Surface diffusion in human serum lipoproteins

BIOCHEMICAL

Vol. 746, No. 3, 1987 August 14, 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1139-1145

SURFACEDIFFDSION IN HDMANSERUMLIPOPROTEINS Robert J. Cushley*, W. Dale Treleaven, Yashpal I. and David B. Fenske

Parmar, Ravinder S. Chana,

Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 156 Received June 16, 1987

SUMMARY.From the viscosity dependence of the 31P RMR signals, the diffusion coefficients DT of phospholipid molecules in the surface monolayer of HDL, LDL and VLDL have been determined. DT for HDL, and HDL, are found to be 2.3~10-~ cm>/5 and 1.8~10-~ cm’/s, respectively. These values are similar to values reported for diffusion of phosphoiipid molecules in phospholipid bilayers above the gel to liquid crystalline phase transition temperature. Viscosity dependence of [16,16,16-ZH,]phosphatidylcholine incorporated into HDL, yielded a value similar to that determined by 31P (D = 1.9x1O-8 cm2/s). Slower diffusion coefficients were measured for LDL, an3 VLDL. VLDL had a value DT = 9.1x10-' cm2/s. The diffusion coefficient for LDL, was 1.4~10-~ cml/s. Thus, diffusion of phospholipids in LDL, is a full order of magnitude slower at 25OC than diffusion of phospholipids in the HDLs. B’ 1987 Acadcmlc Press, Inc. The human serum lipoproteins spherical

particles

phospholipids specific

of an apolar core consisting

composed

esters and triglycerides

enveloped by an amphiphillic

and cholesterol.

into

of cholesterol

monolayer of

Embeddedin the monolayer are proteins

to the various particles.

incorporated

HDL, LDL and VLDL are essentially

the core - this

Some cholesterol

is believed

should indeed. be the case

to be

for particles

the size of VLDL where the volume of the core components would be sufficient al.(l)

to solubilize

significant

have suggested that

the phospholipid partitioned the ratio

- protein

approximately

Eisenberg et

82% of VLDL cholesterol

is within

monolayer while the remaining cholesterol

in the triglyceride-rich of protein

amounts of cholesterol.

to lipid

core. As particle

density

is

increases,

increases at the expense of neutral

lipid.

LDL, low density lipoprotein; Abbreviations: HDL, high density lipoprotein; VLDL, very low density lipoprotein; PC, phosphatidylcholine; SPM, sphingomyelin. *Author to whomcorrespondence should be addressed. 0006-291X/87$1.50 1139

AN

Copyright 0 1987 rights qf reproducfion

by Academic Press, Inc. in any form reserved.

Vol.

146,

BIOCHEMICAL

No. 3, 1987

The major

role

AND

of the serum lipoproteins

from various

tissues.

accomplished

via

between

membrane and lipoprotein

cell

different

high affinity

is

delivery

Thus,

recognize

the

the recognition

is

While myriads of studies on the lipoproteins

measurement of composition and interactions

components, no previous

is interaction

The receptors

surface.

to or

dependent upon the primary amino acid

i.e.

sequence of the apoproteins.

lipids

or lipid-lipid

monolayer.

on the lipoprotein

COMMUNICATIONS

to transport

membrane receptors

to be structure-based

have involved

RESEARCH

In the case of LDL and HDL lipid

apoproteins

believed

BIOPHYSICAL

study of phospholipid

of the surface

surface dynamics has been

reported.

In the present communication, we present evidence, s1P NMR spectroscopy, different

that

surface phospholipid

based on 'H and

diffusion

is significantly

in HDL, LDL and VLDL.

The NMRtechnique we have used is based upon the behavior of the NMR linewidth

as a function

be related

of solvent viscosity.

Av1,2 can

to the second moment M, by (2) rAv1,2

= M,T, + C

where re is the effective

(1)

correlation

the nucleus and C is a constant. the difference axially

The NMRlinewidth

1

symmetric shielding

e'qQ/h is the static bond. The correlation

tensor.

chemical shift For 'H,

quadrupolar interaction time r,

for isotropic

reorientation

For 31P, M, = (4/45)7fH$(o

a - u is the residual II

time

M,

II

- uI)'

anisotropy

term

~~

is the tumbling

= (9/20)(xezqQ/h)l

constant for a carbon-deuterium

(2) time

for the isotropic

rotation

for diffusion

of the particle (3)

R is the particle

radius,

Roltzmann's constant and T is the absolute temperature. time

where

is

?t = (4nqRJ)/(3kT) where 17is the solvent viscosity,

correlation

where

for an

7-l = r-1 + r-1 e t d The

of

k is

The term 7d is the

in the surface plane

Td = R=/6DT

(4) 1140

Vol. 146, No. 3, 1987

BIOCHEMICAL

and DT is the translational obtained from the ratio (Av1,2 - C)-l

AND BIOPHYSICAL

diffusion

coefficient.

