developing a method for measuring the binding of ...

DEVELOPING A METHOD FOR MEASURING THE BINDING OF CHOLESTEROL TO LIPIDS THROUGH THE RELATIVE AMOUNT OF CHOLESTEROL THAT PARTITIONS BETWEEN LUVs AND CYCLODEXTRIN USING THE ANISOTROPY OF FLUORESCENCE Robert Vittoe, Bruce Ray, Stephen Wassall Department of Physics, Indiana University-Purdue University Indianapolis Indianapolis, IN 46202 Abstract The goal of this project is to develop a method for measuring the binding of cholesterol to lipids by using the anisotropy of fluorescence. This will be accomplished through the relative amount of cholesterol that partitions between the large unilamellar vesicles (LUV) and Methyl-β-Cyclodextrin that is added to the LUV. The LUV initially contained a measured concentration of cholesterol. A standard curve was created for the anisotropy from the florescence of a given range of cholesterol from zero mole percent to 30 mole percent in the lipid 1,2 Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) using the fluorophore 1,6 Diphenyl-1,3,5 Hexatrienen (DPH). Then the resulting cholesterol percentage was calculated from the observed anisotropy due to the removal of cholesterol using concentrations of Methyl-β-Cyclodextrin ranging from 0.5 mM to 4.0 mM in a 20 mole percent standard stock of cholesterol in DOPC. The 1.0 mM Cyclodextrin sample gave an anisotropy value that corresponded to 17 mole percent cholesterol on the standard curve and the 2.0 mM Cyclodextrin sample gave an anisotropy value that corresponded to 11.3 mole percent cholesterol on the standard curve. Introduction Fluorescence is a property of certain types of atoms and molecules. It is where an atom absorbs photons of certain wavelength thereby exciting electrons to a higher energy level. After a period of time, the fluorescence lifetime (τ), the electrons fall to a lower energy level emitting photons of a longer wavelength. Fluorescence was first described by Sir George G. Stokes in 1852. He coined the term in honor of the fluorescent mineral fluorite. The first fluorescence microscopes were developed around 1911 by Otto Heimstadt and Heinrich Lehmann. Fluorophores preferentially absorb photons that have an electric field vector aligned parallel to the absorption transition dipole moment of the fluorophore. Excitation of a fluorphore from the ground state and the emission of a photon through fluorescence are measured in quanta. [1]

𝑟=

𝐼‖ − 𝐼⊥ 𝐼‖ + 2𝐼⊥

Where r is the anisotropy, 𝐼‖ is the intensity parallel and 𝐼⊥ is the intensity perpendicular. The G factor is the ratio of the sensitivities of the detection system for vertically and horizontally polarized light. 𝐺=

𝐼‖ 𝑆𝑉 𝐼‖ 𝐼VV 𝑆𝑉 ,𝐺 = = 𝑆𝐻 𝐼⊥ 𝑆𝐻 𝐼⊥ 𝐼𝑉𝐻

Where G is the G factor, 𝑆𝑉 is the sensitivity of the emission channel for the horizontally polarized component, 𝑆𝐻 is the sensitivity of the emission channel for the vertically polarized 𝑆 component, 𝑆𝑉 is the ratio of sensitivities of the detection 𝐻

Fig. 1: Fluorophore DPH Molecule courtesy of chemicalbook.com

According to Planck’s law the energy in a quantum is expressed by the equation. [2] 𝐸 = ℎ𝜈 = ℎ

𝑐 𝜆

Where E is the energy, h is Planck’s constant, ν is frequency, c is the speed of light and λ is wavelength. Isotropy is where light is emitted without preference with regard to direction. Anisotropy is the property of an atom or molecule emitting light preferentially thereby having a different value along a particular direction. According to Lakowicz in “Principles of Fluorescence Spectroscopy”, “Transition moments for absorption and emission have fixed orientations within each fluorophore, and the relative angle between these moments determines the maximum measured anisotropy.” The fluorescence anisotropy (r) is defined by [3]

system for vertically and horizontally polarized light, 𝐼‖ is the intensity parallel and 𝐼⊥ is the intensity perpendicular, 𝐼VV and 𝐼𝑉𝐻 are the polarized intensities for the vertically polarized excitation. The G factor is used to calculate the intensity ratio. The G factor is measured using horizontally polarized excitation.[3] Including the G factor the formula for anisotropy becomes [4] 𝑟=

𝐼‖ − 𝐺𝐼⊥ 𝐼‖ + 2𝐺𝐼⊥

Where r is the anisotropy, 𝐼‖ is the intensity parallel and 𝐼⊥ is the intensity perpendicular and G is the G factor. In order to create unknown concentrations of cholesterol in the lipid DOPC different concentrations of Cyclodestrin were added to the 20 mole percent standard stock. The Cyclodextrin pulls the cholesterol from the solution in a 2 to 1 ratio. That is, two Cyclodextrin molecules will bind with one cholesterol molecule. Then the anisotropy of each sample was measured and compared to the standard curve to determine the amount of cholesterol that remained in the DOPC.

