INTERACTIONS OF N-ALKYL-2-PYRROLIDINONE SURFACTANTS WITH DMPC BILAYERS by YASEMİN KOPKALLI A dissertation submitted to the Graduate Faculty in Chemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy The City University of New York 2006 UMI Number: 3231940 3231940 2006 Copyright 2006 by Kopkalli, Yasemin UMI Microform Copyright All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 All rights reserved. by ProQuest Information and Learning Company. ii © 2006 YASEMİN KOPKALLI All Rights Reserved iii This manuscript has been read and accepted for the Graduate Faculty in Chemistry in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy. Chair of Examining Committee Lesley Davenport, Professor Executive Officer Gerald Koeppl, Professor Supervisory Committee Milton J. Rosen, Professor Emeritus Ruth E. Stark, Professor Brian Williams, Professor THE CITY UNIVERSITY OF NEW YORK iv ABSTRACT INTERACTIONS OF N-ALKYL-2-PYRROLIDINONE SURFACTANTS WITH DMPC BILAYERS by Yasemin Kopkallı Adviser: Professor Lesley Davenport There is great interest in identifying and characterizing new surfactants with predictable solubilization and reconstitution characteristics for use in membrane research. Among them, Triton X-100 and octyl glucoside (OG) are the best-studied surfactants. However, no physical data is available characterizing the interactions of two non-ionic pyrrolidinone surfactants, N-(2-ethylhexyl)-2-pyrrolidinone (C2,6P) and N-octyl-2- pyrrolidinone (C8P), with lipid bilayers. In the present study, their physical effects on dimyristoylphosphatidylcholine small unilamellar vesicles (DMPC SUVs) above the lipid phase transition temperature are described using surface tension, fluorescence spectroscopy, and isothermal titration calorimetry (ITC) methodologies. Surface tension methods show significant deviations from ideal behavior (attractive interactions) for these surfactant/lipid mixtures. In addition, fluorescence intensity studies of DPH and TMA-DPH labeled SUVs suggest that C8P interacts more favorably with v both the acyl chain and head group regions of the bilayer over the corresponding branched chain C2,6P analogue. This is further supported by fluoresecence emission anisotropy measurements (EA). Both surfactants affect the membrane lipid order within the acyl chain region, and the corresponding phospholipid ‘melt’ transition temperature (T m ) values, which decrease linearly with increasing surfactant concentrations. Using the van’t Hoff model, the lipid bilayer-water partition coefficients for these surfactants have been estimated from the depression of the lipid phase transition temperature. Values for the lipid partition coefficient for C8P [(1.22±0.08)× 10 4 ] versus C2,6P [(4.09±0.78)× 10 3 ] further supports a more favorable bilayer association for C8P. ITC data reveals an endothermic association for both C8P and C2,6P together with a somewhat higher binding affinity [(6.78±0.92)×10 -4 M] for C8P compared with C2,6P [(8.46±0.83)×10 -4 M], as predicted from fluorescence studies. We propose that C8P interacts and affects the lipid packing of bilayer membranes to a greater extent than C2,6P. Interactions of C2,6P appears to be more localized within the head group region of the bilayer. We conclude that C8P has great potential for biological applications, particularly membrane solubilization and membrane formation from micellar mixtures. In contrast, C2,6P may have potential for solubilization of extrinsic surface associated membrane proteins. vi To my father-in-law, Veli Kopkallı, who showed me the value of education and taught me to never give up, no matter what the cost. vii ACKNOWLEDGEMENTS I would like to thank Professor Lesley Davenport, my mentor, and Professor Milton J. Rosen, Professor Ruth E. Stark, and Professor Brian Williams, for serving on my thesis committee. I am very grateful to them for having reviewed this project at various stages and for providing valuable input given their individual areas of expertise. This dissertation would not have been possible without the tireless efforts of my advisor, Professor Lesley Davenport. She has taught me to work as an independent researcher and has been a steady source of wise encouragement and support throughout my doctoral studies. Very special thanks go to Professor Milton J. Rosen, director of the Surfactant Research Institute for providing N-alkyl-2-pyrrolidinone surfactants, opening his laboratory for surface