PRESERVATION OF POPC MODEL MEMBRANE INTEGMTY

B Y TREHALOSE. A *H AND 31P NMR STUDY.

'4 Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

b y

STEWART F. HAYNE

In partial hlfilment of requirements

for the degree of

Master of Science

December, 1997

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ABSTRACT

PRESEEtVATION OF POPC MODEL MEMBRANE INTEGRITY BY

TREHALOSE. A *H AND 31P NMR STUDY.

Stewart F. Hayne Cniversity of Guelph. 1997

.A dtisor : Professor K.R. Jeffrey

The role of trehalose in maintaining model membrane inteogity in the presence of

dehydration \la freezing or desiccation is investigated using nuclear magnetic reso-

nance (34IR) techniques. 'H XMR spectra were recorded for DÎO/POPC mixtures

as a function of hydration and temperature to determine the hydration dependence

of the gel to liquid crystal phase transition. 2~ NMR spectra were also recorded for

deuterated head goup POPC in the presence of trehalose as a Function of temper-

ature to investigate the influence of trehalose on the head group. It was found that

adding trehalose resulted in an increase in the alpha position deuterium quadrupolar

splitting. This is due to the trehalose changing the conformation of the headgroup.

resulting in a change in average angle with respect to the surface of the lipid bilayer.

From 31P 'iMR spin-lattice relaxation and a model for the relaxation. an effective

correlation time for the headgroup reorientation was determined as a function of tem-

perature. From t hese data activation energies for the headgroup reorientation were

determined for each hydration. From analysis of the correlation time data it appears

t hat trehalose does influence the headgroup dynamic at low water content.

Acknowledgements

1 would like to thank rny advisor. Dr. Ken Jeffrey for all his assistance. instruction.

and ,guidance throughout the entirety of this project. 1 would also like to thank my

parents. Dr. Ormille and Mary Haqne? for providing the opportunity and encouraging

me to attend university and pursue higher educational goals. Without them I would

have accomplished nothing. I wodd iike to thank Christian Schroeder for coniincing

me to write this t,hesis in the BT~Xpublishing package. and Al Richardson for giving

me a format template From which to start. I would also iike to thank al1 my fellow

students and friends who have helped and entertained me throughout my universit-

career .

Contents

1 Introduction

2 Membranes

2.1 Fluid hIosaic Mode1 . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . 2.2 The Hydrophobie Effect

2.3 Geometrical Packing . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3 Lipid phases and transitions . . . . . . . . . . . . . . . . . . . . . . .

2.5 Trehalose and its role in croprotection . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Aims of this project

3 Theory of Nuclear Magnetic Resonance

3.1 Basic Theory of Suclear Magnetic Resonance . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . 3 2 Xuclear Spin Interactions

. . . . . . . . . . . . . . . . . . . . 3 1 Dipole- Dipole Interaction

3.2.2 .A nisotropic Chernical Shift . . . . . . . . . . . . . . . . . . . .

3.2.3 Nuclear Electric Quadrupole Interaction . . . . . . . . . . . .

3.3 Spin- Latt. ice Relaxation . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 Preklous S tudies

4 klaterials and Methods

4.1 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2 E'cperimental Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4 2.1 Superconducting Magnet . . . . . . . . . . . . . . . . . . . . .

4.2.2 NLIR FT-Spectrorneter . . . . . . . . . . . . . . . . . . . . . . 47

4.3 Pulse Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

5 Experimental Results and Discussion 54

- L .?.I Phase behaviour of POPC . . . . . . . . . . . . . . . . . . . . . . . . aa

3.1.1 L\,-ater content . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

5.2 TheeffectoftrehaloseonPOPC. . . . . . . . . . . . . . . . . . . . . 69

5.3 Spin-Lattice Relaxation Measurements . . . . . . . . . . . . . . . . . 73

-7 ? 5.3.1 Correlationtimes . . . . . . . . . . . . . . . . . . . . . . . . . r (

5 3 2 Activation Energy Calcuiations . . . . . . . . . . . . . . . . . 8 1

6 Conclusions and Future Considerations 90

6 . Conclusions . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . - 90

6.2 Future considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1

Schematic Diagam of Probe Assembly . . . . . . . . . . . . . . . . . 50

Schematic of a typical Ti rneasurement. . . . . . . . . . . . . . . . . 52

Typical T i rneasurement c u v e . . . . . . . . . . . . . . . . . . . . . . 53

Spectra Eor D20:POPC 4:l . . . . . . . . . . . . . . . . . . . . . . . . 59

Quadrupolar splitting as a function of temperature for D20:POPC 4:1 60

Spectra for D20:POPC 1311 . . . . . . . . . . . . . . . . . . . . . . . 61

Q~adrupolarsplittingforD~0:POPC 13:l . . . . . . . . . . . . . . . 62

Liquid crystal to gel transition temperature as a function of hydration

for D20:POPC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

E.xperimenta1 and t heoretical curves showing water absorption of H20

and D20 by POPC at 25 degrees Celsius . . . . . . . . . . . . . . . . 66

Spectra for D20 and POPC (95% RH at 308 K) . . . . . . . . . . . . 67

LineMdth measurements for D20 and POPC (95% RH at 308 K ) . . 68

SpectraforPOPC-d13:trehalose1:295%RH . . . . . . . . . . . . . . i l

Quadrupolar split ting and linewidt h measurements for POPC-d13 : trehalose

1:- 95% RH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Spin lattice relaxation rneasurement of POPC and trehalose 2:1 at

w- 15,57,76and95%RH . . . . . . . . . . . . . . . . . . . . . . . . . . KI

31 Spin lattice relaxation rneasurement of POPC a t 57% RH and POPC

and trehalose 12 at 57% RH . . . . . . . . . . . . . . . . . . . . . . 76

32 Correlation times for 95. 76. 57. 15% RH POPC and trehalose samples 79

33 Correlation times for POPC and POPC: trehalose samples at 57% RH 80

34 Correlation times for POPC/trehalose l:2 equilibrated to 95% RH with

Linear regession and error bars representing 3% error. . . . . . . . . . 84

35 Correlation times for POPC equilibrated at 57% RH with linear re-

session . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

36 Correlation times for POPC and trehalose 1:2 equilibrated at 95% RH

with linear regression . . . . . . . . . . . . . . . . . . . . . . . . . . .

37 Correlation times for POPC and trehalose 1 :2 equilibrated at 76% RH

wit h linear regression . . . . . . . . . . . . . . . . . . . . . . . . . . .

38 Correlation t imes for PO PC and t rehalose 1 :2 equilibrated at 57%' RH

wit h linear regession . . . . . . . . . . . . . . . . . . . . . . . . . . .

39 Correlation times for POPC and trehalose 1:2 equilibrated at 15% RH

with linear regression . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 Introduction

'iuclear magnetic resonance (NMR). was pioneered by gooups lead by Purcell and

Bloch in the 1940's. It has become a rvidely used experimental technique and has

been applied to many areas of scientSc research. Originally used to investigate prop-

erties of nuclear magnetic moments. SMR has since been applied in a wide range of

disciplines. including chernistry. soiid state physics. biochemistry and biophysics. Im-

portant esamples in biophysics include the determination of the structure of molecules

such as proteins both in solution and in a bilayer environment and medical irnaging

via magnetic resonance irnaging (MRI).

One of the most important fields of study in biology and biophysics today is the

study of the biological membrane: one important exampie is the plasma membrane.

A knowledge of biological membrane structure and function is the key to understand-

ing man- biological phenomena associated with the cell. Uany important cellular

functions occur a t an interface such as the plasma or external membrane of a cell. Bi-

ological membranes. amongst other functions. segregate en~ironments with seiectively

permeable barriers. and provide an environment for proteins to exist and function.

Due to the critical role membranes play in cellular physiology and intracellular ac-

tivity. it is necessary to investigate the roles of the dominant constituent parts of

membranes: lipids. protcins. carbohydrates. and water. However. the conformation.

dpamics. and physical properties of biological membranes are not solel y dependent

on these components but also on solutes present in the fluid surrounding the mem-

brane. It is for this reason that much interest has recently been given to the role

of trehalose. (a-D-glucopyranosyl-a-D-glucopyranoside) a non-reducing disaccharide

sugar present in some biological systems. I t has been suggested that trehalose plays

an important role in preseriing membrane integity when the membrane undergoes

dehydration shock from Ereezing or desiccation. Ill. This phenornenon has been

inves tigated using many techniques including nuclear magnetic resonance.

The conformatiori. dnarnics. and the function. of biological membranes are ver-

dependent upon smail changes in their environment. Any tool used to probe the

structure and behaiior of biological membranes should not dismpt the environment in

which the membrane exists. 'iuclear magnetic resonance (XMR) is an ideal candidate

for such a task since it can non-int-wively monitor the molecular conformation and

motion of various components of a mode1 membrane. NhIR also protides us with the

ability to selectively probe the behavior of one portion of a cornplex molecde. This

can be done by taking advantage of the low abundance's of a certain atom in the

biological system. such as phosphow. For example. in a lipid such as POPC there

is one phosphorus atom residing in the head group. NMR can be selectively tuned

to measure the resonance signal from this particdar atom. Through the process

of isotopic siibstitution. h-drogen atoms on one specific part of a molecule can be

selectively replaced by an isotope of hydrogen. such as deuterium. without affecting

the s ~ t e m in any si,@icant way. The resonance frequency of deuterium is unique

from that of hydrogen. and its signal can be selected in the presence of hydrogen atoms

in the system. Similarly the water in the system can be replaced with deuterated

water t hus isolating the water signal

The goal of this project was to

from the rest of the system.

investigate, through NEVIR, the interaction of

2

trehalose with a model lipid membrane as a Çunction of relative humidity and trehalose

content using 'H ?iMR spectra and 3' P XMR spin-lat tice relaxation measurements.

In particular the goal was to determine if trehalose which resides in the water region

alters the order and dynamics of the lipid head grolip in model membranes. Spectra

can show nanonring from their static value which is indicative of molecular motion

fast on the NXIR timescale. Relaxation measurements yield correlation times which

are representative of the molecular motion.

This thesis ivill present a discussion of membranes. the role of trehalose in cry-

opro tection. t heory of nuclear magnetic resonance. as well as experimental techniques.

results. discussion and conclrtsions.

2 Membranes

This chapter nrill introduce the presently accepted fluid mosaic model of a biologi-

cal membrane. The hydrophobie effect which is responsible for lipid self-assembl.

geometrical packing of different types of lipids and the resulting structures Nil1 be

discussed. This chapter d l also address the possible lipid phases and the transitions

which take place between them. In addition. the role trehalose p l a y in maintaining

the essential feat ures of the bilayer structure when freezing and desiccation occurs

;vil1 also be discussed.