A higher concentration of cholesterol in the membrane results in a higher level of order for the molecules in the membrane. A higher level of order for the molecules reduces their freedom of motion resulting in a higher level of anisotropy.

measurements were repeated five times for each sample. Each run lasted 30 seconds with a resolution of 0.02 seconds. An average of the five runs for each mole percent sample was then calculated to determine the standard curve. Methyl-β-Cyclodextrin was then added to the 20% standard stock to create 4 mM, 2 mM, 1 mM and 0.5 mM samples. The samples were allowed to incubate for one hour at room temperature. They were then analyzed on the LS50B using the same procedure and configuration as the standard curve samples.

Fig. 2: Lipid Bilayer Containing Cholesterol and DPH

Experiment The standard curve of anisotropy (r) was determined by preparing five different cholesterol samples ranging from zero to thirty mole percent. Figure 3 shows the formulation used to create each sample. Mole % 0% 5% 10% 15% 30% 20% Standard Ratio 19:1 9:1 17:3 7:3 Stock 0.04 mM 79.5 μl 79.5 μl 79.5 μl 79.5 μl 79.5 μl 318 μl DPH 10 mg/ml 50 μl 50 μl 50 μl 50 μl 50 μl 200 μl DOPC 10 mg/ml 1.30 μl 2.73 μl 4.33 μl 10.54 μl 24.60 μl Cholesterol 1 mg/ml 12.95 μl 27.25 μl 43.30 μl Cholesterol

Results and Discussion Figure 4 shows the chart of the data collected for the standard cholesterol curve at ~24o C. The anisotropy ranged from an average r = 0.0958 with a standard deviation of 0.0079 for zero mole percent sample to an average r = 0.1160 with a standard deviation of 0.0079 for the thirty mole percent sample. 24o C - Anisotropy Cholesterol % 1 2 3 4 5 Average

0% r 0.0940 0.0966 0.0953 0.0956 0.0975 0.0958

SD 0.0080 0.0080 0.0081 0.0079 0.0075 0.0079

5% r 0.0983 0.0980 0.0984 0.0972 0.0972 0.0978

SD 0.0080 0.0073 0.0076 0.0078 0.0077 0.0077

10% r 0.1035 0.1034 0.1026 0.1038 0.0994 0.1025

SD 0.0073 0.0075 0.0077 0.0079 0.0076 0.0076

15% r 0.1066 0.1051 0.1052 0.1044 0.1045 0.1052

SD 0.0087 0.0074 0.0073 0.0080 0.0078 0.0078

30% r 0.1168 0.1150 0.1169 0.1146 0.1168 0.1160

SD 0.0077 0.0078 0.0074 0.0086 0.0078 0.0079

Fig. 4: Data Chart of Standard Curve Results

Figure 5 shows the graph of the data collected for the standard cholesterol curve at ~24o C.

Fig. 3: Mole % Standard Curve Formulation Table

After the samples were mixed they were dried down using Argon gas and then placed under vacuum overnight. Each sample was rehydrated the next morning using 1 ml of NaPO4, pH 7, 10 nM buffer solution. The samples then went through three rounds of a freeze/thaw process alternating between freezing in liquid nitrogen and thawing in a 37 o C water bath. The samples were each then extruded at room temperature, ~24o C, twenty one times through a Nuclepore Track-Etch membrane 19 mm, 0.1 µm to make Large Unilamellar Vesicles (LUV). The samples were then placed in 1 ml cuvettes for analysis. The samples were analyzed at an ambient room temperature of ~24o C in a Perkins Elmer Luminescence Spectrometer LS50B with the excitation wavelength set at 351 nm and the emission wavelength set at 430 nm. The G factor was determined to be 1.65. The excitation slit was set to 15 nm and the emission slit was set to 5 nm. This configuration tended to reduce the noise thereby giving the results with the lowest standard deviation. The anisotropy

Fig. 5: Graph of Standard Curve Results

A standard curve will need to be created each time an experiment is performed to account for changes in temperature and slight variances in concentrations of the mixtures. A different standard curve will also need to be created for each different type of lipid as it is used.