tension measurements and more importantly for introducing me to the fascinating world of surfactants. A debt of gratitude goes to Professor Richard Magliozzo and to people in his laboratory for sharing isothermal titration calorimetry instrument with me in their laboratory. I am grateful to labmates and fellow graduate students Caleen Ramsook and Natalya Voloshchuk for always being there, their kindness and friendship. I want to thank my entire family, especially my mother and father Gülser and Ekrem Tura, for everything they have done for me. They have been by my side, with their unconditional love and support, throughout my entire life. Finally, and most importantly, I want to thank my husband and best friend, Halûk Kopkallı for drawing some figures in this thesis. His support, encouragement, and companionship turned my journey through graduate school into a pleasure. Let it go on record that he promised to read my entire thesis once it is submitted. viii LIST OF ABBREVIATIONS A min minimum area/molecule at the interface a 0 optimal lipid headgroup area C 0 1 molar concentration of surfactant 1 in the solution phase C 0 2 molar concentration of surfactant 2 in the solution phase 12 C molar concentrations of surfactant mixtures in the solution phase C M 1 critical micelle concentration of surfactant 1 in the mixed micelle C M 2 critical micelle concentration of surfactant 2 in the mixed micelle C M 12 critical micelle concentration of surfactant mixture in the mixed micelle C2,6P N-(2-ethylhexyl)-2-pyrrolidinone C8P N-octyl-2-pyrrolidinone C10P N-decyl-2-pyrrolidinone C12P N-dodecyl-2-pyrrolidinone C 12 PC n-dodecyl phosphocholine C 16 PC n-hexadecyl phosphocholine C12SNa sodium dodecanesulfonate C 20 efficiency of surface tension reduction by a surfactant. C 0 L surfactant concentration in the ligand delivery syringe C i,L surfactant concentration in the sample cell, after n i injections C 0 M DMPC SUVs concentration in the sample cell C i,M DMPC SUVs concentration in the sample cell, after n i injections cmc critical micelle concentration cmt critical micelle temperature cp cloud point DHPC 1, 2-diheptanoyl-sn-glycero-3-phosphocholine DMPC L-α-dimyristoylphosphatidylcholine DPH 1,6-diphenyl-1,3,5-hexatriene DPS dimethyl-dodecylamniopropane sulfonate EA fluorescence emission anisotropy F fluorescence emission intensity F 0 fluorescence intensities in the absence of surfactant GUV giant unilamellar vesicle h i heat of reaction HLB hydrophile-lipophile balance number i number of titration ITC isothermal titration calorimetry K binding constant (K a ) K a association constant; 1/K d K d dissociation constant; 1/K a K p partition coefficient k q biomolecular quenching constant ix K SV Stern-Volmer quenching constant [L] free ligand concentration [L] t total ligand concentration l c critical chain length LAS linear alkylbenzenesulfonate LUV large unilamellar vesicles [M] t total macromolecule concentration MLV large multilamellar vesicles MVV multivesicular vesicles n surfactant to lipid molar ratio N aggregation number OG octyl glucoside pC 20 efficiency of interfacial tension reduction PC phosphatidylcholines PE phosphatidylethanolamines POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol Q cumulative heat of binding <r> steady-state fluorescence emission anisotropy r 0 limiting anisotropy R gas constant R 2 correlation coefficient Re sat molar ratio at saturation point Re sol molar ratio at solubilization point SDS sodium dodecyl sulfate S/L surfactant to DMPC SUVs molar ratio SUVs small unilamellar vesicles T temperature T m gel to liquid-crystalline phase transition TMA-DPH trimethylammonium 1,6-diphenyl-1,3,5-hexatriene UV ultraviolet V cell sample cell volume V inj volume of injection V 0 volume of the cell X 1 mole fraction of surfactant 1 in the total mixed monolayer X M 1 mole fraction of surfactant 1 in the total mixed micelles α mole fraction β M molecular interaction parameters for mixed micelle formation β σ molecular interaction parameters for mixed monolayer formation Γ surface excess concentration γ surface tension δhi corrected heat of titrations ∆G free energy of binding ∆G 0 ad standard free energy of adsorption [...]