2.1 Fluid Mosaic Mode1

The currently accepted mode1 of biological membranes was presented by Singer and

Nicholson 121 and is depicted in Fiowe 1. The so-called jluid mosaic model states

that membranes are essentially a two-dimensional fluid of lipid molecules bounded

by water on each side in which membrane proteins are affixed or embedded. Lipids.

therefore. provide the framework and structural integrity of the membrane system.

In this model. al1 lipid molecules are free to translate and rotate in the plane of

the bilayer. and may f ip (very infrequently) to the opposite side of the bilayer [30].

Al1 motions Mthin the bilayer have a characteristic time scale associated with them.

These motions range from the ver'; rapid translation and rotation of lipids to the

very slow motions of protein complexes and collective motions of the membrane as a

whole.

One of the major types of lipid found in membranes are the phospholipids. Phos-

Figure 1: The fluid mosaic model representation of a plasma membrane. (31

pholipids usually consist of two long fatty acid chains esterified to two hydroxy-

groups on a glycerol backbone with the third. phosphate-goup. connected to the

hydrophilic head group. The type of phospholipid used in this model membrane

study was l-palmitoyl-2-oleoyl-sn-g1y~ero-3-phosphochone (POPC). POPC has one

palmitoyl chah of 16 carbons. one oleoyl chain of Id carbons having a double bond

between carbons 9 and 10, and a phosphocholine (PC) head group. where it is the

head group which defines the class of lipid. PC lipids are one of the most abundant

lipids found in plasma membranes of higher plants and animal cells. The cornbina-

tion of a palmitate chain on position one and a oleate chain on position two is a ver-

common configuration. For instance. POPC is the most abundant species in natural

egg phosphatidylcholine.

Phospholipids such as POPC are amphiphilic. meaning that they have a dual

nature in regards to their a&ty for polar solvents such as water. Amphiphilic lipids

typically consist of a polar or hydrophilic head group which has a strong aeinity

for water and non-polar or hydrophobic fatty acid c h a h or tails which have a low

&nit? for water. It is this amphiphilic nature that cirives lipids to spontaneously form

bilayer structures. such as vesicles. the rnost common form of a ceIIular membrane in

water. This particdar part of the interaction of amphiphilic lipids and water. which

is primarily entropv driven. is known as the hydrophobic effect.

2.2 The Hydrophobic Effect

The hydrophobic effect is essential to our understanding of the themodynamics of

self-assernblg. When amphiphiles spontaneously form a Lipid bilayer it is because the

free e n e r g per lipid molecule is less in the aggregated state than in the dispersed state.

This however does not totally ansver the question of why a bilayer form is chosen.

Lipids can spontaneously form many other aggregated structures such as micelles or

hexagonal pliases. .Uso. in what way is the free energy of the lipids lowered by taking

on this state:) We must first consider the structure of water.

Water in its liquid state forms a transient network of hydrogen bonds in which

clusters of water molecules form and break apart spontaneously. Bulk water has a n

average of 2.3 hydrogen bonds per molecule (41 [5] [6]. I t is this formation of transient

hydrogen bond networks that gives water its many unique properties.

When a hydrophobic molecule is introduced into water the water molecules arrange

themselves in a clathrate. or cage-like structure to maintain a similar or sometimes

greater number of hydrogen bonds per molecule. The formation of these hydrogen

bonds is enthalpically driven [SI suggesting that it is overail more favorable for hy-

drophobic molecules to be separated by water molecules. However the entropy of the

resulting clathrate is l e s than that of bulk water. The total Gibbs Free energy. AGt

must be lowered for the system to be stable (61 [7].

If hydrophobie molecules are introduced into the water it is more favorable as

determined by the Gibbs free energy for the molecules to aggregate. The entropy of

a clatherate is inversel'; proportional to the surface area of the clatherate. Molecular

aggegation decreases the combined surface area and increases the overall entropy

of the system. a favorable attribute. The overall Gibbs free energy of the system is

reduced and therefore the aggregated state is more favorable than the dispersed state.

[t is this reduction in the Gibbs Free energy that drives lipids to form aggregated

structiires such as a bilayer or cylindrical structure 16) [SI. The form of the lipid

aggregate depends on many factors but the principle one is geometrical paclàng.

2.3 Geometrical Packing

Lipids which aggregate in water can form various possible structures. One way to

predict the aggregated structure produced by a specific lipid is to consider its geo-

metric shape [51 [SI. The geometnc shape of a lipid is usually characterized by a few

parameters; the optimal surface area required by the polar head group, a,, the maxi-

mum length of the acyl chahs, I , , and the rnolecular volume of the hydrocarbon part

7

of the amphiphile. c. These three parameters are used to form the Critical PacA..g

Parameter [5] [8] :

27 CPP = -

Q J C

The Critical Packing Parameter which can be used t o determine which type of aggre-

gated structure a lipid ma- form. If the lipid has a cone shape. then the C'PP < 1 /3.

and the lipid may form a sphencal micelle. If the CPP = 1 /3 + 112. then the lipid

has a truncated cone shape and may form a cylindrical micelle. -4 CPP of 112 + 1.

refers to a truncated cone of a much lower solid angle approaching a cylinder and the

lipid will form flexible bilayers or vesicles in water. A CPP - 1 refers to a cylinder-

shaped lipid which will form planar bilayers. Finally. if the CPP > 1. the lipid will

have an inverted truncated cone or wedge shape and may form inverted micelles (71.

Large structures are entropically unfavourable. Lipids will tend to aggregate into

the smallest structure that is available. determined bu the C'PP. These results are

summarized in Fig. 2 which is taken directly from reference (81

PC lipids tend to faIl in the range of C'PP = 112 -. 1 and form flexible bilawrs or

vesicles. Followinp the sample preparation procedure outlined in Sec. 4. POPC lipids

form multilameIlar vesicles. or large oonin skin type veçicles 151 [BI. These bilayer

structures. in which many vesicles of increasing dze are concentric. may be as large

as a micron in diameter.

Figure 2: Table of lipid shapes and corresponding packing. 181

2.4 Lipid phases and transitions

Cnder conditions t-pical of croprotection. lipids such as POPC will be found in the

bilayer conformation but can be in one of several phases. Figure 3 shows possible Iipid

phases. Lipid phases are dehed by the long and short range order of the system.

Phases are dependent on hydration and temperature as well as pH? pressure, ionic

strengt h and solute concentration [BI. In this study. only temperature and hydration

were varied. Under physiological conditions. PO PC-water mixtures will be in the

Figure 3: Various phases of lipids in the bilayer conformation. [9]

lamellar liquid crystal phase. buy may also be found in the lamellar gel phase in

conditions relavent to croprotection. The gel to liquid crystal or chain melting

phase transition is of great interest as it could occw under possible conditions for

many lipid systems involved in croprotection. The liquid crystal phase (L,) has

long range order in that there may exist layers of bilayers stacked upon each other.

However the liquid crystal phase has short range disorder. This is due to the high

probability of gauche as weil as tram conformers. rapid rotation about the a i s of

the lipid and fast diffusion throughout the plane of the bilaor. The gel phase also

has long range order as in the liquid crystal phase. but also has short range order.

Lipids in the gel phase are much more likel- to be in the all-tram ~ o n f i ~ w a t i o n and

diffusion throughout the bilayer is very slow.

The gel to liquid crystal phase transition also consists of a lateral expansion and a

t hickness reduction of the Iipid bilayer as well as a overall increase in the total volume

occupied by each lipid. In addition the number of water molecules per lipid bound

to the surface of the bilayer increases.

Gel to liquid crystal phase transitions occur when the entropic reduction in free

energy from chain isomerisrn balances the decrease in bilayer cohesive e n e r s from

the lateral expansion of the bilayer and from the energy cost of converting trarts to

gauche conformers in the hydrocarbon chains [91.

2.5 Trehalose and its role in cryoprotection

Over the past two decades studies have shown that some organisms have the ability

to survive cirtually complete dehydration where the organisms are said to be in a

state of anhydrobiosis. These organisms may persist in this state for decades. and

upon the re-introduction of water. the organisms quickly resume active metabolism.

[t has been found that such organisms often contain large quantities of sugars. up to

20% of the dry weight in trehalose or in some higher piant life. sucrose. It has b e n

over 20 years since it was first sugested that trehalose may replace water around the

polar regions of macromolecular assemblies such as the polar head groups in a bilayer

vesicle. [IO]. The structure of trehalose is shown in f iove 4

Figure 4: Chernical structure of trehalose. (a-D-glucop~anosyl-CL-D-ghcopyranoside)

Pl1

Under the process of freeze-drying or air drying a llipid bilayer vesicle may lose its

integrity [12] (131 [l] (161. LTpon re-hydration of the system the vesicles tend to be

fused together into much larger vesicles. In addition, the contents of the vesicles may

12

have leaked into the surrounding medium. In the past 10 years much evidence has

accumulated that trehalose. and to a lesser extent other sugars. have the ability to

preserve membrane int egrit y agains t liposome fusion and leakage when sub jected to

desiccation shock. Cpon rehydration. systems with trehalose present will r e t m to

their original state. whiIe systems tvithout trehalose d l have undergone fusion and

leakage. Various techniques such as freeze-fracture [131 [201 . XhIR [14] [151 [161. ESR

spin-labeling 1151 [16 1. d o r i met rie [%Il. and resonance e n e r s t ransfer have al1 seen

si milar resul t S.

Crowe et al. proposed the following mechanism for the preservation of membrane

integri tu by trehalose ['LOI. Phosphocholine lipids have approximately 10- 12 water

molecules associated with the headgroup. When these water molecules are removed.

the area pet lipid head group decreases. allotving the packing density of the hydrocar-

bon chains to increase. This allows an increase in van der Waals interactions leading

to an increase in the gel to liquid crystalline phase transition temperature. Essen-

tially a membrane in the presence of water tvhich is in the liquid crystalline phase

at physiologically relevant temperatures will be in the gel phase a t the same temper-

ature range when dry The removal of water also allows the well hydrated vesicles

to become proximate. The result is an increase in the possibility of vesicle fusion or

leakage of vesicle contents into the surrounding medium (241.

In summarl upon the removal of water from the system. the lipids can undergo

a lotropic phase transition from the liquid crystalline phase to the gel phase. Such

a transition will increase vesicle permeabilit- In addition. the formation of some gel

phase lipids during the dehydration process may lead to lateral phase separation cre-

13

ating domains of like lipids ('LU]. Lpon rehydration. vesicles could leak their contents

a t the junctions of these domains. ft is plausible that the mechanisrn by which tre-

halose preserves membrane integrity during dehydration is to replace the water and

maintain the membrane in the liquid crystal phase.