RESEARCH COMMUNICATIONS

The value of DT is

of the slope to the intercept = (3kT)/4M,xqR3 + (6D)/M,RZ

from the relationship (5)

MATERIALS AND METHODS Egg lyso-PC and deuterium depleted water were purchased from Sigma Chemical Co. Aquacide (Molecular Weight 70,000) was purchased from Calbiochem. [16,16,16-aH,]Palmitic acid was purchased from Serdary Research Laboratories. [16,16,16-~H,]PC was synthesized by condensation of egg lyso-PC and [16,16,16-2H,]palmitic acid using l,l'-carbonyldiimidazole by a method described in Thewalt et al. (3). The lipoproteins were isolated from fresh (<3 days old) plasma supplied by the Canadian Red Cross. Isolation was by means of sequential ultracentrifugal floatation: VLDL, dc1.006 g/ml; LDL,, d=1.025-1.063 g/ml; HDL,r d=1.063-1.125 g/ml; HDL,, d=1.125-1.210 g/ml. VLDL fractions from several fresh plasma units were pooled and spun twice more (42,000 rpm; 5OC) for 30, then 20, min and the top one-quarter fraction discarded both times. [16,16,16-ZH,IPC was incorporated into HDL, by addition of solid (5 mol % with respect to lipoprotein phospholipid) to the HDL, solution followed by three one-minute sonications at 42OC using a Biosonic III probe-type sonicator . The HDL, purity was checked by immunoelectrophoresis (4). Protein was determined by the method of Lowry (5) as modified by Kashyap et al. (6). Phospholipid was determined as described by Ames (7). Cholesteryl ester and total cholesterol were enzymatically determined using Boehringer Mannheim clinical test kits. Prior to NMRexperiments all lipoproteins except HDL, were dialysed into 0.15M NaCl, 2mtj EDTA, pH 1.5. HDL, was dialysed into deuterium depleted water. The lipoproteins were concentrated by ultracentrifugal floatation (VLDL), treatment with Aquacide (LDL,), or Millipore CX30 submersible ultrafiltration units (LDL,,HDL,). The 31P NMRexperiments were performed at 102.2 MHz, without proton decoupling, using a 5.9 Tesla Nalorac superconducting magnet and a home-built spectrometer. Collection and Fourier transformation of the free induction decays was performed using a Nicolet BNC-12 computer. Spectra were obtained using a one-pulse sequence with phase alternation in order to minimize baseline distortion. Except for HDL,, temperature was controlled at 25f0.5Y by a home-built solid state controller/home-built variable temperature probe. 2H NMRspectra were determined as above at 38.8 MHz. The spectra for VLDL, LDL, and HDL, were analyzed using an iterative least squares fitting routine to Lorentzian lineshape functions. The plots in Fig. 1 were analyzed using a weighted least squares routine courtesy of Dr. Ian Gay, Simon Fraser University. Mean sizes of VLDL and LDL, were determined using a model 270 Nicomp Submicron Particle Sizer. Electron micrographs of HDL, were obtained using a Philips EM300 Electron Microscope at 80 kV. Electron microscopy samples were stained with 2% ammoniummolybdate at pH 8.0 and applied to 200 mesh Formvar carbon coated grids, then air dried. Solvent viscosities were measured using an Ostwald viscometer. RESULTSAND DISCUSSION ,lP NMRspectra were obtained for function

of solvent viscosity.

HDL,, HDL,, LDL, and VLDL as a

Computer generated spectra composedof the 1141

Vol. 146, No. 3, 1987

BIOCHEMICAL

AND BIOPHYSICAL

25

LDL

(3’P

RESEARCH COMMUNICATIONS

NMR)

20. 15.

0

20

601HDL,

9 x -i 0

40

(3’P

NMR)

5

10

60

80

0’0

100 -1

20 ‘LDL

(“P

40

60

80

100

1

60

80

100

12

NMR)

30.