... Titration of DPH labeled DMPC SUVs with C8P at 30°C 112 Fluorescence emission spectra: Titration of DPH labeled DMPC SUVs with C2,6P at 30°C 113 Fluorescence emission spectra: Titration of DPH labeled DMPC SUVs with buffer at 30°C 114 25 Titration of DPH labeled DMPC SUVs with surfactants at 30°C 115 26 DPH embedded in DMPC SUVs, correction of Figure 25 for dilution effect by subtraction of control... presence of C2,6P (■) and C8P (∆) 116 Fluorescence emission spectra: Titration of TMA-DPH labeled DMPC SUVs with C8P at 30°C 119 Fluorescence emission spectra: Titration of TMA-DPH labeled DMPC SUVs with C2,6P at 30°C 120 23 24 27 28 xix Figure 29 Page Fluorescence emission spectra: Titration of TMA-DPH labeled DMPC SUVs with buffer at 30°C 121 30 Titration of TMA-DPH labeled DMPC SUVs with surfactants. .. embedded in DMPC SUVs, correction of Figure 30 for dilution effect by subtraction of control from samples In presence of C2,6P (■) and C8P (∆) 123 DPH labeled DMPC SUVs phase transition temperatures with no surfactants as a control 127 Effect of C8P surfactant on the phase transition temperature of DPH labeled DMPC SUVs 128 Effect of C2,6P surfactant on the phase transition temperature of DPH labeled DMPC. .. Corrected heat of reaction (δhi) per injection in kilocalories per mole of C8P surfactant versus molar ratio (C8P /DMPC SUVs) 161 Corrected heat of reaction (δhi) per injection in kilocalories per mole of C2,6P surfactant versus molar ratio (C2,6P /DMPC SUVs) 162 Proposed model of the interactions of C8P and C2,6P surfactants with DMPC SUVs 171 48 49 50 51 52 1 CHAPTER 1: INTRODUCTION 1.1 Aim of the Research... temperature of TMA-DPH labeled DMPC SUVs 136 Effect of surfactant on the phase transition temperature (Tm) of DMPC SUVs as reported from the steady-state emission anisotropy of TMA-DPH (∆) C8P; and (■) C2,6P 137 Plot of the depression of the phase transition temperature - T, against the concentration of added surfactant obtained with DPH as the probe (□) C2,6P; (▲) C8P 144 Plot of the depression of the... steady-state emission anisotropy of DPH 131 Effect of surfactant on the phase transition temperature (Tm) of DMPC SUVs as reported from the steady-state emission anisotropy of TMA-DPH 138 Partition coefficients ( K p ) of C8P and C2,6P surfactants between water and DMPC vesicle membranes obtained from plots of depression of the phase transition temperature - T versus the concentration of added surfactant 143... labeled DMPC SUVs 129 Effect of surfactant on the phase transition temperature (Tm) of DMPC SUVs as reported from the steady-state emission anisotropy of DPH (∆) C8P; and (■) C2,6P 130 TMA-DPH labeled DMPC SUVs phase transition temperatures with no surfactants as a control 134 Effect of C8P surfactant on the phase transition temperature of TMA-DPH labeled DMPC SUVs 135 Effect of C2,6P surfactant on the... the mixedmicelle process, three kinds of interactions exist between the monomers of two unlike surfactants, i.e., favorable interactions, nonfavorable interactions, or ideal mixing (no interaction) 4 Fluorescence spectroscopy provides a sensitive tool for the examination of the resultant effects of surfactants on the physical properties of lipid membranes and bilayers [27-55] To achieve our aim fluorescence... coefficients of other non-ionic surfactants for lipid vesicles estimated from the depression of phase transition temperature of vesicle membrane 146 11 Thermodynamic parameters of partitioning C8P and C2,6P surfactants into DMPC SUVs as reported from the isothermal titration calorimetry at 30°C 163 xvi Table 12 13 Page Thermodynamic parameters of partitioning OG, C8P and C2,6P surfactants into DMPC vesicles... τ Φ enthalpy of binding entropy of binding depression of the phase transition temperature molar extinction coefficient wavelength with maximum emission intensity hydrocarbon volume effectiveness of interfacial tension reduction lifetime quantum yield xi TABLE OF CONTENTS Abstract iv Dedication vi Acknowledgements vii List of Abbreviations viii Table of Contents xi List of Tables xv List of Figures xvii . Titration of TMA-DPH labeled DMPC SUVs with buffer at 30°C. 121 30. Titration of TMA-DPH labeled DMPC SUVs with surfactants at 30°C. 122 31. TMA-DPH embedded in DMPC SUVs, correction of Figure. Titration of DPH labeled DMPC SUVs with C2,6P at 30°C. 113 24. Fluorescence emission spectra: Titration of DPH labeled DMPC SUVs with buffer at 30°C. 114 25. Titration of DPH labeled DMPC. INTERACTIONS OF N-ALKYL-2-PYRROLIDINONE SURFACTANTS WITH DMPC BILAYERS by Yasemin Kopkallı Adviser: Professor Lesley Davenport There is great interest in identifying and characterizing new surfactants