Temperature sensitivity of fluorescence probes in the presence of model membranes and mitochondria

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Temperature sensitivity of fluorescence probes in the presence of model membranes and mitochondria
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  Volume 22, number 1 FEBS LETTERS April 1972 zyxwvuts TEMPERATURE SENSITIVITY OF FLUORESCENCE PROBES IN THE PRESENCE OF MODEL MEMBRANES AND MITOCHONDRIA J.M. VANDERKOOI and B. CHANCE zyxwvutsrqponmlkjihgfedcbaZYXWV Department zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA f Biophysics and Physical Biochemistry Johnson Research Foundation University of Pennsylvania Philadelphia Pennsylvania 19104 USA Received 18 February 1972 1. Introduction Biological membranes have been hypothesized to undergo phase transitions as a requirement of their physiological functions [ 1,2] . Various techniques have been applied to study the proposed phase transitions in both model and natural membranes, including X-ray analysis [3], electron microscopy [4,5], thermal ca- pacitance measurements [6] and recently use of spin labeled probes [7] and fluorescent probes [8,9]. Use of fluorescent probes appears especially promising in the detection of changes in membrane structure in view of the well established response of fluorescence probes to the energy state of biological membranes [lo] and their extreme sensitivity to environment zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH [l l] In this report phospholipid dispersions obtained by ultrasonication were used to calibrate fluorescence polarization responses observed in natural membranes. Fluorescent probes demonstrated by X-ray analysis to be located in different regions of model membranes [ 121 were chosen for this work. These include &ani- lino-1-naphthalene sulfonate (ANS), which on the basis of model systems is demonstrated to be a probe of the aqueous interface; dansylphosphatidylethanol- amine (DPE) and octadecyl naphthalene sulfonate (ONS), shown to be probes of the polar head group region, and 12(9-anthroyl)-stearic acid (AS) which is a probe for the hydrocarbon region of the membrane. Each of these dyes was found to be sensitive to the melting of the hydrocarbon chains of dipalmitoyl lecithin, evidenced by a discontinuity in the Perrin plots at 40”. Incorporation of cholesterol in a 1: 1 molar ratio with dipalmitoyl lecithin completely abol- ished the discontinuity in the Perrin plot of AS polar- North-Holland Publishing Company - Amsterdam ization. Perrin plots of AS or ANS fluorescence polar- ization in the presence of pigeon heart or rat liver mitochondria, or in the presence of egg lecithin were linear under the conditions used, in contrast to the biphasic curve obtained with pure dipalmitoyl lecithin. 16y /‘I I I/P 12 c ID I I 8 123456 I 2 3 4 5 6 T/q x IO-’ Fig.1. Perk plots of AS in the presence of artificial mem- branes. All samples were sonicated in the presence of 10 PM AS and 1 mM phosphate buffer, pH 7.2. Excitation was at 380 nm; emission measured at 450 nm. A 1 mg egg lecithin/ ml; B 1 mg dipalmitoyl lecithin/ml; C 1 mg dipalmitoyl lecithin/ml and 0.5 mg cholesterol/ml; D 1 mM dipalmitoyl lecithin and 0.1 mM coenzyme Q,. 23  Volume 22, number I FEBS LETTERS ONS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA PE 0 2 4 0 2 4 6 T/q x lO-4 Th x lO-4 Fig.2. Polarization of ONS and DPE incorporated into dipal- mitoyl lecithin micelles. Dipalmitoyl lecithin (1 mg/ml) was sonicated with 10 PM ONS (left) or 10 MM DPE (right) in the presence of 0.5 mM phosphate buffer, pH 7.2. Excitation for ONS, 350 nm; emission, 430 nm. Excitation for DPE, 340 nm; emission, 490 nm. T/n was varied by increasing (0) or decreasing (o) the temperature. 