An example of how this is achieved has been presented by Crowe et al. (201. A

mixture of POPC and phosphatidylserine (PS) in a ratio of 9:l \vas freeze-dried and

the dry mi-xture was found to have its major calorimetnc transition corresponding to

the POPC transition at 330 I< and a smaller transition corresponding to the PS at

250 K. We believe however. that this is due to the existence of a two phase region

in the sample. The same mixtiire with the addition of 1.0 g of trehalose per gram of

iipid. approxirnately a 9:1 trehalose to lipid molecular ratio. was found to have single

transition at 265 K. a 65 I< decrease in the gel to liquid crystalline phase transition

temperature. Therefore under physiologically relevant temperatures. the vesicle will

remain in the liquid crystalline phase even when dried and therefore will not undergo

a phase transition upon rehydra tion: thus eiiminating liposome fusion and leakage.

These results are summarized in figure 5 . This figure shows freeze-fracture im-

ages of four situations. Section A shows freshly prepared well hydrated unilamellar

POPC-PS vesicles. Section B shows the same sjstem dried \vithout trehalose and

freae-fractured dry. The vesicles in t his situation have t ransformed into large mul-

tilamellar structures and have also leaked their contents to the surrounding medium.

Section C shows POPC-PS vesicles dned in the presence of trehalose, and they exist

discretely embedded in a solvent of trehalose. Section D shows the vesicles that were

previously dried in the presence of trehalose and rehydrated. There is however. no im-

14

age provided showing vesicles rehydrated in the absence of trehalose. but it is stated

that upon rehydration. the lipids form multilamellar and oligolamellar vesicles much

larger than the original vesicles. It can be seen from these micrographs that there

is a marked change in the dehydrated behaviour of the lipid system when trehalose

is present. It can also be seen that the micles return to their hydrated state &en

re hydra t ed .

Other studies have shown similar results. Anchordoguy et al. [lil investigated

the abili ty of various compounds. glycerol. dime t hyl sufoxide. sarcosine. sucrose and

trehalose. to inhibit liposome fusion during freeze-thaw c-les by resonance energy

transfer methods. The effects of the rate of freezing were also investigated. Rapid

freaing \vas shown to be the least disruptive and siow cooling the most disruptive.

Trehalose and sucrose showed ii t tle sensi tiklty to the freezing rates whereas glycerol.

dimet hyl sufoxide. and sarcosine shoived great dependence on the cooling rate.

Anchordoguy et al. 1171 used the percent of probe intermixing as a measure

of membrane leakage. Samples ivere prepared with particular flourescent materials

inside the vesicles and other flourescent rnaterial outside the vesicle. The arnount of

probe intermixing gives a measure of the amount of vesicle ieakage. With increasing

concentrations of t re halose or sucrose. percent probe intermixing declined steadil y

and reached a minimum value of 0.2 b1 and had little dependence on cooling rate.

The other compounds. glycerol for example. had a much larger dependence on cooling

rate and d id i t reach a minimum value of probe intermixing until about 1.0 M.

Anchordoguy et al. [171 corne to the conclusion that the interaction between

molecules like glycerol and the iipid bilayer is weaker than that of trehalose or sucrose.

15

lt is well knotvn that europium. as well as most trivalent cations form ionic bonds

with the phosphate of phospholipids. This is used by Strauss et al. [lôl, (161 and

Anchordoguy et al. [17] to test if a croprotectant is interacting with the phosphate

group of a phospholipid. Strauss and Huser in [l5] and [161 show that europium in

high enough concentrations abolishes the croprotective abilit- of sucrose on EggPC.

Anchorduoguy et al. [171 used this effect to determine that proline does not interact

with the head goup but with the hydrophobie tails. I t is from the arnount of reduction

in the croprotectant effect by europium t hat the conclusion was dratvn that trehalose

and sucrose interact rnuch more strongly with the head group than other molecules

Molecular modeling has been used to investigate the somewhat anomalous prop-

erties of trehalose. Chandrasekhar et al. [181 investigated the possible mode of interac-

tion OF trehalose. and sucrose with a mode1 1.2-dimyristoyl-sn-glycero-3-phosphatidylcholine

(DàIPC) monolayer in the absence of water: the objective being to maximize hydrogen

bonding between the two systerns. A DXIPC crystal unit ceIl consists of four molecules

arranged in two tail to tail pairs. Each pair consists of two conformationally diRerent

molecules. .L\ and B. Conformers A and B differ primarily in the conformation of the

PC headgroup. where the headgroup of B is more extended than A. This results in

an undulating surface along the bilayer. The objective was to dock the sugar ont0

the hydrogen bond acceptors of the zwitterionic phosphatidylcholine moiety. the neg-

atively charged non-esterified phosphate oxygens. and maximize the hydrogen bond

formation between the non-esterified phosphate oxygens and the hydro-y1 oxygens of

trehalose. Results of the minirnization show that trehalose is particularly suited for

16

the interaction. The interaction of trehalose with DMPC lipids is s h o w in figure 6.

In this figure. the positions of the five PC headgroups are designated bp A. For the A

headgroup conformer. specifically the phosphate group. and B for the B headgroup

conformer. Labels for the relavent oxygen and hydrogen atoms have been added to

the diagram. Phosphorus atorns are labeled PB for the phosphorus nucleus in the B

headgroup conformer. and PA for the A headgroup conformer. Dashed lines represent

hydrogen bonds. The model shows that the first glucose monomer interacts strongly

with the membrane surface via hydrogen bonds and with non-bonded interactions.

Investigations into the ~i~gpificance of the a-a linkage of trehalose Found that s - a -

thetic a-3 and 0-8 linkages place the second glucose monomer in a very unfavourable

position. The a-û linkage provided a bridge over the A conformer of DMPC to allow

interactions of the second glucose monomer with the B conformer of DMPC in the

DSIPC crystal structure. The croprotective ability of sucrose vas compared to tre-

halose bu substituting for trehalose into the model. It was found that the exocyclic

arm of the five membered fructose moiety behaved similar to the esocyclic hydroxy

oxygeen of the trehalose molecule to form the boundary of the pocket in which the glu-

cose ring sits. However. the distance between the glucose ring and the choline rnethyls

was found to be larger by approximately 1.0 Angstroms. resulting in less favourabie

interactions. A second study in which Rudolph et al. [19] investigated the effect of

trehalose. sucrose and glucose where the system was expanded and a more complete

treatise was given. The second study included trehalose, sucrose and glucose. It was

found that the saccharide-lipid interaction energies become less favourable for the

sucrose case. and even more less favourable with glucose.

17

Much evidence has been accumulated that it is the direct interaction between the

sugar hydrogen bonding to the polar head groups of POPC that is responsible for

the croprotective nature of sugars. However. evidence does e.xist that it is merely

the glass formation. or vitrification of sugars which is responsible. The glass state is

one in which the molecules do not enter a crystaline form, but entered a very \ïscous

disorded state.

Koster et al. (231 performed a study in which the ability of sugars to prevent

increases in the gel to fluid transition temperature of POPC was examined. In ad-

dition the titrification of sugars as a function of Iiydration \vas determined. Finally

the phase behaviow of POPC \vas characterized as a function of hydration in the

presence of various sugars. Four sugar samples were used. The first from dehy-

dration tolerant seed embryos. consisting of sucrose and raffiose (83: 15. w/w) . A

aample From non-dehydration tolerant seed embryos. consisting of glucose and sucrose

(7525) . and finally two control samples. one pure sucrose and one pure sorbitol. It

was lound that al1 three sugar samples diminish the increase in the gel to Ruid phase

transition temperature of POPC upon dehydration. DifferentiaIly scanning calorime-

t,ry (DSC) scans show that the Tm of full- hydrated POPC was 270 K. When POPC

was dehydrated in the absence of any sugar the Tm rose to 334 K. When POPC was

dehydrated in the presence of sugars . Tm only rose to a maximum of 279 I< (for ?iT

sugars). The conclusions dram are that the croprotective nature of sugars is largely

due to the osmotic and volumetric properties of the sugars, since al1 sugar samples

produced similar changes in the transition temperatures.

Crowe et ai. (241 1221 made atternpts to clarify the contributions of giass formation

18

and direct interaction with the head p o u p via hydrogen bonding. I t was found that

de-xtran and hydroxyet hyl starch. which are both good plass formers are not effective

in preventing leakage kom eggPC during dehydration. In addition. these polymers

show no evidence of direct interaction with the headgroup phosphates nor an ability

to lower the gel to liquid phase transition temperature of the Lipid. It was Found

however. that the' do prevent fusion of liposomes during dehydration. a property

they attribute to the good glass forming nature of these polymers. Further evidence

is that trehalose is a good glass former and also shows direct interaction with the

headgroups of Lipids. Glucose lowers the Tm of the eggPC. a collection of PC lipids

from egg o lks . and shows interaction with the headgroup but does not stabilize

liposomes.

Al1 of these finding support a combined role of glas formation and interaction

Mth the head group of the lipid. Trehalose has the ability to do both and is therefore

one of the better croprotectants.

Crowe et al. (251 showed that trehalose while having a very large croprotective

ability does not have an anomalous effect. but merely lies a t the end of a continuum

of materials which have a croprotective nature. In addition to the many previous

studies. they suggest that it is the ability of trehalose to sequester water away from

the headgroup once it has been re-introduced in the form of a dihydrate. that assists

in its croprotective ability. It is the removal of water by trehalose ivhich aliows the

sugar to rernain in a glassy state a t ambient temperatures, thus keeping the viscosity

high. which helps in the reduction of liposome fusion.

Finally, Lee et al. (141 investigated dry trehalose and 1.2-dipalmitoyl-sn- phos-

19

phatidylcholine (DPPC) mixtures using 31P and 2H XMR techniques. They found

that 31P spectra are consistent with a rigid headgroup above and below the calori-

metric transition for both dry DPPC and dry 2 1 treha1ose:DPPC mixtures. 'H XMR

spectra of acyl chains labeled at position 7 from the carboxy end (A7) showed es-

change narrowed linewidths of 120 kHz for dry DPPC correspondhg to a two site

jump model. Dry treha1ose:DPPC m~utures showed linewidths of 29 kHz indicative

of a four site jump model. This is very similar to disordered acyl chains commonly

fo~md in the liquid crystal phase of hydrated systems. Lee et al. conclude that sam-

ples containing DPPC and trehaiose exist above their transition temperatures in a

new phase termed A. similar to the liquid crystalline phase of hydrated systems. It

is this new phase and its dy-namic properties that are the means by which membrane

integrity is preserved upon dehydration.

