THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
VoI. 261,No. 14,Issue of May 15,pp 6222-6229 1986
Printed in L?S.A.
0 1986 by The American Society of Biological Chemists, Inc.
Terbium Probe of Calcium-binding Sites on the ProthrombinMembrane Complex*
(Received for publication, November 20, 1985)
Leslie E. SommervilleS, Robert M. ResnickS, David D. Thomas§, and GaryL. NelsestuenST
From the Departments of Biochemistry, University of Minnesota, $St. Paul, Minnesota 55108 and
$Minneapolis, Minnesota 55455
Terbium was used as a probe of Ca2+-bindingsites brose et al., 1979; Jackson et aL, 1979). Based on other
on the prothrombin-phospholipid complex. Stoichio- membrane binding studies, another model suggests that Ca2+metric titrations of prothrombin binding to phospho- mediates binding by nonionic interactions (Resnick and Nellipid vesicles with either Tb3+or Ca2+ showed that a sestuen, 1980). Another prothrombin-membrane binding
minimum of 8 metal ions were needed for binding model postulates that the Ca2+-stabilized conformation of
prothrombin to vesicles (3Mn2++ 5 Ca” for prothrom- theseproteinshas
an exposed hydrophobic regionwhich
bin or 8 Tb3+for F-1). When Ca2+ alone was used, a interacts with the membrane (Lecompte and Miller, 198%
total of about 11 metal ions were needed for complete Madar et aL, 1982; Rhee et aL,1982). Onedifficulty with this
binding. These stoichiometries indicated 3 classes of
metal ions: one class needed to induce the conforma- latter proposal is that itdoes not containa description for the
tional change, a second required for protein-membrane role of negatively charged phospholipids.
Studies have indicated that Ca2+ andprothrombin will
contact, and a third class bound at other sites on the
cause PS1 to cluster (Mayer and Nelsestuen, 1981). With
protein that arenot involved in membrane binding.
By adding Tb3+to solutions containing both protein phospholipid monolayers, conditions were found where only
and phospholipid, undesirable Tb3+-induced events, slight perturbation of the phospholipid occurred upon protein
such as irreversible aggregation of prothrombin or binding (Mayer et al., 1983). Measurements of the on/off rate
vesicle fusion, were avoided. Protein-vesicle binding constants for prothrombin binding to phospholipid vesicles
apparently prevented protein aggregation or vesicle indicated that a population of Ca2+bound to theprothrombinfusion. The protein-vesicle binding affinity was sev- phospholipid complex was exchanging at a rate comparable
eralfold greater in the presence of Tb3+compared to to prothrombin (Wei et aL, 1982). All of these results have
been interpreted as additional support for a population of
Ca”.
CoEDTA quenching of Tb3+bound to the prothrom- Ca2+forming a bridge between the protein and phospholipid.
bin-phospholipid complexes indicated that all metal
Ca2+has few spectral properties which can be used to study
ions were at least partially exposed to the quencher. its environment. Lanthanides, which are spectroscopically
Some populations of Tb3+showed lower quenching con- active, have been shown to be able to substitute for Ca2+in
stants when all of the prothrombin was bound. Tb3+ membrane binding (Nelsestuen et al., 1976; Furie et al., 1976).
emission lifetimes revealed that some Tb3+ions in the Gadolinium has been shown to support the prothrombinase
protein-membrane complex were in a different envi- reaction in the presence of phospholipids although the initial
ronment from those bound to the protein alone. The velocity of the reaction is much less than that obtained for
results indicated that the metal ions in the prothrom- calcium (Furie et aL, 1976).Two luminescent lanthanides,
bin-membrane complex are relatively open to the sol- Tb3+ and Eu3+ have been used to probe metal ion binding
vent yet do affect the characteristics of the protein- sites of many different biological systems (for review see
membrane binding equilibrium.
