TY - JOUR
T1 - Detection of Hindered Rotations of 1,6-Diphenyl-1,3,5-Hexatriene in Lipid Bilayers by Differential Polarized Phase Fluorometry
AU - Lakowicz, J. R.
AU - Prendergast, F. G.
N1 - Funding Information:
We thank the Freshwater Biological Research Foundation, and especially its founder Mr. Richard Gray, Sr., without whose assistance this work would not have been possible. Additionally, we acknowledge the generous support of the American Heart Association, Mayo Foundation, and the National Institutes of Health grant ES GM 01238-OlAI to J.R.L. and CA 150 83-00 to F.G.P. J.R.L. is an Established Investigator of the American Heart Associatiori. Receivedforpublication 8 December 1977.
PY - 1978
Y1 - 1978
N2 - Differential polarized phase fluorometry has been used to investigate the depolarizing motions of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the isotropic solvent propylene glycol and in lipid bilayers of dimyristoyl-L-α-phosphatidylcholine (DMPC), dipalmitoyl-L-α-phosphatidylcholine (DPPC), and other phosphatidylcholines. Differential phase fluorometry is the measurement of differences in the phase angles between the parallel and perpendicular components of the fluorescence emission of a sample excited with sinusoidally modulated light. The maximum value of the tangent of the phase angle (tan Δmax) is known to be a function of the isotropy of the depolarizing motions. For DPH in propylene glycol the maximum tangent is observed at 18°C, and this tangent value corresponds precisely with the value expected for an isotropic rotator. Additionally, the rotational rates determined by steady-state polarization measurements are in precise agreement with the differential phase measurements. These results indicate that differential phase fluorometry provides a reliable measure of the probe's rotational rate under conditions where these rotations are isotropic and unhindered. Rotational rates of DPH obtained from steady-state polarization and differential phase measurements do not agree when this probe is placed in lipid bilayers. The temperature profile of the tan Δ measurements of DPH in DMPC and DPPC bilayers is characterized by a rapid increase of tan Δ at the transition temperature (Tc), followed by a gradual decline in tan Δ at temperatures above Tc. The observed tanΔmax values are only 62 and 43% of the theoretical maximum. This defect in tanΔmax is too large to be explained by any degree of rotational anisotropy. However, these defects are explicable by a new theory that describes the tan Δ values under conditions where the probe's rotational motions are restricted to a limiting anisotropy value, r∞. Theoretical calculations using this new theory indicate that the temperature dependence of the depolarizing motions of DPH in these saturated bilayers could be explained by a rapid increase in its rotational rate (R) at the transition temperature, coupled with a simultaneous decrease in r∞ at this same temperature. The sensitivity of the tan Δ values to both R and r∞ indicates that differential phase fluorometry will provide a method to describe more completely the depolarizing motion of probes in lipid bilayers.
AB - Differential polarized phase fluorometry has been used to investigate the depolarizing motions of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the isotropic solvent propylene glycol and in lipid bilayers of dimyristoyl-L-α-phosphatidylcholine (DMPC), dipalmitoyl-L-α-phosphatidylcholine (DPPC), and other phosphatidylcholines. Differential phase fluorometry is the measurement of differences in the phase angles between the parallel and perpendicular components of the fluorescence emission of a sample excited with sinusoidally modulated light. The maximum value of the tangent of the phase angle (tan Δmax) is known to be a function of the isotropy of the depolarizing motions. For DPH in propylene glycol the maximum tangent is observed at 18°C, and this tangent value corresponds precisely with the value expected for an isotropic rotator. Additionally, the rotational rates determined by steady-state polarization measurements are in precise agreement with the differential phase measurements. These results indicate that differential phase fluorometry provides a reliable measure of the probe's rotational rate under conditions where these rotations are isotropic and unhindered. Rotational rates of DPH obtained from steady-state polarization and differential phase measurements do not agree when this probe is placed in lipid bilayers. The temperature profile of the tan Δ measurements of DPH in DMPC and DPPC bilayers is characterized by a rapid increase of tan Δ at the transition temperature (Tc), followed by a gradual decline in tan Δ at temperatures above Tc. The observed tanΔmax values are only 62 and 43% of the theoretical maximum. This defect in tanΔmax is too large to be explained by any degree of rotational anisotropy. However, these defects are explicable by a new theory that describes the tan Δ values under conditions where the probe's rotational motions are restricted to a limiting anisotropy value, r∞. Theoretical calculations using this new theory indicate that the temperature dependence of the depolarizing motions of DPH in these saturated bilayers could be explained by a rapid increase in its rotational rate (R) at the transition temperature, coupled with a simultaneous decrease in r∞ at this same temperature. The sensitivity of the tan Δ values to both R and r∞ indicates that differential phase fluorometry will provide a method to describe more completely the depolarizing motion of probes in lipid bilayers.
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U2 - 10.1016/S0006-3495(78)85357-0
DO - 10.1016/S0006-3495(78)85357-0
M3 - Article
C2 - 708824
AN - SCOPUS:0018022237
SN - 0006-3495
VL - 24
SP - 213
EP - 231
JO - Biophysical Journal
JF - Biophysical Journal
IS - 1
ER -