2. Materials and methods AS, ONS and DPE were the gracious gifts of Drs. AS. Waggoner and L. Stryer [ 131. ANS was obtained from Eastman Organic Chemicals and was recrystal- lized twice from hot water as the ammonium salt. Coenzyme Q and egg lecithin were the gifts of Drs. K. Folkers and K. Gulik, respectively. L-alpha-lecithin dipalmitoyl (synth) was obtained from Schwarz/Mann. Aqueous suspensions of phospholipids were dispersed ultrasonically with a Branson sonifier prior to each experiment for 1 min. 6- I/P 4- 2- Y-2-G-T T/v x IO-’ Fig. 4. Interaction of ANS with mitochondrial lipid. Mixture contained about 4 mg mitochondrial lipid/ml, 5 mM PO, buff- er, pH 7.4 and 30 nM ANS. Excitation, 360 nm; emission, 470 nm. 24 8 i I/P 4 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON   Is 2 4 T/11 April 1972 ZI 16 . . . l/P 8 ANS 2 4 T’1, Fig. 3. Polarization of AS and ANS in the presence of pigeon heart mitochondria. Left: Mitochondria (1 mg/ml) were incubated in the pres- ence of 10 MM AS, 0.225 M mamritol, 0.075 M sucrose and 0.05 M morpholinopropane sulfonate, pH 7.2 for 1 hr. Excitation, 380 nm; emission, 450 nm. Right: Mixture contained 1 mg mitochondrial protein/ml, 30 yM ANS, 225 M mannitol, 0.075 M sucrose, 0.05 M mor- pholinopropane sulfonate and 0.2 mM KCN. Excitation, 360 mn; emission, 470 mn. Due to low fluorescence intensity, unpolarized light was used for excitation. Fluorescence was measured at 90” from the excit- ing beam using a Hitachi MPF-2A fluorescence spectro- meter. Fluorescence polarization was measured with the aid of a 105PB Polacoat lens inserted between the exciting beam and the cuvette and between the cuvette and the photodetector. 5------ 3 4 5 T/T) x IO4 Fig. 5. Polarization of AS in the presence of mitochondrial lipid. Mitochondrial lipid (about 4 mg/ml) was sonicated in the presence of 10 rcM AS and 5 mM PO, buffer, pH 7.4. Excitation, 380 nm; emission, 450 nm.  Volume 22’ number zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA   FEBS LETTERS April 1972 Polarization zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA   was defined as r) _ ZII - zl -z,, z, where Zli and Z1 are corrected light intensity parallel and perpendicular to the exciting beam. Results were plotted according to the Perrin equation [ 141. Except where noted, polarized light was used for excitation. Pigeon heart mitochondria were prepared according to Chance and Hagihara [ 15 ] and rat liver mitochon- dria essentially according to Schneider [ 161. Mito chondrial lipid was extracted with 90 acetone [ 171. 3. Results and discussion 3.1. Fluorescence polarization of dyes incorporated into artificial membranes A plot of reciprocal polarization versus temperature over viscosity is linear for AS incorporated into egg lecithin (fig. 1A). In contrast, the Perrin plot for AS polarization in the presence of dipalmitoyl lecithin shows a sharp discontinuity at around 40°, at which temperature other studies have indicated melting to occur [ 181. Incorporation of cholesterol in a 1: 1 molar ratio to dipalmitoyl lecithin abolishes the dis- continuity in the curve and results in lower polariza- tion values for AS below the melting point of dipalm- itoyl lecithin and higher values above the melting point (fig. 1C). These results suggest that cholesterol disrupts the crystalline array of lecithin fatty acid chains, but under conditions when the chains are melt- ed, reduced mobility occurs in the presence of cho- lesterol. A similar conclusion was obtained using spin- labeled fatty acid in the presence of saturated lecithins [ 191. Incorporation of oxidized coenzyme Q, which is known to quench AS fluorescence both in model systems and in mitochondrial membranes [2], into dipalmitoyl lecithin micelles, results in an increased polarization of AS below SO”, but at the concentra- tions of Q used (1: 10 molar ratio with dipalmitoyl lecithin) the discontinuity in the Perrin plot persists (fig. ID). It is of interest to note that the change in hydro- carbon fluidity at the melting point of dipalmitoyl lecithin, inferred from AS depolarization, is also ob- served using ONS (fig. 2A) or DPE (fig. 2B) as flucl rescent probes. A similar result was obtained with ANS (not shown) in support of previous findings [2 1,221. Our results concerning DPE polarization are essen- tially in agreement with those of Lusson and Faucon [23] ; however, their failure to observe changes in ANS polarization at the melting point of dipahnitoyl lecithin is unresolved. 