Horrocks and Albin, 1984). The luminescent properties of
these two lanthanides are sensitive to theenvironment (Horrocks and Albin, 1984). Eu3+ has been used to study both
prothrombin
(Rhee et al., 1982) and factor Xa (Rhee et al.,
Ca2+-mediated membrane binding of the vitamin K-dependent blood clotting factors is essential for blood clotting. 1984) and in each case Eu3+-induced precipitation of the
How Ca2+mediates this membrane binding is not well under- proteins was reported. T b 3 + has been used to study prothromstood. It is known that acid phospholipids are required for bin (Brittain et al., 1976; Sommerville et ab, 1985), factor IX
binding and the proteins must be in a specific conformation, (Morita et aL,1984),and proteinC (Johnson et ul., 1983)with
induced by binding Ca2+ (for review see Nelsestuen, 1984). no Tb3+-inducedprecipitation reported. For this reason Tb3+
Various models for Ca2+-mediated membrane binding have was chosen as the probe for the Ca2+-binding sites in the
been proposed. One model proposes that ionic interactions protein-phospholipid complex.
between the Ca2+,protein, andphospholipid are thedominant
The abbreviations used are: PS, bovine brain L-a-phosphatidylforces involved in Ca2+-mediatedmembrane binding (Dom-
* This work was supported by National Institutes of Health Grants
HL 15728 (to G. L. N.) and GM 27906 (to D. D. T.). The timeresolved spectrometer was purchased by National Institutes of Health
Shared Instrumentation Grant RR01439. The costs of publication of
this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “aduertisement” in
accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.
n To whom correspondence should be addressed.
L-serine; Gla, y-carboxyglutamic acid; F-1, the amino-terminal 1-156
residues of bovine prothrombin; PC, egg L-a-phosphatidylcholine;
K2PIPES, dipotassium piperazine-N,N’-bis(2-ethanesulfonic acid);
NBD-PE, N-4-nitrobenzo-2-oxa-1,3-&azole-~-a-phosphatidylethanolamine; LRBS-PE, lissamine rhodamine B sulfonyl L-a-phosphatidylethanolamine; CoEDTA, the Co(II1) complex with ethylene&aminetetraacetic acid TbEDTA, the Tb(II1) complex with EDTA;
DNP-PE, 1,2-dipalmitoyl-sn-glycerol-3-(~-2,4-dinitrophenyl-aminoethyl) phosphate.
6222
6223
Tb3+Probe of Ca2+-binding Sites
The studies presented here indicate that, in addition to the
metal ions required to cause a protein conformational change,
5 2 1 additional metal ions are necessary for prothrombinmembrane binding. The mininum number of metal ions associated with each prothrombin-membrane complex was 811. The metal ions in the complex are allpartially accessible
to a collisional quencher in solution. The results suggest a
relatively open and accessible protein-membrane complex
with all of the metal ions clustered near the membrane surface.
EXPERIMENTAL PROCEDURES
Materials
emission spectrum and the emission spectra for the mixtures of
LRBS-PE and NBD-PE containingvesicles were uncorrected spectra.
All of the spectra were collected on a Perkin-Elmer MPF-44A spectrofluorometer. The T b 3 + emission intensities were measured with
excitation a t 488 nm or at 295 nm. The emission was scanned from
530 nm to 560 nm by methods previously outlined (Sommerville et
al., 1985).
Tb3+Emission Measuremnts-Tb3+ was excited using the 488-nm
line from Coumarin 500 (in dioxane/MeOH, 95/5, v/v) pumped
through an NRG dye laser. A 488 & 0.6-nm band pass filter was used
to eliminate 2nd and 3rd harmonics from the exciting light. A Lambda
is 308 nm) was used to excite the dye
Physik Excimer Laser X,(
laser. The Tb3+emission was detected a t 90" to theincident beam of
light through a Spectra Film band pass filter (Amm is 546 +. 10 nm).