2.6 Aims of this project

The goal of this project was to inves tigate. t hrough Xuclear Magnetic Resonance. the

interaction of trehalose with a model lipid membrane as a function of relative humidity

and trehalose content using 'H NMR spectra and 31P ShIR spin-lattice relaxation.

These two tools measure the dynamics of the system. We first wished to measure the

effect of hydration on the liquid crystal to gel transition temperature of POPC. By

observing the *H resonance from the *H20 in POPC-water miutures. it is possible

to ascertain the transition temperature. *H NMR spectra of deuterated headgroup

POPC with trehaiose will protide evidence of the effect of trehalose interaction at the

headgroup-solvent interface. X series of spin-lattice relaxation time measurements of

POPC were performed as a function of hydration and trehalose content. 31P NMR

spin-lattice relaxation times can directly be converted into correlation times which

in turn. give a rneasure of the rate of movement of the PC headgroup. It is our

hypot.hesis that the rate of motion of the PC headgroup of a dehydrated sample Nill

be slower than a hydrated sample and the rate of motion of the PC headgroup of

a sample containing trehalose wiil be similar to a hydrated sample not containing

trehalose. It is believed that trehalose interacts directly with the PC headgroup to

prok-ide a water like environment.

Figure 5: Four freeze-fracture images showing vesicles in hydrated and non hydrated

states with and without trehalose. A. Hydrated lipid vesicles. B. Dehydrated lipids

C . Dehydrated vesicles wi t h trehalose. C . rehydrated vesicles a i t h t rehalose. 1201

F i e f i Scliemnt ic of t lie interaction of t relialow atid D l I PC' Iieadgroups. :\ 1 t cred

from Il$/

3 Theory of Nuclear Magnetic Resonance

This chapter will give a brief description of the fundamental theory of nuclear mag-

netic resonance necessary to understand the experiments described in this thesis. It

will also cover some of the nuclear spin interactions other than the Zeeman interac-

tion which are relevant to this thesis. These include the dipole-dipole interaction. the

anisotropic chemical shift and the nuclear electric quadrupole interaction.

3.1 Basic Theory of Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) is derived [rom transitions between nondegen-

erate Zeeman energy levels of the nucleus. Any nucleus which has a non-zero spin

angular rnornenturn Th will have a rnagnetic moment jl associated with it in the

following way:

where A is Planck's constant divided by 2lr and -, is t.he so-called gyromagnetic ratio

whose value varies from one nucleus to another and whose sign refers to the direction

of Ci relative to f If the non-zero magnetic moment is placed in a static rnagnetic

field. Ëo there will be an interaction between the magnetic field and the nuclear

magnetic moment given by the Hamiltonian [261 [271:

If \ve define the 2-auis to be dong the direction of the magnetic field and define the

Larmor precessional frequency to be = ;Bo t hen equation 3.2 becomes.

where Iz has the quantized values rnh where rn = 1. (1

energy levels of the s-tern are given bu:

(3.3)

1). . . . . -1. Therefore the

and are the so called Zeeman energy levels $261 (271. The energy level scheme for I= 1 is

shown in figure 7. Resonance occurs when electromagnetic radiation of energ ;yual

\ j, m r l

Figure 7: Zeeman Energy Level Diagram for 1=1

to the energy difference between Zeeman levels. AE, = ?ABo. is incident on the

sample and is absorbed promoting a spin from the lower energy level or "spin down"

to a higher energy level or -spin up". or stimulatinp a spin to drop from the excited

state to the ground state. Einstein showed that the coefficients for absorption and

25

stimulated emission were equal and as such. in the presence of incident radiation equal

to the difference in Zeeman levels. there nill be an equal number of spin excitations as

demotions. This however is net the cornplete case. Since there is a e n e r 3 difference

between the two Zeeman levels. there will be different spin populations on each of

these levels given by the standard Mauwell-Boltzmann statistics. For the sake of

simplicity we ivill investigate the spin 1/2 system. which gives: 1261 p71

ivhere :V- and .V- are the populations of the two energy levels. v is the Erequency

of the radiation. I< is Boltzmann's constant. and T is the absolute temperature of

the surroundings called the lattice which is considered to be a thermal reservoir [261.

For standard laboratory magnetic fields the frequencies of the transitions between

the nuclear Zeeman levels are in the range 1 .\[Hz to 1000 SIHz. XhIR is therefore

a branch of spectroscopy in ivhich transitions occur in the Radio-Frequency range.

ShIR may also be treated semi-classically [26] and therefore we may think of a net

4

magnetization vector I I that is the vector sum of the spins in the excited and ground

and its m a b ~ t u d e being;

where the total number of spins. N = AL + hi- - k i n g 3.6 i t can be shotvn that :

We can therefore define a rate equation of the form:

where CC- is the probability per second that a transition from a lower e n e r 3 level to

a higher energy level occurs. and similady. Cb*: is the probability per second of the

opposite transition occurring. If we define n = :V- - X . it can then be s h o w (271

that:

and if ive define

and

we can imite;

Equation 3.9 states that the magnitude of the net magnetization of the sarnple is

proportional to the difference in the populations of the excited and ground states

of the system. Equation 3.15 shows that once perturbed away from its equilibrium

value. the net mapetization vector will e-xponentiaiiy approach its equilibrium value

with a time constant. Ti. The time constant Tl is known as the spin-lattice relaxation

time.

A N'Vf R e-xperiment consists of stimulating transitions betwen the ground state

and an excited state. Semi-classicalIy. this refers to perturbing the net magnetization

vector a w from its equilibrium value. directed dong the Z-axis and tipping it into

the X-Y plane. This is done by an applied alternating magnetic field. An alternating

field applied for a characteristic time rvhich tips the net magnetization vector into

the ,Y-Y plane is cailed a 90" pulse. In the case of XMR. it is convenient to consider

the linearly polarized field as the sum of trvo fields. equal in magnitude but circularly

polarized and rotating in the opposite direction. One field rotates in the same sense

as the precession of the magnetic moments and the other in the opposite direction.

The field which rotates in the opposite direction has a negligible effect on an NMR

erperiment. Thus t,he applied field may be then written as 1271 :

IF ive define ?Bi then the Hamiltonian. including the Zeeman interaction and

the effect of the applied rotating field is;

Once the nuclear magnetization is perturbed away from its equilibrium value. it

experience a torque induced by the magnetic field. B. The torque experienced is

equal to the time rate of change of its an,gular momentum therefore:

Combining the precession described by Eq. 3.18 with the spin-spin and spin-lattice

relaxation effects rields the standard Bloch Equations pi ! :

In the X-Y plane. we introduce T2. the spin-spin relaxation time. T2 being different

From Tl in that there is no loss of energy of the spin system associated with the decay

of the magnetization. but a dephasing of the spins in the X-Y plane. [271. Each

nucleus h a a local magnetic. Hl,. field set ~ i p by the orientational distribution of the

neighboring dipole moments. This creates a spread in the precession rates t hroughout

the sample. IF al1 nuclei were precessing at the same rate and in phase at t=O. then

at a time ~ H i o , r 2 1 there would be a siohficant dephasing p271.

3.2 Nuclear Spin Interactions

This section d l address some of the important nuclear spin interactions such as

the dipole-dipole interaction. the anisotropic chemical shift and the nuclear electric

quadrupole interaction.

3.2.1 Dipole-Dipole Interaction

The classical interaction energy between two magnetic dipoles and g2 is given by

[Z] :

where F is the vector From Ci; to The general dipolar contribution to the Hamil-

tonian for 3' spins is then: [27]:

For the quantum mechanical Hamil tonian we wnte f i Mt h yihI;. Where if ive mi te

and F2 in terms of raising and lowering operators. and convert to spherical coordinates

we can ivrite the Hamiltonian in the following form [171:

where

When ive are only concerned with spectra. this expression can be simplified by

dropping terms C.D.E. and F. as they contribute only to extra absorption peaks a t O

and 2;.b and are relatively very weak. T h e remaining terms. A and B. the so-called

secular terms. can be cornbined to give a moditied dipolar Hamiltonian given by (271:

Thus the total Harniltonian. containhg the Zeeman interaction and the dipole-dipole

interaction is.

The remaining nonsecdar terms. C.D.E. and F are the source of spin-lattice and

spin-spin relaxation. Terrns C and D involve the raising and lowering operators where

terms E and F involve double raising and lowering operators.

3.2.2 Anisotropic Chemical Shift

To fully appreciate the " P spectra from nuclei in a phosphoiipid bilaor. we must

have an understanding of the anisotropic chemical shift in addition to the Zeeman

and dipolar interactions. Chemical shifts are set up by- the precession of the sur-

rounding electrons producing a magnetic field at the location of the nucleus which

is in opposition to the applied static magnetic field. In a chemical bond. the effect

of the surroundhg electrons is non-isotropic and therefore the chernical shielding is

dependant on the orientation relative to the applied magnetic field and is described

4

bj- the chemical shielding tensor. 5 in the principal axis system:

The form of the anisotropic chemical shift Hamiltonian is:

(3.27)

We can convert to the lab frame by a rotation of the coordinate system to give the

where

= o, sin2 0 cos' CD -t obb sin' 0 sinZ CP + O,, cos2 0 (3 -29)

where 9 and O are standard polar angles describing the orientation of the principal

axes of the chemical shift anisotropy tensor with respect to the applied magnetic field.

3.2.3 Nuclear Electric Quadrupole Interaction

To this point we have only considered the effects of the applied static magnetic field.

local magnetic fields set up by the arrangement of neighboring spins and an applied

alternating magnetic field. However. these are not the only contributing factors in

an NhIR experiment. Nuclei tvith spin values geater or equal to 1 have a electric

quadrupole moment. EQ. which i d 1 interact with the eIectric field gradient (EFG)

of the local charge distribution which is determined bu the structure of the matter

being examined. The interaction energy of a distribution of charge and an electric

potential.

where d r = dxdgdz . If we consider the center of the nucleus to be 7 = O. This energy

can be expanded in a standard Taylor series about the ongin in the following way.

where a, 4 = x, y, z and

a2 r/- c,, = - dctdB

The first term represents the interaction of a point charge with the electric field which

is independent of the orientation of the nucleus. The integral in the second term is

zero. because the electric dipole moment of the nucleus is zero. The third term

represents the n~iciear quadrupole interaction. Higher order terms are i n ~ i ~ d f i c a n t .

[27]. Defining

The Hamiltonian for the quadrupolar term then becomes:

I t can be shown. [27]. with the use of the Wigner-Eckart

lar Hamiltonian can be written as;

(3.34)

Theorem. that the quadrupo-

in the principal avis system. where only txo parameters are needed to characterize

the derivatives of the potential. the asymmetry parameter. q. and the field gradient.

q. given by the following equations:

ln an arbitrary coordinate system. it is convenient to mi te the WamiItonian in terms

of spherical tensors. For the components of the EFG (271;

'ion. the form of the quadrupolar

1271 ;

Hamiltonian expressed in spherical tensor form is

Csually it is permissible to treat the quadrupolar interaction as a perturbation on the

Zeeman energy.

