lee.;rz
and
l€fth€l
attd
R. Bentjcg
e.Jil-eQS
f\riAa({
P.*,l\o/tl, q-a,6.
S. -Daw;al5
1rnal1/r^t4'6
Prolein
pa*oi
@tsp^*.)
,, 6Lood
fn,
adsorption
at
the
solid-liquid
interface
measured by
ellipsometry
P.A. Cuyperso', H.C. Hemker*, w.Th.
of
Departments
Centre,
Biophysicso
University
of
Hermenso
and
Linburg ,
Biochemistry*,
6200
MD
Biomedical
IIAASTRICHT, The
Netherlands.
Organi.sation of
rn
r974
the
university
mechanism of
chosen
as
centraL
The process of
actions
ting
another
and
process was
this
research
biochemistry,
physiology
the
work
ceL_
close
to-
research possibilities.
number
their
in
consist
of
of
sequence,
a
zymogen to
cLotting
and their
interactions
on the
last
are
part
complex
These
com_
phospholipirl
vesser. warrs.
A number of
to
be
serine
proteases
(zymogen) forrns.
of
the
which
The clot_
enzyme activations
activated
and
zymogen acti_
protease.
purified
studied
of
conponents.
inter_
circuJ_ating
series
are
by
rnembranes) are
an active
factors
controlled
and
the
unactivated
this
is
of
platelet
reside
Different
been spent
and
have been found
in
process
rn
biophysics,
calcium
blood
blood-rand/or
each step
vates
great
proteins,
proteins
research.
bl-ood cLotting
a
(e.9.
are present
in
of
i.e.
structures
founded
bloodcLotting
between
ponents,
the
of
Iike
was
the bloodclotting
multidisciplinary
regulation
the
of
electronmicroscopy
gether offering
in
praastricht
field
departments,
biology,
The
of
the regulation
a
different
the universitv
this
in
(I,2t3).
proces,
our
departments
Much effort
the
has
conversion
46
of
prorhrombin
rofe
of
focus
the
(II)
to
the phospholipid
of
(IIa).
thrombin
surface
More specifically
rn thi.s process
our
research.
In
order
to
different
proteins
of
this
prothrombinase
the phospholipi-ds we modified
ellipsometry,
tlon
of
in
or
of
complex with
optical
mono-
the main
interaction
technique,
we couLd foflow
p h o s p h o l .i p i d
on
the
an existing
such a way that
proteins
study
is
rhe
the adsorpmultilayers
(4,6,1 ,g).
Ef I ipsometry
The ellipsometer
is
an optical
changes in polarization
of
instrument
light
that
due to reflection
The changes are measured by two adjustable
cated
as
conslsts
to
polarizer
of
finding
mi-nimal
Iight
(p)
and
the
positions
intensity
change in polarization
optical
1s
properties
covered
protein
surface
results
with
1ayer,
are
mass from
the
thickness
can be calculated.
the
the adsorbed rayer
refractive
where derived
of
of
films
or
a
the reflecting
p and A.
refractive
n,
the surface
optical
The formulas
index
rf
polymer
in
The
dependent on the
surface.
change
and the
photodiode.
is
properties
the positions
indi_
The measurement
the
due to reflection
This
t).
p and A corresponding
of
the reflecting
(fiq.
polarizers
(A).
reaching
then the optical-
in changes in
adsorbed layer
analyzer
phospholipidlayers,
changed.
and A varues
the
of
measures the
properties
From these p
index
to
of
the
carcurate
and thickness,
and experimentaLry
d,
of
validat-
ed (6).
A
complete
method of
description
of
the
computation is given
instrument
in ref.
(6,7\.
as
hrell
as
the
47
stepprng stepprn9
motor
motor
of
Schematic representation
Fj-gure l-
the automated ellipso-
meter.
of
Preparation
the
proteins
of
Adsorption
was measured on
sLide placed in a well
glass
cuvette
brith
fi1led
chromium slides
layers
This
sample (4)
buffer.
were
temperature controlled
most of
with
coated
chromiun
our
stacked
these
studies
multi-
mono- or
phospholipids.
of
stacking
was done by using
the method of
to
Different
types of protein
pects of
the adsorbed layer.