Figure 6 shows the chart of the anisotropy data collected from the samples prepared from the twenty mole percent stock with Cyclodextrin added. The 0.5 mM Cyclodextrin sample had an average r = 0.1093 with a standard deviation of 0.0080 which corresponded to ~20 mole percent. No cholesterol was observed to be removed from this sample by the Cyclodextrin. The 1.0 mM Cyclodextrin sample had an average r = 0.1069 with a standard deviation of 0.0080 which corresponds to ~17 mole percent. The 2.0 mM Cyclodextrin sample had an average r = 0.1030 with a standard deviation of 0.0081which corresponds to ~11.3 mole percent. The 4.0 mM Cyclodextrin sample had an average r = 0.0945 with a standard deviation of 0.0079 which corresponds to zero mole percent. All the cholesterol was observed to be removed from this sample by the Cyclodextrin.

portion of the DPH is being pulled out along with the cholesterol. [8] Cyclodextrin has been shown to remove DPH in a 1 to 1 ratio. Conclusion The experimental results for the measured anisotropy seem to confirm that Cyclodextrin is removing cholesterol from the LUV. However, the discrepancy between the kx measured in this experiment and the values obtained by other methods suggests that the anisotropy for DPH doesn’t accurately reflect the amount of cholesterol left in the LUV. More experimentation needs to be performed to determine if Cyclodextrin is in fact removing a portion of the DPH from the LUV. References

24o C - Anisotropy mM Cyclodextrin 1 2 3 4 5 Average

0.5 r 0.1092 0.1105 0.1097 0.1085 0.1085 0.1093

SD 0.0080 0.0081 0.0082 0.0076 0.0081 0.0080

1.0 r 0.1073 0.1083 0.1062 0.1052 0.1075 0.1069

SD 0.0083 0.0076 0.0079 0.0080 0.0082 0.0080

1. 2.0

r 0.1022 0.1037 0.1027 0.1026 0.1040 0.1030

SD 0.0075 0.0079 0.0084 0.0084 0.0085 0.0081

4.0 r 0.0949 0.0940 0.0942 0.0952 0.0941 0.0945

SD 0.0083 0.0079 0.0083 0.0075 0.0074 0.0079

2.

3.

Fig. 6: Data Chart of Cyclodextrin Results

4.

5.

6.

Fig. 7: Graph of Cyclodextrin Results

The partition coefficient, kx was calculated using the formula. [5-7] 𝑘𝑥 =

7.

[𝐿𝑈𝑉 + 𝑐ℎ𝑜𝑙]([𝑐𝑦𝑑𝑒𝑥] − 2[𝑐𝑦𝑑𝑒𝑥 + 𝑐ℎ𝑜𝑙])2 ([𝐿𝑈𝑉] + [𝐿𝑈𝑉 + 𝑐ℎ𝑜𝑙])[𝑐𝑦𝑑𝑒𝑥 + 𝑐ℎ𝑜𝑙]

Where LUV is large unilamellar vesicles in mM, cydex is Cyclodextrin in mM and chol is cholesterol in mM. The partition coefficient kx = 4.93 for the 2 mM Cyclodextrin sample and kx = 5.93 for the 1 mM Cyclodextrin sample. These values are less than the values (mid 20’s) measured by other methods. One possibility for this discrepancy is that a

8.

Brian Herman, V.C.F., Josph Lakowicz, Douglas Murphy, Kenneth Spring, Michael Davidson. Basic Concepts in Fluorescence. [web page] 2012 [cited 2013 May 26]; Available from: http://www.olympusmicro.com/primer/techniques/fl uorescence/fluorescenceintro.html. Planck, M., On the Law of Distribution of Energy in the Normal Spectrum. Annals of Physics, 1901. vol. 4: p. p. 553 ff Lakowicz, J.R., Principles of Fluorescence Spectroscopy. 1st Edition ed1983, New York, NY: Plenum Press. 496. William Stillwell, T.D., Alfred Dumaual, Thomas Crump, Laura Jenski, Cholesterol versus alphaTocopherol: Effects on Properties of Bilayers Made from Heteroacid Phosphatidylcholines. Biochemistry, 1996. 35: p. 13353-13362. Alekos Tsamaloukas, H.S., Peter J. Slotte, Heiko Heerklotz, Interactions of Cholesterol with Lipid Membranes and Cyclodextrin Charactgerized by Calorimetry. Biophysical Journal, 2005. 89(August 2005): p. 1109-1119. Justin Williams, Cynthia Wassall, Maureen Kagimbi, Christopher Eslinger, Marvin Kemple, Stephen Wassall, Cholesterol-Lipid Affinnty Determined by EPR. Biophysical Journal, 2012. 102(3): p. 293a-294a. Justin Williams, Cynthia Wassall, Marvin Kemple, Stephen Wassall, Dependence of CholesterolPhospholipid Affinity on Degree of Acyl Chain Unsaturation as Determined by EPR. Biophysical Journal, 2013. 104(2): p. 588a. G. Pistolis, A.M., Nanotube Formatioin between Cyclodextrins and 1,6-Diphenyl-1, 3, 5-Hexatriene. Journal of Physical Chemistry, 1996. 1996(100): p. 15562-15568.