For the case of axial symmetry where C ;, = l yy equation 3.39 becomes:

where 8 is the angle between the applied magnetic field and the avis of syrnrnetry of

the electric field gradient. The addition of the Quadrupolar interaction to the Zeeman

interaction results in a shift of the energy levels. In the case of a spin 1 nucleus. such

as deuterium. this changes the single resonance iine corresponding to a change in Tu;,

to two lines separated by 6A where A has the following definition ( See Fig. 8) .

In the case of the Quadrupole interaction applied to deuterons in a carbon deu-

terium (CD) bond. the electric field gradient is approximatel y axiall y symmetric along

the bond axis.

Zeeman ûwdnrpolar

F i e 8: Zeeman and quadrupolar e n e r c level diagram for I=1

where the angle Q is the angle between the applied magnetic field and the CD bond.

See Fig. 9. In the case where there is rnolecular motion on the time scale of the XbfR

Figure 9: Definition of angle 0 in quadrupolar splitting

esperiment. LW must consider the effect of the change of the angle between the CD

bond and the static magnetic field. Lipids residing in a b i laor have an inherent axial

symmetry about the bila- normal. This slmmetry simplifies the system. In this

case equation 3-42 must be expanded to the following form using the symrnetry of .

the system.

where the angled brackets represent a time or ensemble average and angles are defined

in Fi,me 10. It is convenient to define an order parameter for the C-D bond with

Figure 10: Angles in quadrupolar splitting rvhere molecular motion has been taken

into account

respect to the bilayer normaI as

3 cos' a - S C - D = ( ')-

In a lipid dispersion. al1 values of 8 are realized. resulting in what is called a Powder

Pattern Spectrum. (See Fig. 11)

I I I I

Figure 11: Powder pattern spectrum frorn a spin 1 system with axial synmetry 1301

3.3 Spin-Lat t ice Relaxation

Phosphorus 3 1 spin-lat tice relaxation measurements of phosp holipid membranes in-

volve two important processes. the anisotropic chernical shielding and the 31P - 'H

dipolar interaction. E-xpressions for relaxation times can explicit ly be calculated for a

given molecular motion. however due to the complexïty of the motion of phospholipids

in a bilayer membrane. previous studies have assumed isotropic motion. Expressions

for Tl relaxation times can be calculated from the chemical shielding and dipolar

Hamil tonians. For the anisotropic chemical shift .

and for the dipolar interaction between the 31P and surrounding protons:

where 7 p and are the gyromagnetic ratios for phosphorus and hydrogen respec-

t ively and where the funct ions J ( u ) are isotropic spectral dens i t .~ functions given

where r, is the correlation time. The total Tl relasation mechanism is the sum of the

two components

3.3.1 Previous Studies

Prevbus work has been done by Milburn and JeBey (291 to investigate the depen-

dence of Tl on temperature and resonant frequency for egg yolk phosphocholine lipids

38

(eggPC). EggPC has the same PC headgroup as POPC but has a mixture of acyl

chain lengths and degree of saturation. The largest component however is POPC.

The 31P Tl results should be similar for the tnro materials.

hlilburn et al. investigated the spin-lattice relaxation time of the 3' P nucleus in

the phosphate group in eggPC in multilammelar dispersions at four resonant frequen-

cies. 38.9. 81.0. 108.9. and 143.7 l W z and in a temperature range of -30 to 60 degrees

Celsius. Results show that both dipolar and anisotropic chemical shielding are irn-

portant mechanisms. However at low resonance frequencies. the di polar interaction

was fo~md to dominate where at h g h resonance frequencies the anisotropic chemical

shielding interaction was found to dominate.

The phosphorus resonant frequency in this study was 11 7.8 MHz and so the

anisotropic chemical shielding will be the dominant mechanism for T relaxation.

Figure 12 shows Ti measurements as a function of temperature at the four resonance

frequencies. The value of Ti a t the minimum was not highly frequency dependant

but a large frequency dependence is seen on either side of the minimum. TI min-

imum values occurred a t temperature ranging from -10 to 10 degrees Celsius and

had Ti values at the minimum ranging from 900 ms to 2200 ms depending of the

f r equenc~ At 108.9 4IHz the minimum occurred a t 3 degrees Celsius and had a value

of approsimately 900 ms.

F i e 12: 31P Tl relaxation time of eggPC as a hnction of resonant frequenry and

temperature. (291

Figure 13: Theoretical cuves for chernical shielding and &polar contributions to Tl

fit to experimentd data above 265 K. 1291

Fiames 13 11 show the theoretical contributions to the overall Tt relaxation

for four resonant frequencies calculated by blilburn et. al 1291. Data was fit above

and below 265 K separatel. The resonant frequency of 108.9 MHz is closest to the

one used in this study of 117.9 XIHz and it can be seen that the chemical shielding

interaction clearly dominates.

Figure 14: Theoretical cuves for chernical shielding and dipolar contributions to T

fit to experimentd data belon. 265 K. 1291

4 Materials and Methods

This section will include a description of the e-yenmental methods emplqved in this

project. This consists of sample preparation techniques. a discussion of the design

of the home-built FT-N'rlR spectrometer. a discussion of pulse sequences used to

measure Tl and obtain spectra. and a n a l l i s techniques.

4.1 Sample Preparation

The samples used in this study w r e made from four main sample components. Proto-

nated and partially deuterated 1- palmi toyl-2-oleo+sn-glqTero-3-phos phochone ( POPC).

trehalose (a- D-glucopj-ranos yl-a- D-glucopyranoside) and water. POPC is a 2 acyl

chain phosphocholine (PC) lipid in which the oleoyl acyl chain has 18 carbons with a

double bond between carbons 9 and 10. and the palmitoyl acyl chah has 16 carbons.

The acyl chains are conriected to the PC headgroup as depicted in fiorne 15. Purelx

protonated POPC. in which al1 hydrogen atoms on the POPC lipid are present and

1. 1.2.2-dl-S.N.5-trimethyl-dg POPC (POPC-dl,) in which al1 hydrogen atoms on

the PC headgroup have been replaced with deuterium were used. Protonated and

partially deuterated (POPC-di3) lipid samples were purchased from .\tanti Polar

Lipids Incorporated ' and were used with no further purification. POPC lipids were

purchased both dissolved in methylene chloride and in powder form and were stored

at -15 degrees Celsius until needed for sample preparation.

For 31P NMR Tl relaxation studies. approximately l5Omg of pwely protonated

'-4vanti Polar Lipids Inc. 700 Industrial Park Drive Alabaster Alabama USA 55007

Fioure 13: POPC lipid molecule [311

POPC (molecular weight 760.1) was weighed and placed in a round bottom flask with

twice the number of moles of trehalose. (MW 378.3) approximately EOrng, purchased

from Sigma Chernical Company 2 . The two components were then dissolved in a

1: 1 weight ratio of water:methanol. The dissolved mixture was then vortex mixed

and dried under a stream of very dry nitrogen gas at 313 K for 3 to 5 days. Cpon

completion of drying. samples were equilibrated with various salt solutions to humidify

the sample to the appropriate relative humidity. Samples were equilibrated with the

various solutions for 3 days at 308 K. Samples used for 3 ' ~ NMR Ti relaxation

studies consisted of approximately 150 mg of POPC and lJOmg of trehalose. giving

a molecular ratio of 12 P0PC:trehalose. Four relative humidities were usedr 95%

RH. equilibrated with potassium sulfate (K2S04). 76% RH. equilibrated wi th sodium

chloride (NaCl). 57% RH equilibrated with sodium bromide (NaBr), and 15% RH,

equilibrated with lithium bromide (LiBr) [32].

?sigma Chernical Co. P.0 Box 14508 St. Louis MO USA 63178

Samples which were purchased dissolved in rnethylene chloride underwent two

preliminary preparation steps. Ampules of POPC dissolved in methylene chioride

were transferred into a round bottom flask and rinsed with more methylene chloride

to ensure total sarnple transfer. Samples were then dried under a steady stream of

verv - drv - ni trogen gas at 308 K until the majority of the solvent \vas removed. Samples

were then placed in a vacuum and evacuated at torr for approximately 24 hours

and then underwent the same preparation steps as the powder POPC samples.

Once the samples had equilibrated at the appropriate relative humidities. the

lipid-sugar-water mixture \vas removed from the round bot tom flask and transferred

to a 0.2 ml plastic tube. The tubes were then sealed and kept at 238 K when not in

use.

Samples iised in the 'H NSIR spectra studies were prepared in a similar fash-

ion. Samples for 'H 4'vIR spectra studies consisted of approximately 50 mg of

deiiterated POPC-d13 (SIIV 7'73.1) and 50 mg of trehalose in a molecular ratio of

l::! P0PC:trehalose.

4.2 Experimental Setup

This section will describe experimental setup used in this study including the 'IMR

spectrometer. superconducting magnet . and support hardware.

4.2.1 Superconducting Magnet

Xh1R spectroscopy relies on the lifting of the degeneracy in nuclear energy levels. This

is achieved by placing the sample in a magnetic field. which produces the Zeeman

46

splitting. There are some desirable attributes that the magnetic field should have.

3 S It should have a large m a o ~ t u d e . which improves the signal to noise ratio. :, - B;

[33] and it should be homogeneous and stable across the sample volume. ,411 these

requiremeuts are best met by a superconductiong magner. Experiments performed

herein al1 employed a Bruker 7 . X superconducting magnet. This field resillts in

a Larmor precessional frequency of 14.6 SIHz for 'H nuclei and 117.8 MHz for "P

nuclei .

4.2.2 NMR FT-Spectrometer

To be able to work at a large number of Frequencies. the previousl- built spectrom-

eter was designed such that the majority of the spectrometer components ran at an

intermediate frequency ol 10 M i z . With this set-up only the preamplifier. sample

coil. and the frequency generator had to be adjusted or changed in order to change

the resonance frequency of the ent ire spectrometer .

The intermediate frequency is gated and phase shifted to create puises of four

necessary phases. 0. 90. 180. and 270 degrees corresponding to x. -x. J-. -y pulses in

the rotating Frame of the pulses. A stable frequency generator supplies the reference

signal a t the desired frequency v, - 1OMHz which is then used wit h a combination of

m~uers and the gated intermediate frequency to create the superposition of the two

frequencies at v, and v, - 20 MHz from which the Larmor precessional frequency is

selected. The RF pulses were then amplified by a tuned amplifier to an approximate

output of 1 kW into the 50 R load of the probe. Fiome 16 shows a schematic of the

spectrometer.