3.2. Fluorescence polarization of ANS and AS in the presence of mitochondria The Perrin plots of AS and ANS in the presence of pigeon heart mitochondria (fig. 3) or in the presence of lipid extract of mitochondria (figs. 4 and 5) are linear. Similar results were obtained when rat liver mitochondria were substituted for pigeon heart mitochondria. While polarization of AS and ANS decreases with increasing temperature, the absence of a discontinuity in the curve from 0 to 40” is indicative that a structural change such as a phase transition does not occur under these conditions. In contrast to the lack of any real evidence for a phase transition occurring in mitochondria as detected by ANS or AS, the spin-labeled compound 12-nitroxide stearate has been reported to be sensitive to phase transitions in a variety of mitochondria. Reasons for the discrepancy between the results observed with AS or ANS and the spin-labeled probe include the possible different binding properties of the various molecules of the membrane, the possible perturbation of the system by any of the probes resulting in alteration of the membrane which masks structural changes, or in the the case of the fluorescent probes, scatter of the experi- mental points may have obscurred a small but signifi- cant deviation from linearity in the Perrin plots. While discontinuities in the Perrin plots are gener- ally thought to reflect changes in the microviscosity of the dye environment, and polarization values are roughly correlated to “mobility” of the fluorescent dye, changes in fluorescence lifetime of the excited state as a function of temperature will also affect polarization values. A systematic study of fluorescent lifetime of the dyes used in this study has been under- taken, and a correlation between polarization values and the microviscosity of the probe environment should be possible to make. Acknowledgements This research was supported by the United States Public Health Service grant GM-1 2202-08. 25  Volume 22, number I FEBS LETTERS April 1972 eferences [1] J.P. Changeux, J. Thiery, Y. Tung and C. Kittel, Proc. Natl. Acad. Sci. U.S. 57 (1967) 335. [2] J.A. Lucy, J. Theoret. Biol. 7 (1970) 360. [3] V. Luzzati and F. Husson, J. Cell. Biol. 12 (1962) 207. [4] W. Stoeckenius, J. Cell. Biol. 12 (1962) 221. [5] E.L. Benedeiti and P. Emmelot, in: The Membranes, eds. A.J. Dalton and F. Haguenau (Academic Press, New York, 1968) p. 33. [6] J.M. Steim, M.E. Toartellotte, J.C. Reinert, R.N. McElhaney and R.L. Rader, Proc. Natl. Acad. Sci. U.S. 63 (1969) 104. [7] J.K. Raison, J.M. Lyers, R.J. Mehlhorn and A.D. Keith, J. Biol. Chem. 246 (1971) 4036. [8] J. Vanderkooi and A. Martonosi, Arch. Biochem. Biophys. 133 (1969) 153. [9] M. Kasai, J.P. Changeux and L. Monnerie, Biochem. Biophys. Res. Commun. 36 (1969) 420. [10] A. Azzi, B. Chance, G.K. Radda and C.P. Lee, Proc. Natl. Acad. Sci. U.S° 62 (1969) 612. [11 ] M. Shinitzky, A.C. Dianoux, C. Gitler and G. Weber, ~iochemistry 10 (1971) 2106. [12] W. Lesslauer, J. Cain and J.K. Blasie, Proc. Natl. Acad. Sci. U.S. in press. [13] A.S. Waggoner and L. Stryer, Proc. Natl. Acad. Sci. U.S. 67 (1970) 579. [14] F. Perrin, J. Phys. Radium 7 (1926) 390. [15] B. Chance and B. Hagihara, in: Proc. Vth Int. Congress Biochem., Moscow 1961, ed. A,N.M. Sissikian (Pergamon Press 5, 1963) p. 3. [16] W.C. Schneider, J. Biol. Chem. 176 (1948) 259. [17] R.L. Lester and S. Fleischer, Biochem. Biophys. Acta 47 (1961) 358. [18] D. Chapman, R.M. Williams and B.D. Ladbrooke, Chem. Phys. Lipids 1 (1967) 445. [19] G. Radda, B. Chance and M. Erecinska, unpublished results. [20] E. Oldfield and D. Chapman, Biochem. Biophys. Res. Commun. 43 (1971) 610. [21 ] J.M. Vanderkooi and A. Martonosi, Arch. Biochem. Biophys. 144 (1971) 87. [22] H. TrOuble, Naturwissenschaften 58 (1971) 6, 277. [23] C. Lussan and J.F. Faucon, FEBS Letters 19 (1971) 186. 26
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