Effect of 2,4-dichlorophenol on biological model membranes [Elektronische Ressource] / vorgelegt von Agnes Csiszár

Klauth

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2003
• Nombre de lectures: 18
• Poids de l'ouvrage: 5 Mo

Extrait

Effect of 2,4-dichlorophenol on biological model
membranes



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der
Rheinisch-Westfälischen Technischen Hochschule Aachen zur Erlangung des
akademischen Grades einer Doktorin der Naturwissenschaften genehmigte
Dissertation

vorgelegt von

Diplom-Chemikerin

Agnes Csiszár
aus Szombathely






Berichter: Prof. Dr. Andreas Schäffer (RWTH Aachen)
Univ. Dozent Dr. Attila Bóta (TU Budapest)

Tag der mündlichen Prüfung: 14.11.2003.






Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

Content

1. Introduction................................................................................................ 1
2. Theoretical background; model membranes and their investigations............... 3
2.1. Biological membranes.....................................................................................................3
2.2. Lipids and model membranes..........................................................................................5
2.3. Structures of lyotrope mesophases..................9
2.4. Phase transitions in lyotrope systems .............................................................................14
2.5. Effect of guest molecules on model membranes..............................................................17
2.6. Some physicochemical methods for investigation of liposome systems ............................19
3. Goal of this work.......................................................................................26
4. Experimental part.27
4.1. Preparation of DPPC/water system...............................................................................27
4.2. Preparation of DCP/DPPC/water systems.....27
4.3. Experimental condition of DSC measurements...............................................................27
4.4. Experimental conditions of X-ray diffraction measurements............................................28
4.5. Experimental conditions of freeze-fracture/TEM investigations........28
4.6. Experimental conditions of FT-Raman spectroscopy stuies ............................................29
4.7. Computational methods and stategy of normal mode assignments...29
5. Results and discussions .............................................................................30
5.1. Investigation of the pure fully hydrated DPPC/water system...........31
. -5 5.2. Effect of DCP on DPPC liposmes in the DCP/DPPC molar ratio range from 4 10
. -3 up to 4 10 mol/mol.............................................................................................................47
. -2 5.3. Effect of DCP on DPPC liposmes in the DCP/DPPC molar ratio range from 2 10
. -1 up to 1 10 mol/mol.............................................................................................................53
. -1 5.4. Effect of DCP on DPPC liposmes in the DCP/DPPC molar ratio range from 2 10
. -1 up to 4 10 mol/mol.............................................................................................................63
. -5 5.5. Effect of DCP on DPPC liposmes in the DCP/DPPC molar ratio range from 6 10
up to 1.2 mol/mol ................................................................................................................69
5.2. Conclusions..................80
6. Summary...................................................................................................83
7. References................................85
List of figures .................................................................93
List of tables.................................97

1. Introduction

Biological membranes simultaneously serve as a barrier for the intracellular
environment and for the transduction of energy as well as signals between the cell and the
environment. The determination of the role of membrane components is important both for
fundamental scientific research and for practical applications. The concepts of the functions
and structures of the two main membrane components, proteins and lipids, have drastically
changed since their discovery in the last century. Early studies from the seventies considered
the lipid bilayer to be solely a matrix, in which the proteins were embedded. These molecules
allow the selective uptake and excretion of solutes and play a crucial role in the energy status
of the cell and in the regulation of the intracellular environment. Subsequent studies from last
decades revealed that the structure of the bilayer matrix is of great significance because its
physicochemical properties can strongly influence the functions and structure of the
embedded components.
Because of the complexity of understanding and investigation of the functions of
biological membrane components - like proteins or the lipid matrix - model systems have
been developed. To study the properties of lipid bilayers without the confounding effects of
proteins or other membrane components liposomes are frequently used. Liposomes, formed
by various lipids in aqueous solution, are ideal model membranes. They can consist of one
lipid bilayer membrane enclosing water in the middle (unilamellar vesicle) or of concentric,
onion-like shells of altering lipid bilayers and water layers (multilamellar vesicle). Liposomes
mimic the structure of biomembranes and they are able to model physicochemical properties
of biological membranes, for example structural changes depending on temperature and
pressure, thermal behaviors of phase transitions, and diffusion processes through the
+membrane. In fact, the transport processes of small ions and molecules such as, H, H O, 2
+ + 2+metal ions (e.g. Na , K , Ca (Roux and Bloom 1990)) have been successfully characterized
using liposomes. Recent studies demonstrated that model membranes are particularly useful
for studying the toxic effect of pollutants, xenobiotics, and pharmaceutical compounds. The
major advantage of these approaches is that they yield information on the mechanisms and
dynamics of the damaging processes in the cell membrane.
Chlorophenols are organic recalcitrant compounds which are weakly soluble in water
and may occur from contaminated soil to groundwater. In contaminated industrial areas (such
as manufacturing plants, wood-treatment facilities, municipal waste discharges and in
1
receiving waters adjacent to these sources) chlorophenols are frequently present at
concentrations that are known to have diverse biological effects (Jensel 1996).
Chlorophenols are lipophilic molecules and their toxicity is primarily connected to the
effects on cell membranes. However, it is still unclear whether the primary site of action is the
lipid matrix or the membrane bound proteins. Previous studies suggested that the
accumulation of chlorophenols in membranes increases the fluidity of the lipid matrix leading
+ +to an inhibition of membrane bound proteins such as the Na /K - ATPase and the glycose
transport system (Cascorbi and Ahlers 1989). Chlorophenols are also thought to uncouple the
cellular energy production by dissipating the chemiosmotic proton gradient (Escher 1996,
Argese 1999).
Biological membranes also play a vital role in the removal of chlorophenols from the
environment. In ground waters and soils aerobic and anaerobic biodegradation is the main
route of removal for chlorophenols, primarily via oxidative dechlorination and hydroxylation
(Kaufman D.D. 1978) which are catalyzed by biomembrane-associated enzyme systems.
Despite the biological, ecological and economical importance, less data is available about the
direct actions of chlorophenols on the physicochemical properties of biological membranes.
Based on the aforementioned studies it can be hypothesized that chlorophenols perturb
the lipid matrix as well as the proteins. The changes of the lipid bilayer structure induced by
chlorophenols may be drastical causing perturbing effects on protein activity and on the
transport processes through the membrane. To obtain a better understanding of the mechanism
of the effect of chlorophenols on the lipid bilayer the interactions between the 2,4-
dichlorophenol (DCP) and 1,2-dipalmitoyl-sn-glicerophosphocholine (DPPC) liposomes were
investigated. The thermal behaviors of liposome systems were studied by differential
scanning calorimetry (DSC). To investigate structural properties, such as layer formation,
lamellarity, and molecular order in the layer, small- and wide-angle X-ray scattering (SAXS
and WAXS) were used. The surface patterns of lipid bilayers were investigated by freeze-
fracturing combined with high resolution electron microscopy (TEM). To obtain information
about the conformational changes and the reoriented chemical bonds of lipid molecules
molecular spectroscopy was used.
2
2. Theoretical background
2.1. Biological membranes

Before life was possible, some barrier was required between the life processes and
their environments in which those processes were occurring. The plasma membrane provided
the necessary bo