Fibrinogen
Adsorption
The cuvette
of
In
covered
according
ties
stirred,
a
the
was fiLled
Langmuirtrough
Langmuir and Bl-odgett.
adsorptions,
on hydrophil,ic
with
buffer
chromium surface'
measured. After
a preparative
(9)
structural
as-
Chromium.
and the
positions
of
about 200 seconds fibrinogen
optical
proper-
P and A'
was added.
were
48
After
the
moment of
change fast
conds
for
preted
in
shown in
during
anaLyzer
terms of
Fig'
behaviour
130 A'
and an end feveL
both
the
addition,
2'
a
index is
mass shows the same behaviour
index
saturation
stabirizes
then n = r-3g.
as the
change much
folr.ows the
and
thickness
rnrer_
and mass are
incex does not
adsorption
analyzer
about 2000 se_
The results
refractive
The thickness
monorayer
and
reached after
The refractive
The refractive
mass va.Iue is
is
polarizer
and polarrzer.
thickness.
adsorption.
of
the
around
fhe calcufatecl
does.
The end
0 . 4 7 l t c / c m 2.
xfi3
,!195
i . J
Figure
surface.
of
tire
2
Adsorption
Buffer
fibrinogen
of
fibrinogen
10J sec.
on hyrrrophir-ic
was 10 pq/m1.
.
that
durinq
constant
chromium
0,01 M Tris_HCl- pH=7.0. Bufferconcentration
the
refractive
first
I50
seconds the
index n = 1.g.
Fig.3
thickness
snows
grows at
Then the refractive
rndex
49
drops
the
while
grows until
= 1.48.
still
about 68 A while
From this
point
increases.
the
on the
denser. The layer
cally
refractive
gets
layer
which reaches
thickness
index drops
to
n
and opti-
thinner
monolayer
maximum before
its
The
at d = 35 A and n = I.'12.
stabilizes
mass shows a regular
The caLculated
tion
thickness
the
type
adsorp-
Layer gets
thinner
m=o.56vg/cn2.
Figure
3
Adsorption
of
as in
surface exp. conditions
Adsorption of
cl-otting
fibrinogen
factor
fig.
on hydrophobic
chromium
2.
va on dops,
etacked
on chro-
mlum.
Fig.
4 shows that
during
during
the
the
first
f irst
the refractive
5000
seconds.
index
The
2000 seconds. Af te.
is
changing slightLy
thickness
l - h ' ci ^ ^ i n f
increases
rha
thick-
)t,
u88
Figure
4
stacked
Adsorption
on
chromiun
NaCl.
Buffer
ness
decreases
of
the
calcufated
mass
adsorption.
The refractive
after
the
total
vaLue m=0.16
constant
indicating
tion
anC
penetration
0.05
m.
of
factor
shows
a
index of
mass adsorbed
vg/cm2.
of
n=2.3 after
a
a
index,
reorganisation
very
of
high
a
of
into
of
Tris_HCl
pH=7.5,
V. was 2
us/nr.
DOPS
0.1
I{
The
regular
monolayer
cype
increases
even
the rayer
reached
the
an
equilibrium
refractive
index
of
104 seconds.
an adsorption
changing
the
refractive
the proteins
layer
increases.
examples show respectively
refractive
one
index
has
The value
is,
on
refractive
adsorbed
the aclsorbed layer
These three
V.
buffer
concentration
and
factor
0 . 1 6 pg/a2
refractive
molecules
index
with
after
n=2.j
the phospholipid
a
index
adsorp_
indicatrng
layer
(9).
Binding
of
constants
to phospholipids
the proteins
membraneq
(4,5,10).
FiS.
5
containing
values
of
the
ciation
6.
prothrombin
on
808 DOPS/202 DOPC. By measuring
the
adsorbed quantity
f as a function
shows the
the
prothrombin
constant
From these
concentrations
of
adsorption
of
prothrombin
concentration
c
the
in
Ka can be obtained.
data
it
up to I0
equilibrium
buffer,
This
is
that
was calculated
a monolayer
the
shown in
for
ug/m1 the adsorption
assoFig.
prothrombln
process on 100?
!!:
,ili
ilr:
f 'v"1"''
Fig. 5 Stepwise adsorption
prothrombin
DOPC.
o
on 808 DOPS/208
F id.
!
6
v
r Y .
Scal-chard
DIOt
v v ! . e v l r q L v
the
adsorption
bin
on
I
of
f or
prothrom-
DOPS/DOPC mixtures.
O=100% DOPS, 0=40% DCPS;
*=20?
DOPS.
DOPScan be described
Ka =
with
(2.01
0.15 + 0.0r
extra
of
+ 0.02)
a
*
singl_e class
lcB
vg/cm2. For high
amount of
DOPC in
by
the
prothronbin
prothrombin
is
monolayer
is
initiarry
more
increased,
values
of
fo.*.