Frequency Synthesizer

I Gatin g

Vo+lO MHz Signal * Spiiner

I

l Programmer

Single- Sideband Generator

Broadband Amplifier

Pre-Amp

Figure 16: Block Diagram of Home-Built FT NMR Spectrometer.

The function of the probe is to place the sample in the most hornogeneous part of

the magnetic field. irradiate the sample with the RF pulse. and to collect the signal

From the Free induction decay. The probe contains two capacitors. C; and used to

tune the probe to electrical resonance and the 500 impedance of the coaxial cable to

ensure maximum power transfer. Two sets of crossed diodes alloiv the transmitter and

receiver to be isolated from each other. 5 t Ien the transmitter is active. the diodes

conduct and the shorted quarter wavelength cable acts like an i d n i t e impedance

allowing maximum power to be delivered to the sample coil. m e n the transmitter is

not active. the signal produced by the nuclear precession is no t strong enough to allow

the diodes to conduct so the transmitter is essentially isolated and the nuclear signal

is transmitted to the pre-amp and receiver assembly. Fioure 17 shows a schematic

of the probe assembly.

The design used has a single solenoidal coil which serves to deliver the field

set up by the RF pulse and to detect the signal set up by the rotating magnetic field

after the application of the RF puise. The signal induced by the precessing nuclear

moments is then amplified by a home built. tuned pre-amp. The nuclear signal pnor

to the pre-amplification is very iveak and h a poor signal to noise ratio. For this

reason it is important to have a high gain. low noise amplifier. The amplified signal

is then converted to the intermediate frequency of 10 MHz. Quadrature detection is

used to extract two signals out of phase with respect to one another by 90 degrees.

-At this point the signal. S = S, + iS,. is digitized by a digital oscilloscope and can

be recorded for further analysis.

The probe also is part of the temperature control systern. The lower part of

49

Reciever Transmitter Probe

Figure 17: Schematic Diagam of Probe Assembly

the probe contains an oven and cooling coi1 as well as two therrnocouples used in

a feedback loop to control the voltage sent to the oven. For temperatures above

room temperature. the cornputer sets a reference voltage corresponding to the chosen

temperature. This voltage is compared to the voltage read from the thermocouple

placed near the oven. Depending on whether the voltage clifference is positive or

negative. the heating coi1 surrounding the sample was tumed on or left off. To achieve

temperatures below room temperature a cooling system was used. The cooling systern

consisted of a flow of nitrogen gas. passed through a bath of liquid nitrogen. The

heating feedback system then ran on top of the base temperature set up by the cooling

system to achieve the desired temperature.

4.3 Pulse Sequences

Tivo simple 33IR pulse sequences were used in this project . The h s t pulse sequence

was used for acquiring spectra. Due to the excellent homogeneity of the static mag-

netic field and the short recovery time of' the spectrometer. a simple 90 degree pulse

Followed by a data acquisition pulse was used to collect the free induction dec- The

signal was then collected and digitized by the digital oscilloscope and stored bu a

computer.

A typical Tl measurement uses a 180-7-90 pulse sequence in which an initial 180

degree pulse tips the magnetization vector into the negative Z direction where the

magnetization will then start to decrease in mamgpitude. pass through zero and then

gow in the positive Z direction until i t reaches its thermodynamic equilibrium value

with a characteristic time TL. If a 90 degree pulse is applied a t some tirne T Iater.

then the value of .\7, wili then be tipped into the x'-y' plane and the magnitude of

- the free induction decay that folloas is a measurement of :\lz ( t ) . The growth of :GZ

can be described by the following rate equation.

and using the initial condition that :LIz = -Mo at t = O then we can obtain:

For Ti measurements in this study there nTas a slight modification where a standard

saturation pulse sequence was used. This consisted of a train of approximately 5 or 6

30 degree pulses until the f r e induction decay nras zero by the end of the last pulse.

51

.4chieving the effect of a 90 deqee pulse or complete saturation of the magnetization.

By wing shorter pulses and therefore pulses of a greater spectral width complete

saturation tvas achieved. The delay time T nras set by the cornputer and varied to

achieve data points aiong the Ti relaxation curve. Schematic of the saturation pulse

sequence is shown in fi-gure 18. -4 typical Tl measurement is shown in figure 19.

Figure 18: Schernatic of a t-pical Tl measurement.

The numerical f-alue of T I can then be determined by a least squares fitting

routine. Tl measurements in this study were fitted to a single e-xponential b c t i o n

of the following form.

Typical T, Measuiement T,=868.5 ms

1000 2000 3000 4000

Tau (ms)

Figure 19: Typical TI measurement c u v e

5 Experimental Results and Discussion

This section will present all the relevant e-yenmental results obtained in this s t u d .

Three t-ypes of experiments were performed. The fkst determined the gel to liquid

crystal phase transition temperature of POPC as a function of hydration. The second

e-uperiment investigated the effect of trehalose on the POPC/nater system using

deuterated POPC. while the third experiment probed the influence of trehalose on

the dpamics of the system. There are tivo main types of results to present. 2H

spect ra. and " P spin-Iat tice relaxation time measurements.

Spectra were acquired using the experirnental techniques outlined in section 4.

Figure 11 shows a t~.pical 'H N3IR spectrum from a lipid water mixture. The sepa-

ration between the peaks in the powder pattern spectrum is termed the quadrupolar

split ting. \Ve will begin ivith a short discussion on the significance of the value of the

quadrupolar splitting. The value of the quadrupolar splitting for H in an electric

field gradient oriented dong the C-D bond is given by:

where

3 cos2 û, - CD = ( l ) (5.2)

where CI is the angle between the major avis of symmetr- the bilayer normal. and the

C-D (or O-D for water) bond direction. The effect of molecular motion which is fast

on the Nb1 R timescale. about IO-' seconds. is to change this value. This is achieved

y an averaging of the quadrupolar interaction tensor. There are two methods by

which the average of the interaction can be altered. The magnitude of the motion

54

can become greater. or the average angle about which the motion takes place can

change. For a Lipid system in the L , phase. there is a ~i~gnificant amount of rapid

motion on the 'iMR tirnescale. .As a resdt. the observed quadrupolar splitting is

typically between 1 and 20 percent of the static value.

5.1 Phase behaviour of POPC

This experiment investigated the effect of hydration on the gel to liquid crystal phase

transition temperature of POPC using 'H NMR. Spectra ivere obtained over a tem-

perature range of 250 I< to 313 K. Spectra nrere only accepted if the temperature

was stable to within 0.2 degrees of the desired temperature over the data collection

period. For each set of spectra. turo independent measurements of the quadrupo-

lar splitting were averaged. The error was then estimated based on the deviation

of the average value from the two measurements. Error values were rvell ivithin 5%

and were typicallv on the order of 1%. There was also some deviation associated

with the temperature deviation during the data collection time. This error is more

difficult to estimate. However. since spectra were only accepted if the temperature

drift was smali. it is assumed the error associated with this is negligible. However.

in the folloiving plots of the quadrupolar splitting vs. temperature or line width vs.

temperature the error bars are not shown.

The goal of the first experiment was to folIow the liquid crystalline to gel transition

t,emperature as a Function of hydration. Previous work done by Swayne [341 was used

dong with experiments done for this thesis. Spectra were collected for POPC and

D20 over a range of hydrations and temperatures. D20 and POPC were cornbined

in specific molecular ratios. Samples were then weighed and sealed in glass tubes.

Spectra from samples with a molecular ratio of 3:l D20:POPC. 4:1. 5:l. 6:l. 7:l. 9:l.

and 13: 1 were collected over a range of approximately 250 to 3 13 K.

Spectra for 4: 1 D20:POPC are shown as an example of typical spectra in fieme 20.

At high temperature there is a typical motionally averagd powder pattern. As the

temperature decreases. the splitting decreases. At approximately 290 K the splitting

goes to zero as the line becomes isotropic- As the temperature continues to decrease

from t his point. the split ting increases.

The quadrupolar splitting is plotted as a function of temperature for 4:l D20:POPC

in a molecular ratio of 4: 1 in figure 2 1. Twvo data collection runs. increasing and de-

creasing in temperature are shown. At high temperature the quadrupolar splitting is

approximately 1.6 kHz. As the temperature decreases. the split ting slowlp decreases.

At 293 1< the splitting decreases dramatically until a minimum is reached at about

289 K. -4s the temperature decreases From the minimum. the quadrupolar splitting

increases dramatically until about 283 K. and then increases slowly as the tempera-

t ure continues to decrease until a maximum recorded value of 1.9 kHz is reached. The

difFerence between the two runs map be due to error associated with the temperature

control system.

By way of contrast the results for the 13:l molecular ratio of D20:POPC sample

w i l be examined. Fi-me 22 shows the evolution of the spectra as a function of

temperature. Fimure 23 shows the measurement of the quadrupolar splitting as

a function of temperature. Starting at high temperature. the spectra are t-ypical

56

motionally averaged powder patterns. characteristic of an auiaily s ~ i m e t r i c system.

indicative of the liquid crystal phase. with a quadrupolar splitting of 0.73 kHz a t

304.5 K. This phase persists until the region of 265.4 K to 261.9 K. however. there

is some ambi,guity due to reproducibility in the temperature. Spectra taken a t 261.9

I< show splittings characteristic of both the gel phase. such ,as that at 256.6 K. and

of the liquid crystalline phase similar to spectra taken at 263.4 K. This is due to a

two phases coexisting Mthin the sample. This is designated by the region between

the two dashed lines in fi-we 23. ,4t 465.2 K Ive see t,hat the line is isotropic which

is characteristic of there being no bound water a t the surface of the bilayer which is

know to be a phase transition effect (351. As the temperature continues to decrease

the systern enters the gel phase and the quadrupolar splitting slowly increases to a

value of 2.1 kHz.

A comparison of the data in figures 21 and 23 show the quadrupolar spiitting

is lower than in the 4:l sample indicating a lower order parameter with increasing

hydrat ion.

The quadrupolar splittings were plotted as a function of temperature for each

hydration. Based on previous work by Bryant et al. and references therein [35].

the transition temperature is taken to correspond to the temperature at which the

minimum split ting occurs.

The transition temperatures were recorded for all hydrations, 3: 1 to 13: 1 D20:POPC

and plotted in fiove 24 to show the effect of hydration on the transition temperature.

