Orientational distribution of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles as determined by global analysis of frequency domain fluorimetry data

Abstract

Polarized differential phase and modulation ratios were obtained for 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles by using multifrequency phase fluorometry. Data were analyzed in terms of both empirical sums-of-exponentials modeling and directly in terms of the orientational distribution functions. The orientational analysis model was used to recover the angular distribution of DPH and the rotational diffusion coefficient in the various membrane model systems throughout the phase transition. A global analysis methodology was utilized to obtain an internally consistent set of parameters that fit all of the data simultaneously. The rank order parameters (P2) and (P4) were extracted from the experimental data, and the angular distribution functions of DPH were calculated. When the time-zero anisotropy (r0) of several sets of data taken at various temperatures were linked in a single global analysis, better recovery of the fourth rank order parameter (P4), diffusion constant D, and r0 was obtained with respect to the unlinked analysis. From these recovered values, a detailed picture concerning the orientational distribution of DPH in membranes as a function of temperature was obtained. The results suggest that a single population of DPH molecules was present in the bilayers with their orientational distributions dependent upon the physical state of the membranes in the pure phases. During the phase transition, a superposition of two populations corresponding to the population of the pure phases was present. As the temperature increased in the transition region, one population was increasing at the expense of the other.

Global Analysis of Unmatched Polarized Fluorescence Decay Curves of Systems Exhibiting a Residual Anisotropy at Long Times

“…Therefore, this model does not seem to be appropriate. This is in agreement with the results reported by Wang et al, 10 Davenport et al, 51 and Lakowicz et al, 54 although other authors found that a multiexponential expression in addition to r ∞ was required for an adequate description of the data. 14,15 The number of components in an experimentally collected anisotropy decay can be influenced by the time-resolved method used, the analysis programs for the data, and the experimental method used for preparing the samples.…”

New Insights on the Biophysical Interaction of Resveratrol with Biomembrane Models: Relevance for Its Biological Effects

“…The membrane location of resveratrol was assessed by steady-state fluorescence quenching and lifetime measurements using DPH and TMA-DPH probes with a known membrane position and depth [33][34][35] 15 . In order to study if the quenching mechanism was static or dynamic, the dynamic quenching constant (K D ) was determined from the slope of the Stern-Volmer plots obtained by lifetime fluorescence measurements (τ o /τ) 15 .…”

“…DPH is deeply buried in the hydrocarbon core of the lipid bilayer aligned parallel to the acyl chains 33,34 and TMA-DPH is anchored in the polar head region of phospholipids due to its charged group and therefore is located closer to the lipid/water interface 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 13 increasing concentrations of resveratrol (quencher). Similar plots were obtained for the other membrane systems with both probes DPH and TMA-DPH, at the same physiological conditions.…”

Spectroscopic innovations at the Laboratory for Fluorescence Dynamics

“…This mode of operation degrades the spatial resolution of the MCP some what, but signal averaging and spatial correction algorithms reduce this difficulty [9] rotational dynamics of the enzyme [13]. Other applications involve determinations of orientational order parameters in membranes, the orientational distributions [14]. The linked global analysis methods are also powerful in analyzing excited state reactions and energy transfer.…”