.27±0.03
0.151
60.0
1.32±0.03
0.155
70.0
1.16±0.04
0.165
'
0.125
^ The uncertainty in temperature is ±1.0°C. ^ The fluorescence lifetime was measured as described in Chapter II with SLM 4800C fluorometer. c The uncertainty in steady-state anisotropy is ±3%.
137
Table 4.20: Steady-State Anisotropy and Fluorescence Lifetime of MC 540 in DSPC Vesicles
Temperature (°C), ^
Xf, ^ (ns)
r, ^
00
l.53±0.02
0.159
50
l.57±0.02
0.129
10.0
l.76±0.02
0.115
15.0
1.59±0.02
0.175
20.0
1.79+0.02
0.189
25.0
1.74±0.07
0.187
30.0
1.65±0.02
0.185
35.0
1.77±0.06
0.185
40.0
1.58±0.02
0.169
45.0
1.58±0.02
0.147
50.0
1.41±0.04
0.116
55.0
1.62±0.02
0.121
60.0
1.48±0.02
0.121
70.0
1.26±0.02
0.138
^ The uncertainty in temperature is ±1.0°C. ^The fluorescence lifetime was measured as described in Chapter II with SLM 4800C fluorometer. ^ The uncertainty in steady-state anisotropy is ±3%.
138
Table 4.21: Steady-State Anisotropy and Fluorescence Lifetime of MC 540 in Mixed DSPC and DOPC Vesicles
Temperature (°C), '
xj, ^ (ns)
r, ^
0.0
2.37±0.02
0.107
50
2.39±0.02
0.115
10.0
2.14±0.04
0.108
15.0
2.20±0.02
0.103
20.0
2.00±0.03
O.l 10
25.0
2.02±0.02
0.106
30.0
1.89±0.03
0.104
35.0
1.90±0.03
0.118
40.0
1.76±0.02
0.130
45.0
1.73±0.02
0.144
50.0
1.59±0.03
0.149
55.0
1.47±0.03
0.150
60.0
1.32±0.02
0.158
70.0
1.21 ±0.02
0.167
^ The uncertainty in temperature is ±1.0°C. ^'The fluorescence lifetime was measured as described in Chapter II with SLM 4800C fluorometer. ^ The uncertainty in steady-state anisotropy is ±3%.
139
1
r~—
T
1
1 A
, A
T
I
A
A
1
1
A A
Cl. Urn
0.16
-
8 O o o O
o
I
A
>>
C/5
-
O
0.14
OS
-
•
A
o 'c