20. 4

lo-

&

07 0 14’

HDL,

(2H

15

20

25

20

:

40

NMR)

120. 1 oo80. 60~ 40. 0

5

10

15

20

q-1 (Pa.&) FIGURE1. Plots of reciprocal NMRlinewidths versus reciprocal of solvent viscosity at 25OCfor, clockwise from bottom left, 'H NMR linewidth of [16,16,16-zH,]phosphatidylcholine in HDL,; SIP NMR linewidths of phospholipids in HDL,; HDL,; LDL,; VLDL. The solid lines in the plots for HDL,, LDL, and VLDLare weighted least squares fits to the data points. The constant C was taken as 15 Hz for 3lP and 2.5 Hz for =H.

superposition

of two Lorentzian

were used to fit

the experimental

contained approximately noise-decoupled

functions,

a signal

spectra in most cases. HDL, and HDL,

20% and 15% SPM respectively,

31P NMRspectroscopy,

proton noise-decoupled 31P NMRof extracted

linewidths

as shown by proton

and the linewidths

equal. LDL, and VLDL contained approximately

Necessary conditions

each for SPM and PC,

of the signals are

40% and 14% SPM as shown by lipids

in CHCl,/MeOH (2:l

for convergence of the computer routine

of the SPM and PC signals be made equal and that

chemical shift

remain unchanged at 0.6 ppm. 1142

were that

v/v). the

the SPM-PC

BIOCHEMICAL

Vol. 146, No. 3, 1987

Fig. viscosity

AND BIOPHYSICAL

1 is a double reciprocal (see eqn. 5). The plots

mL3, HDL, containing respectively.

-5

A plot

mol%

plot

RESEARCH COMMUNICATIONS

of NMRlinewidth

verSuS solvent

are determined based on the 31P NMRof

[16,16,16-~H,]PC,

LDL,, and VLDL,

was also determined from the 2H NMRlinewidth

dependence of the labelled

HDL, sample upon solvent

viscosity

(Fig.

1,

lower left). The sizes of the LDL, and VLDL particles determined by light the Tablel.The light

scattering

used in the NMRstudies were

and the mean particle

HDLs are too small to give reliable

scattering

and since HDL, is reported

radii

E are given in

size measurements by

to have a diameter of 8.5-11 nm

(81, a mean radius of E = 5.0 run was assumed. The size of HDL, was determined by electron From the plots

microscopy. in Fig.

1, diffusion

coefficients

DT for phospholipid

molecules in the surface monolayer of the lipoproteins

were calculated

at

25OC and are presented in the Table. From 31P NMRDT for HDL, and HDL, were calculated

to be 1.8x10-*

approximately

and 2.3x10-*

the sameas that

where values for DT range

from

reported 1x10-*

respectively.

cm*/s,

These values are

for PC in phospholipid to

7~10-~

cm2/s

The fact

(9).

= 1.8x10-8 cm2/s for HDL, when determined by 2H NMRindicates

TABLE

1

DIFFUSION COEFFICIENTS OF PHOSPHOLIPIDS IN THE OUTER MONOLAYER OF HUMAN SERUM LIPOPROTEINS Lipoprotein

HDL2

c Nucleus

D (cm2

R (nm)

. s“)

3’P

4.0a

2.3

2 0.8

x 10-8

3’P

5.ob

1.8

20.3

x 10-8

2H

5.0b

1.9 to.3

x10-8

LDL2

3’P

12.oc

1.4 kO.5

x 10-9

VLDL

3’P

18.4c

9.1 + 1.0

x 10-g

a. Measured by electron microscopy b. Sizes reported with R=4.3 - 5.5 nm (P.J. and Cell Biol. 6_3, 850 (1985) c. Measured by quasielastic light scattering

1143

Dolphin,

Can.

bilayers

J. Bich.

that

that the

DT

BIOCHEMICAL

Vol. 146, No. 3, 1987

phospholipid envisige

AND BIOPHYSICAL

molecules undergo diffusion

each phospholipid

as rigid

(7<5xlO-*s)

entities.

molecule moving as a cylinder

across the surface of the lipoprotein. is rapidly

RESEARCH COMMUNICATIONS

reorienting

The phospholipid

Thus, we randomly walking

molecule, of course,

about the long axis of the cylindrically

symmetric molecule. The larger diffusion.

particles,

LDL, and VLDL, have distinctly

The value of DT = 9.1x10-'

cm*/s for phospholipid

the surface monolayer of VLDL is at least corresponding diffusion

diffusion

is the fact

slower diffusion

compared to HDL can be attributed (10). The ratio

of cholesterol

in the two HDLs it

at 25OC, phospholipid

found in VLDL, it

of phospholipid

to phospholipid

to phospholipid

can explain,

only in part,

acyl chain heterogeneity, the gel to liquid

transition

display

significantly

rate

phospholipids

greater

(see

occuring SPMs, as a result

of

complicated thermal behaviour and,

crystalline

phase transition

temperature is

occuring PCs (11). Howwer, we are

monolayer for LDL, at 25Y,

as it

from LDL, do not display

has been shown

a thermal

over the range 20-45OC (12,13).