-4s the hydration increases. the phase temperature decreases. Physically this can be

interpreted in the following way. As the number of water molecules available to the

57

headgroup increases. the effective area of the headgroup. and in conjunction. the acyl

chains. increases. This increase in the effective area of the acyl chains reduces the

steric interactions of the acyl chah region. This reduction NiIl allow the chains to

melt a t a lower temperature. This trend shotdd continue until t here is bulk water as a

separate phase in the system. Once this point is reached the addition of water would

not affect the effective area of the acyl chains and wodd therefore not affect the tran-

sition temperature. The error bars are based on the shift due to the reproducibility

of the temperature in the system. typically about kl degree. This curve agrees Mth

the results from Koster e t al. [231 in which they measured the pl to liquid transition

temperature of pure POPC by DSC techniques. The- found a transition temperature

of 334 K for dry POPC and 270 I< for POPC with a 20% weight percent water which

corresponds to 8.6 H 2 0 molecules per lipid. which is in agreement with our data.

Figure 20: Spectra for D20:POPC 4: 1 1341

2 H Quadrupolar Splitting 4:l D,O: POPC (m:m)

Fipure 21: Quadrupolar splitting as a Function of temperature for D20:POPC 411

P41

D,O: POPC 13:1 'H NMR

Fiowe 22: Spectra for DîO:POPC 13:l

2 H Quadrupolar Splitthg

l3:l D,O :POPC (m:m)

Temperature (K)

Figure 23: Quadrupolar splitting for D20:POPC 13: 1

Phase transition temperature as a function of number of H,O molecules per POPC molecule

Number of H20 moleailes per POPC rndewle

Figure 24: Liquid crystal to gel transition temperature as a function of hydration for

D20:POPC

5.1.1 Water content

Klose et al. [36] conducted a study in which the hydration and swelling properties

of POPC in H20 and 'H20 were examined to clarify the influence of the deuterium

isotope. In this study the water absorption isotherms were measured for POPC and

a plot was made shonring the relative humidity for a POPC sample equilibrated at

298 K vs. water to POPC rnolecular ratio. Both experimental and theoreticai cuves

are shown in fi,pre 25. From these data we can get a measure of the number of

molecules of water per POPC molecule that correspond to a relative humidity as a

percent. From the graph we see that with 100% relative humidity a t 298 K there are

about 18 rvater molecules per POPC molecule. .\ ratio of about 13: 1 corresponds to

approximately 95% relative hurnidity a t 298 K.

Xs stated. samples used later in this study were equilibrated with various salt

solutions to bring them to a specific relative humidity. These samples. however. were

equilibrated at 308 K. There is more water content in the samples equilibrated at

308 K then those equilibrated with the same salt solution at 298 K. Increasing the

equilibration temperature increases the sorption of water by POPC and thus shifting

the curve up higher on the y avis in fibgure 25. A cornparison of the water content of

these samples to those conducted preklously samples with a specific molecular ratio

was desired .

A POPC and D20 sample was equilibrated vrith potassium sulfate. K2SO4. a t

308 I< and spectra were recorded as a Function of temperature and are presented

in £iepre 26. At high temperature there is a very srnall quadrupolar splitting. As

the temperature decreases. this splitting becomes zero as the line becomes isotropic

From 285 K to 263 K. At approxirnately 265 K the quadrupolar splitting returns and

increases as the temperature decreases to a maximum value of 1.2 kHz.

FiOgue 27 shows a plot of the FWHM linewidth as a Function of temperature. At

low temperature the linewidth is approxirnately 2 kHz and falls as wve approach the

phase transition temperat ure at which point the rate of narrowing decreases drarna t-

ically and then slowly begins to broaden again as we approach high temperature.

These spectra suggest that the water content when equilibrated at 308 K is greater

than a molecular ratio of 13:l water to POPC. To clarify the question of water content.

it would seem that the easiest approach to determine water content of a sample is by

weight. Hydrated samples could be dried under dry nitrogen gas and then placed in

a vacuum to remove any remaining solvent. The difference between the n-eight of the

hydrated sample including container. minus the weight of the dry sarnple including

container would yield the weight of the water in the hydrated system. This approach

unfortunately is not feasible. Some samples contained as little as an estimated O-lmg

of water. The accuracy of scales awilable to us would not >ield results that would be

accurate for the small amount of water that is in a tvpical sample.

Fiewe 23: Absorption of H 2 0 and D 2 0 by POPC at 298.2 I< [361

Quadrupolar Splitong FWHM measurement *H NMR POPC only in D,O 95% RH

O Cd 4 vs Spütting (kHz) I Cd4VoFWHMw)

260 280 300 320

Temperabire (K)

Figure 26: Spectra for D 2 0 and POPC (95% RH at 308 K)

Figure 27: Linewidth measurements for D20 and POPC (95% RH at 308 K)

5.2 The effect of trehalose on POPC

In this experiment, the interaction of the trehalose Fvith the POPC headgroup was

investigated. The sample used consisted of deuterated headgroup POPC-dl, with

trehalose at a molecular ratio of 12. P0PC:trehalose. The sample was equilibrated

to 95% relative humidity a t 308 K and then sealed. Figure 28 shows representative

spectra as a hnction of temperature. At high temperature a quadrupolar splitting

Frorn dl deuterium species is evident . The alpha deuterons show a spli tting of about

7 kHz. beta deuterons. 3.3 kHz. and gamma or methyl deuterons have a splitting of

about IktIz. As the ternperature decreases. the splitting for al1 three species increases.

Below 280 K the splitting from the a deuterons disappears and D deuterons splitting

disappears at about 273 K. Spiitting from the methyl deuterons disappears a t about

263 K as the line becomes isotropic. .At low temperature. 248 K. the line shape is

relatively isotropic and no quadrupolar splitting is mident.

Fi,we 29 shows the splitting for al1 three types of hydrogen atoms as well as the

full width at half maximum (FWHM) linewidth as a function of ternperature. We

see that there is a possible phase transition at the temperature where the splitting

first ernerges a t approximately 263 K. There is also a gneral decrease in quadrupolar

splitting as a function of temperature for al1 three deuterons as well as a decrease in

line width as the ternperature increases. however the rate of line narrowing is much

larger before the phase transition.

Bechinger et al. 1371 studied the interactions of polyhydroxyl compounds. includ-

ing trehalose. with mode1 POPC membranes. Alpha and beta deuterated POPC lipid

dispersions in an aqueous environment were investigated as a Function of trehalose

content at 293 K. They Found the quadrupole splitting of the alpha deuterons to be

6.74 kHz when the sample had a 65% ( a / v ) trehalose content. which is equivalent

to a molecular ratio of 1: 1.3 P0PC:trehalose. In the absence of trehalose. they ob-

served a 6.01 kHz splitting. At 294 Kelvin we recorded an alpha hyirop11 splitting

of 4.00 kHz at a trehalose content of 1:2 molecular ratio P0PC:trehalose. Bechinger

et al. plot the split ting as a Function of trehalose content including a linear regres-

sion line. A quadrupolar splitting of 7.00 w o d d correspond to approximately 90%

(wv/v) trehalose content which would be equivalent to a molecdar ratio of 1 :1.74

POPC: trehalose. This value is consistent wit h our results.

Seelig et al.. [391 and references therein. investigated the effect of the conformation

of the headgoup via 2H 'ihIR. [t was found that changing the headgroup conforma-

tion was reflected in a change in the alpha splitting. Fully hydrated PC headgroups

lie essentially parallel (within 30 degrees) to the surface of the lipid bilayer. The

addition of trehalose has the effect of increasing the angle of the headgroup from the

surface of the lipid bilayer. This increases the quadrupolar splitting from 6.01 kHz in

the case of pure POPC to 7.00 kHz with the addition of trehalose in a molecular ratio

of 1 2 P0PC:trehaIose at 293 K. There is doubt therefore that trehalose interacts

directly with the headgroup of the phospholipids vîa by changing the conformation

of the headgoup relative to the surface of the lipid bilayer via steric interaction.

Figure 28: Spectra for POPC-d13:trehalose 12 95% RH

2~ Quadrupolar Splitting and FWHM 1 :2 D-13 POPC: Trehalose 95% RH

51

220 240 260 280 300 320

Temperature (K)

Fieme 29: Quadrupolar splitting and linewidth measurements for POPC-

di~:trehalose 1:2 95% RH

5.3 Spin-Lat t ice Relaxation Measurements

In order to determine the influence of trehalose and dehydration on the dynamics

of the lipid headgroup. a series of spin-lattice relaxation time (Tt) measurements

were made. The motivation for this study was to determine if trehalose alters the

environment of the headgroup as seen by changes in the Tl value. 31P XMR Ti

measurements were camed out on samples consisting of trehalose and POPC in a

molecular ratio of 2:l. Four samples were used. Each ivas equilibrated to a specific

relative hurnidit.~ 95.76.57. and 15% RH. at 308 K and then the samples were sealed.

Tl measurements were taken as a function of temperature between 248 K to 313K

and are shoivn in figure 30. There was also one set of measurements taken on pure

POPC at 57% RH and show in fieme 31 where it is presented with the measurement

of the POPC and trehalose sample also equilibrated at 57% Tl values were only

accepted if the sum of the squares of the deviation of the calculated curve from the

experimental points was less than 9 IO-^. which corresponds approximately to a 3%

error. Typically values ranged from 1 10-' to 6

From figure 30 it can be seen that there are only small differences between TI

values as the relative humidity decreases from 95 to 76%. There is however evidence

that there is a broad Tl minimum at both these temperatures. When the hydration

is reduced to 57%. the range of Tl values increases. and the Tl minimum appears to

occur at a higher temperature. m e n the hydration is reduced Further. to 13% RH.

there is a si,~ficant change. In figure 31 it is seen that except for some deviation at

low temperature. t.here is very little change in the Tl value of pure POPC hydrated

to 37%, RH as compared to POPC in the presence of trehalose also equilibrated at

57% RH. The effect of the trehalose at low temperature is to allow a very similar

amount of movement in the POPC headgroup.

Tl measurernent for P0PC:trehaîose (1 :2) equlibrated at 95,76,57, 15% relative humidity

Figure 30: Spin lattice relaxation measurement of POPC and trehalose 2: 1 at 15.57.76

and 93% RH

T, measurement for POPC and P0PC:trehalose 1:2 both equilibrated to 57 % Relative Humidity

a POPC O POPC and tretlalose

2500 O

-

Figure 31: Spin lattice relaxation measurement of POPC at 57% RH and POPC and

trehalose 12 at 57% RH

5.3.1 Correlation times

Milburn et al. (291. investigated the 3' P spin-lat tice relaxation in egg phosphatidyl-

choline lipid bilayers. 'ililbuni showed t hat at higher frequencies. such as 1 1 7.8 MHz.

the dipolar interaction becomes less important as the chemical shielding interaction

becomes more dominant. W e r e the complete expression for Ti is:

where

and

where the spectral density function of the following form was used.

