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Comments on 1,6-diphenyl-1,3,5-hexatriene fluorescence decrease at critical cholesterol concentration in phospholipid membranes.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Parasassi, Tiziana Gratton, Enrico |
| Copyright Year | 1996 |
| Abstract | Biophysical Journal Volume 70 March 1996 1560-1562 Comments on 1,6-diphenyl-1,3,5-hexatriene Fluorescence Decrease at Critical Cholesterol Concentration in Phospholipid Membranes We were intrigued by the recent publication by Tang et al. (1995) describing unusual properties of 1,6-diphenyl-1,3,5- hexatriene (DPH) fluorescence intensity in dimirys- toylphosphatidyl-choline (DMPC) as a function of choles- terol concentration in the phospholipid. A vast literature exists on phospholipid membrane properties viewed by DPH fluorescence parameters, reporting intensity, polariza- tion, lifetime, and anisotropy data of the probe in phospho- lipids (for recent reviews see Lentz, 1995; Gratton and Parasassi, 1995). Among other researchers, we have per- formed several experiments using DPH to study the prop- erties of lipid bilayers, also in the presence of cholesterol (Fiorini et al., 1988). Although the effect reported by Tang et al. occurs in a very narrow range of cholesterol concen- tration that can be easily missed, an anomalous DPH inten- sity behavior was not noticed by us or others. To verify this inconsistency, we performed new experi- ments using DMPC vesicles labeled with DPH at choles- terol concentration from 17 to 24 mol %, where a profound dip of the DPH fluorescence intensity should occur accord- ing to Tang et al. (1995), and reproducing all the experi- mental conditions described by Tang et al. (1995). Our results are shown in Fig. 1, where the data from Tang et al. are also superimposed. Our results show a constant value of the DPH fluorescence intensity. We also measured DPH intensity decay in the same samples, and the data were analyzed using a model of two distributed lifetime components (Parasassi et al., 1991). The decay parameters do not show anomalous behavior versus cholesterol concentration (Fig. 1). In our opinion, the hypothesis of a regular distribution of cholesterol in the bilayer matrix at peculiar concentrations is quite interesting. Several recent papers reported evidences for such superlattice organizations of guest molecules in phospholipid membranes (Tang and Chong, 1992; Chong, 1994). We reported intensity (Parasassi et al., 1994) and generalized polarization (Parasassi et al., 1995) measure- ments of 2-dimethylamino-6-lauroylnaphthalene in various phospholipids as a function of temperature and of choles- terol concentration, indicating the existence of regular mo- lecular arrangements of cholesterol in the bilayer at peculiar guest molecule concentrations. In the data of Tang et al., what is surprising to us is the magnitude of the variation of DPH fluorescence intensity. A decrease of fluorescence intensity of -80% implies that -80% of DPH molecules are totally quenched, because the fluorescence lifetime of the remaining 20% DPH molecules Received for publication 27 September 1995 and in final form 20 Decem- ber 1995. ( 1996 by the Biophysical Society is not affected by the presence of cholesterol (Fig. 1). A membrane structure where most DPH molecules are quenched is difficult to imagine. Water is a well established quencher for DPH in the membrane and is the only possible quenching agent in the Tang et al. experiments. However, DPH in the presence of water develops a short lifetime component of - 1 ns, rather than being completely quenched (Parasassi and Gratton, 1995). Because of the magnitude of the effect reported, water should permeate the bilayer. To calibrate the magnitude of intensity changes, consider the variations of DPH intensity and lifetime values during the phospholipid phase transition, which are --15% (Fiorini et al., 1988; Parasassi et al., 1984). The gel to liquid-crystal transition involves major changes in the mem- brane organization and water penetration. Based on the changes reported by Tang et al., the 20 mol % cholesterol concentration should introduce in the bilayer even greater perturbation (in regard to DPH location and water penetra- tion) than the gel to liquid-crystal phase transition. From a methodological point of view, an important procedural question was, in our opinion, overlooked. To evaluate cho- lesterol effect, only the variation of fluorescence intensity in the individual samples was measured. Actually, other pa- pers reporting studies on regular molecular arrangements are also based only on fluorescence intensity data (Chong, 1994). The excimer/monomer ratio measurements of pyrene-PC (Tang and Chong, 1992; Chong, 1994) also depend on absolute concentrations. The reproducibility of intensity-based measurements is questionable, because of individual variability in sample preparation. In the data reported in this letter (Fig. 1), our best care could not avoid a 10% variability of DPH intensity! To reduce variability, in our studies of regular molecular arrangements we use gen- I-- CD 5r. 8 -X> intensity~ t 7 * ., - -.i.Y....M. a N, 1.0 cts CO 0.4 C/) I 0.2 a1) ) C. T Fraction ... ..M I. U . ....M U..... F--l L- intensity Tang et al. Cholesterol (mol%) O.0 DPH fluorescence parameters in DMPC vesicles at 24°C as a function of cholesterol concentration. Cl (-) and Fl (-), center and fraction of the main distributed component of DPH continuous lifetime distribution. Also reported is the DPH total fluorescence intensity data in our samples (X) and in those reported by Tang et al. (1995) (C]). |
| File Format | PDF HTM / HTML |
| DOI | 10.1016/S0006-3495(96)79720-5 |
| PubMed reference number | 8785314 |
| Journal | Medline |
| Volume Number | 70 |
| Issue Number | 3 |
| Alternate Webpage(s) | https://cloudfront.escholarship.org/dist/prd/content/qt2zh135x2/qt2zh135x2.pdf?t=oewbjc |
| Alternate Webpage(s) | https://doi.org/10.1016/S0006-3495%2896%2979720-5 |
| Journal | Biophysical journal |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