2. Core-Monolayer Interactions. cholesteryl

the slower diffusion

in LDL, is so slow:

higher than for naturally

the isolated

in LDL, is the same as

molecules in the LDL, monolayer. We can suggest three

not proposing a gel-like that

content

in our VLDL is 0.27 whereas

ratio

above) than does VLDL or the HDLs. Naturally

significantly

molecules in VLDL

to the much higher cholesterol

reasons why the diffusion

in general,

is in the HDLs.

of phospholipid

1. Monolayer Composition. LDL, has a SPM content

fatty

cm2/s) is

is X0.15.

Since the cholesterol

possible

that,

one order of magnitude slower than it

The slightly

that

in the surface monolayer of the

in the surface monolayer of LDL, (DT = l.l~lO-~

approximately

molecules in

two times slower than the

rate of phospholipid

HDLs. Even more significant,

slower surface

The LDL, core is composedpredominantly

esters while the VLDL core is mostly triglycerides. 1144

It

is

of

Vol.

146,

No. 3, 1987

possible

that

phospholipid just

into

their

AND

BIOPHYSICAL

interactions

is

slowed.

RESEARCH

COMMUNICATIONS

in LDL, are of a nature

At 25OC the core

phase transition

diffusion

that

CT,-20-40°C)

components

(13)

by means of interdigitation

that

of LDL, are

and the solid-like of phospholipid

core

acyl

chains

core.

3. Protein Weight

core-monolayer diffusion

may impair into

BIOCHEMICAL

LDL, contains

Composition.

512,000).

phospholipid

Perhaps

there

and apoB which

a single

strong

is a unique,

causes

protein

apoB (Molecular

interaction

the phospholipid

diffusion

between rate

to slow

significantly. Clearly,

more work

component diffusion however,

any explanation

communication must also

in

(14,15),

must be done to explain lipoproteins. of surface

possibly

accommodate the surface

In light

the differences

of our diffusion

in surface measurements

phenomena such as protein-protein

necessary dynamics

for

efficient

presented

receptor

binding,

here.

REFERENCES L Eisenberg, S., Bilheimer, D. W., Levy, R. I. and Lindgren, F. T. (1973) Biochim. Biophys. Acta 326, 361-377 2, Abragam, A. (1961) Principals of Nuclear Magnetism, pp 424-427, Clarendon Press, Oxford & Thewalt, J. L., Wassall, S. R., Gorrissen and Cushley, R. J. (1985) Biochim. Biophys. Acta 817, 355-365 4. Hatch, 5. T. and Lees, R. S. (1968) Adv. Lipid Res. 6, l-35 5, Lowry, O.H., Rosebrough, N. J., Parr, A. L. and Randall, R. J. (1951) J. %ol. Chem. 193, 265-275 & Kashyap, M. L., Hynd, B. A. and Robinson, K. (1980) J. Lipid Res. 21, 491-495 7. Ames, B. N. (1966) Methods Enzymol. 8, 115-118 z Pownall, H. J. and Gotto, A. M. (1983) Phospholipids and Atherosclerosis (Avogaro, P., Mancini, M., Ricci, G. and Pavletti, R, eds) pp 99-114, Raven Press, New York k Mackay, A. L., Burnell, E. E., Nichol, C. P., Weeks, G., Bloom, M. and Valic, M. I. (1978) FEBS Lett. 88, 97-100 & Cullis, P. R. (1976) FEBS Lett. 70, 223-228 11. Barenholz, Y., Suurkuusk, J., Mountcastle, D., Thompson, T. E. and ztonen, R. L. (1976) Biochemistry 15, 2441-2447 12. Small, D. M. (1977) J. Colloid Interface Sci. 58,581-602 13. Deckelbaum, R. J., Shipley, G. G. and Small, D. S. (1977) J. Biol. Chem. 252, 744-754 14. Devaux, P. and McConnell, H. M. (1972) J. Am. Chem. Sot. 94 ,4475-4481 15. Schlessinger, J. and Elson, E. L. (1981) Membrane Receptors; Receptors xd Recognition (Jacobs, S. and Cautrecasas, P. eds) Series B, Vol. 11, pp 159-170, Chapmanand Hall, New York

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