The adsorption
layers
is
stilI
somewhat biphasic
with
If
the
this
pronounced
=
percentage
biphasic
with
an
beha_
clecreasing
on 20g DOpS/g0B DOPC mono_
Ku = (l.lf
vaLues of
sites
concentratrons
adsorbed.
becomes
binding
(mean + sD) and I'-r"
lt-I
vrour
cribed
of
but
can gt6UaLly
+ 0.34)
be des_
x 107 M-l and I*.*
= 0.25 + 0.02 ug/cm2.
AdsorptionFig.
and desorption
7 shows the
function
of
time
concentrations
seconds.
physico
the
of
Fig.
order
f*u*
Iayer
is
sional
Tris-tlcl.
=
the
reLative
adsorption
rate
and the
adsorption,
the binding
After
at
these
about
300
importance
transport
the
an unstirred
of
kinetics
the
of
rate
data
of
layer
of
up to
vg/cm2.
The
the
addirion
30? of
0.I
protein
i-s in
adsorbed
m a x i m u mb i n d i n g _ c a p a _
thickness
iso-electric
of
prothrombin
of
amount of
d = 5 Urn. The adsorption
limited
AII
in
hre analysed
model with
DOPS as a
from the same data.
limited
0.38
of
value
establish
independent
diffusioqal
layer
concentrations.
equilibrium
a diffusional
beginning
city
an
to
d obtained
on a double
di-fferent
during
B shows that
j..e.
for
chemicaf
7 with
thickness
adsorption
reach
prothrombin
fig.
the
In
rates
of
of
the
fibrinogen
point
M NaCL or
unstirred
is
pH=6 in
changing
diffu_
0.0I
M
the pH to
?3.
ug/ml
10.
eg/nl
5.
eg/nl
?'
eg/ml
.75 ug/nt
aoo
t lme ( s )
Adsorption of Prothrombin
Fig.7
( .05M pH=7.5 ) , NaCI
Tris,/Hcl
0.00
0.o5
0.10
on
(.IM),Ca
0.15
++
0.2o
?*l n.I
Dq
Buffer:
(1.5 mM)
o.25 2 o.3o
(uslcm-)
/
Fig.8
Adsorption
B u ff e r :
T r i s , / H C I ( . 0 5 t ' l p H = 7 . 5 ) , N a c l ( . l M ) , c a + + ( 1 . 5 m t l ).
velocity
of
Prothrombin
on
2*18:1
PS.
54
pH=7.5 the diffusionaf
tlon
was never diffusional
until
recently
surfaces
are
A.
fimitations
we mainly
or metal
starting
in
Bantles
of
Netherlands
covering
parameters
proteins
adsorption
on metal
Twente University
polymer
on
of
refractive
index
in
interactions.
with
the effect
Feyen and
Technofogy
protein
surfaces
J.
Now we
homolog
of
By
series
of
po}ymer
didferent
and amount of
the
different
adsorbed.
We are
their
system.
the groups of
with
investigate
on the
this
coated vrith phospholipids.
reflecting
polyrners we will-
in
protein
studied
cooperation
research
our
Iimited
surfaces
the
vanishes. Al-buminadsorp-
also
starting
a
cooperation
with
industry
to
test
polymers on the foLl-owing aspects.
1) nffect
on
bfoodclotting
different
clotting
factors
vation
clotting
factor
2) fne
telns
of
general
in
in
XfI
adsorptioncharacteristics
and
particular,
the
effect
e.g.
on
the acti-
(Hagemanfactor).
of
different
plasma
pro-
on these polymers.
3) ffre effect
of
polymer
toxicity
on different
cel-cultures
a n d c e l - a d h e si o n .
These aspects
ments of
are
studied
biochemistry
in
cooperation
with
the
depart-
and celbiology.
Summarizing:
I)
Our
institute
coagulation.
is
specialised
in
the
mechanism of
blood
55
2) Ellipsometry
tion
on
faces,
the
the
offers
a good technique
interactj-ons
proteins
such as adsorbed amount and the
adsorbed protein
t]-on constants
This
of
to
and
information
without
the fiefd
testing
tems for
using
of
1ayer
the
is
adsorption
obtained
any labets
biomateriaf
biomaterials
(refractive
and
biocompatibili.ty.
or
obtain
with
informa_
polymer
water
content
index),
and desorption
continuously,
further
of we are
developing
rates.
in
srru,
So in
interested
of
of
assocra_
protein-modifications.
research
sur_
in
testsys_