On the basis of measurements of four frequencies. hlilbux-n et al. [29] determined

2 a value of 1.6. 10-"or &~o' and 2.65 - 10' for 6 (Y) . These values are not

e-xpected to change significantly for pure POPC. The correlation time is a measure

of the time for the position of the POPC headgroup to become uncorrelated from its

initial position at some time t=O. The correlation time is essentially then a measure of

the rate of mowment of the POPC headgroup. The correlation times were calculated

using equations 5.3 to 5.6.

L i e unable to fit our data with the parameter values determined by Milbum et

al. Two approaches could be taken to correct this problem. increase the dipolar in-

77

teraction strength or. increase the magnitude of the chemical shift tensor. Since the

chemical shift tensor is determined by the arrangement of chemical bonds surrounding

the phosphorus atom. there is little reason to think this d u e wodd change s i o ~ f i -

cantly hom that determined by Milburn. I t is possible that there is some change in

bond angles in our system, which c o d d result in a change in the chemical shift tensor.

The dipolar interaction constant could change i f the average distances between hy-

drogen and phosphow atoms in this system changed slightly tiom the eggPC systern

studied by Milburn. both possibilities were tested and it was foundthat there \vas

very little qualitative difference between the two situations. It is not possible in this

case to determine the correct scenario. W e increased the magnitude of the dipolar

interaction constant used by hIilburn et al.. 2.65 - 10S to 8.15 - to compensate

for the lower Ti values rneasured in this study Because of the $ dependence of the

dipolar interaction on the distance. this change in the dipolar constant represents a

change of only 18% in r. the average distance between hydrogen nuclei and phospho-

rus nuclei over the time of the NMR experïment. Fimgne 32 shows the correlation

times for POPC and trehalose samples in a l:2 molecular ratio for 95. 76. 37. and

15% relative humidities. Fi,we 33 shows the correlation time measurement for both

POPC and POPC/trehalose sarnpies at 57% RH.

Correlation tirnes for P0PC:trehaiose (1 :2) equilibrated at 15, 57, 76, 95% RH

Fiove 32: Correlation times for 95. 76. 37. 13% RH POPC and trehalose samples

Correlation time for POPC and P0PC:trehalose (1 :2) Both equilibrated at 57% RH

..

POPC I 1 O POPC and trehalose 1

Fieme 33: Correlation times for POPC and POPC: trehalose samples at 57% RH

5.3.2 Activation Energy Calculations

Milburn et al. assumed an Arrehenius dependence of the correlation tirne on temper-

ature that is

Using a graph of the natural logarithm of correlation time vs. 1000/absolute temper-

ature. one can calculate the activation energy. E, from the dope of the line of best

fit. Seeiig at al. [381 found an activation e n e r s for DOPC of 17.1 kJ/mole over an

approximate temperature range of 250 I< to 35OK. Milburn's data had an apparent

discontinuity around 263 K. This was assumed to be the gel to liquid crystal phase

transition. hiilburn divided the data into two segments and obtained activation ener-

gies in the two regions. Above 265 K . E. = 16.9 kJ/mole. and below 265 K. E, =32.3

kJjmole. In this study five samples were analyzed to determine the activation ener-

$es. POPC equilibrated at 57% RH correlation time had a single dope associated

with it. as shown in fioure 35. P0PC:trehalose 1:2 (m:m) samples equilibrated at 95.

i 6 . 3 7 and 15% RH each had an apparent transition. Fi,we 34 shows POPC/trehalose

(1:2. m:m) sample equilibrated to 95% RH at 308 K correlation times as a function of

temperature with linear regression and error bars representing 3% error which was the

upper bound of acceptable error. In the case where there is an apparent transition.

the data above and below the transition temperature was fit separatel- These resuits

are shown in figures 36. 37. 38. and 39. Activation energies calculated for this

study as well as two others quoted from the literature are presented in the foUowing

table. Al1 activation energy values are presented in kJ/mole. The activation energies

calculated are consistent with the eners- associated with breaking a hydrogen bond.

1 Low T 1 Migh T / temperature

Activation energy

This S t u d ~ 1 POPC 57% RH

Discontinuity

POPC + trehalose 95% RH

/ POPC + trehalose76%,RH) 11.1 1 2.3

Fi,we 36 shows that at high temperature the slope (m) of the regression fine is

0.17. and low temperature the slope is 1.52. As the hydration is reduced to 76% RH.

the dope of the high temperature line increases. and the dope of the low temperature

line decreases. There is also a shift of the discontinuity to a higher temperature.

This trend continues into the 57% RH case where the idection of the curve has been

inverted. The discontinuity seen in the plots of the correlation time as a Function of

temperature is not completely understood. Reduction of the hydration to 15% RH

increases this effect. The negative slope of the regression line at low temperature

is an unphysical situation and is a result of the failure of the model. One possible

explanation of this is that there is an additional relaxation process taking place where

there is a coupling between the hydrogen atoms of the trehalose molecule and the

12.6

286.5 K

LIilburn (291

Seelig [39]

1.4

egg PC fdly hydrated

DOPC full? hydrated

281.7 I<

32.5

17.1

16.9

17.1

265K

none

phosp horus nucleus.

The discontinuity does not occur at the gel to liquid crystal phase transition.

The presence of trehaiose shifis the gel to liquid crystal phase transition temperature

down from the pure POPC case at the same hydration 1201 [23/. The temperature at

whch the discontinuity occurs. for a given hydration in this case. occurs at a higher

temperature than in the pure POPC case. Values of the activation e n e r s seem to

be reasonable in cornparison with the other values presented in the above table from

klilburn et al (291 and Seelig et al. [381.

Correlation time with 3% error bars for 95% RH POPCltrehaiose

3.0 3.2 3.4 3.6 3.8 4.0

Temp 1000iK

Fiame 34: Correlation tirnes for POPC/trehalose 1:2 equilibrated to 95% RH with

linear regression and error bars representing 3% error.

Correlation time and linear regression for POPC equilibrated at 57% RH.

Figure 33: Correlation times for POPC equilibrated at 57% RH with linear regression

Correlation time and linear regressions for P0PC:trehaiose 1 :2 equilibrated at 95% RH. Separated about an apparent transition

Conelationtime - IOW T: m 4-52. b=-25.0 high T: m=O.f 70, b=-20.2

Figure 36: Correlation tirnes for POPC and trehalose 1 2 equilibrated at 95% RH

wi t h linear regression

Correlation time and linear regression for P0PC:trehalose 1 :2 equilibrated at 76% RH. Separated about an apparent transition

Correlation time law T: m4.33, b-24.3 hign T: m =O.2?3, b=-20.6

Fi,we 37: Correlation times for POPC and trehalose 1 2 equilibrated at 76% RH

wi t h linear regression

Correlation time and linear regression for POPC and trehaiose 1 :2, equilibrated at 57% RH

Figure 38: Correlation tirnes for POPC and trehalose 12 equilibrated at 57% RH

wi t h linear regression

Correlation time with lear regression for P0PC:trehalose 1 :2 equilibrated at 15% RH, sparated about an apparent transition

O Conelationtime low T: m4.429, b=-17.6 high T: m4.82, b=-31.9

Figure 39: Correlation times for POPC and trehaiose 12 equilibrated at 15% RH

wit h linear regression

6 Conclusions and Future Considerations

6.1 Conclusions

The effect of hydration on the POPC Liquid crystal to gel phase transition temperature

u-as investigated by 'H 'TXIR spectra. it was found that as the hydration increased.

the transition temperature decreased. This will continue until there is bulk water in

the systern. The values obtained are in agreement with other studies conducted such

as Koster et al. (231.

I t was also found that equilibrating POPC at 95% relative hurnidity at 308 I<

resulted in an excess of water in the system.

The effect of trehalose on the POPC headgoup was investigated. I t was found that

adding trehalose resulted in an increase in the alpha position deuterium quadrupolar

splitting from 6.01 to 7.01 kHz which is in agreement with other studies (371. This

is due to the trehalose changing the conformation of the headgroup, increasing its

average angle with respect to the surface of the lipid bilayer. This shows that there is

indeed interaction between trehalose and the phospholipid headgroup, but does not

determine whether this is a hydrogen bonding effect. or is due to trehalose forming a

glass.

Spin-lat tice relaxation measurements show that there was little change from the

high water content system (95% RH) to the partially dehydrated system equilibrated

at 76% RH. There was some change in Tl as the water content was reduced further

to 57% RH, where the range of TI d u e s increased over the same temperature range.

At the lowest water content (15% RH), there nias a more significant change. From

90

this no conclusions can be d ram regarding the effect of trehalose or hydration on the

rate of motion of the PC headgroup.

There was also little change h o m a partially hydrated (57% RH) POPC system

to that of a POPC system with trehalose equilibrated to the same relative hurnidity

At low water content. the trehalose allows a similar amount of movement of the PC

headgroup to a high water content situation.

Activation enerses were calcdated and compared with other studies. Values were

found to be consistent with the energy associated with breaking a hydrogen bond.

The reason for the apparent transition in the activation energ? is unknown. The

negative dope observed in the 15% RH case corresponding to a negative activation

energy is an unph'ical situation. This is indicative of the relaxation mode1 breakinp

down. One possible esplanation is there is an additional relaxation mechanism taking

place. This ma? be due to a direct coupling of the hydrogen atoms of the trehalose

molecide and the phosphorus nucleus.

6.2 Future considerations

To fully understand the effects trehalose has on a lipid system more control studies

wiH have to be done. Due to time constraints. these studies will have to be left to be

done at a later date.

There are many st.udies that are needed to be done for a full treatise of this

problem. Spectra from a 18: 1 D20:POPC sample which corresponds to 100% relative

humidity is desirable. .A similar study with trehalose present is desired. This would

allow us to see the effect of hydration on the gel to liquid crystal phase transition

temperature of POPC in the presence of trehalose, allowing for cornparison with the

pure POPC case.

A cornparison of the water content in samples equilibrated at 308 K vs. known

water content is also desired.

There is also a need for additional 'H spectra to be collected of deuterated POPC-

d13 samples as a function of hydration with and nit hout trehalose. This would show

directly the effect of the trehalose with the head group. This should also be conducted

in the absence of water to see its full effect. However. the availabilit,~ of headgroup

deuterated POPC at a feasible price is iimited.

TI measurement,~ of POPC at 95. 76. 15% RH are needed to allow one to compare

the case of the POPC alone and ivith trehalose as a function of hydration. it is

anticipated that a greater effect will be seen at the lower water contents. t,hat there

will be a shift of the Tl values of P0PC:trehalose samples at 15% RH Mth respect

to pure POPC samples as the trehalose has an opportunity to interact directly Rith

the POPC headgroup.

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