Multiple (20,000 to 50,000) transients were collected at 40 to 80 Hz
with 10 ps/channel and 1024 channels. Emission detection, collection,
and analysis were done as described previously (Sommerville et al.,
1985). These experiments were conducted at ambient temperature.
CoEDTA Quenching of T b 3 + Emission-CoEDTA can quench Tb3+
emission by dipole-dipole energy transfer. The rate of energy transfer
is
The hexahydrates of TbCl3, Lac&, HOC13, Co(I1) sulfate, MnClZ,
and CaC12were obtained from Aldrich in the highest purity available
and were used without further purification. KZPIPES, NazEDTA,
bovine brain PS, and egg PC were purchased from Sigma. NBD-PE
and LRBS-PEwere obtained from Avant; Polar Lipids. Sephadex G50 was from Pharmacia. The AG 1-X8 anion-exchange resin was
kt = 1/70 ( R o / ~ ) ~
(1)
from Bio-Rad. All other reagents were reagent grade or better. All
solutions were prepared with distilled and deionized water. The
sulfated Sephadex G-50 column was prepared as described previously where 7 0 is the lifetime of the donor in the absence of acceptor, Ro is
the distance at which the efficiency of energy transfer ( E )is 0.5, and
(Miletech et al., 1980).
CoEDTA was prepared by HzOz oxidation of Co(I1) to Co(II1) in r is the distance between the acceptor and donor (for reviewsee
the presence of EDTA (Yeh and Mears, 1980). The CoEDTA showed Stryer, 1978). Ro can be estimated by
a characteristic absorption spectrum (Haner et al., 1984) and was
Ro = 9.79 X io3 ( K ' ~ - ~ + J ) ' / ~
(2)
quantitated by absorption spectroscopy using a molar extinction
coefficient of 326 a t 535 nm (Haner et al., 1984). The lanthanide where K is the dipole orientation factor (K' is assumed to be %, n is
stock solutions were quantitated by EDTA titration using Xylenol the refractive index of the medium through which the energy transfer
Orange as theindicator (Birnbaum and Sykes, 1978).
is occurring (nis assumed to be 1.33, n-4 = 0.32), 6 is the quantun
Single bilayer vesicles were prepared with 20% PS, 80% PC, or yield of the donor in theabsence of the acceptor, and J is a function
with 10% NBD-PE, 20% PS, 70% PC, or with 10% LRBS-PE, 20% which describes the overlap of the acceptor's absorption band with
PS, 70% PC by a modification of the method of Huang (1969) as the donor's emission bands (see Fig. 1 for this overlap). Ro for all
described previously (Nelsestuen and Lim, 1977). Phospholipid con- of the experiments was taken as 21 A (Yeh and Meares, 1980). This
centrations were determined by phosphorus analysis (Chen et al., estimated distance, due to thesixth-root dependence of Ro on 6, will
1956) using a phospholipid/phosphorus weight ratio of 25.
not be significantly effected by small changes in thelifetimes such as
Prothrombin (Nelsestuen,1976), factor X (Nelsestuen et al., 1976), those observed in the studies presented below. The efficiency of
and protein C (Stenflo, 1976) were isolated from citrated bovine energy transfer is equal to
plasma as described. Factor X was used after one 'additional ionexchange chromatography on DEAE-Sepharose similar to the final
E = k T / ( k T + ko)
(3)
step reported by Stenflo (1976). Protein C was separated from final
traces of prothrombin by chromatography on sulfated G-50. This where ko = 1 / 7 0 . If T = (kT + h)-',
the lifetime of the donor in the
replaced the heparin-agarose column used by others (Esmon et al., presence of an acceptor, then Equation 3 becomes the following.
1983). The sulfated G-50 column was initially equilibrated with 0.1
M sodium phosphate, pH 6.0, containing 0.1 M NaC1. Protein C
E = 1 - (T/TO)
(4)
always eluted inthe void volume while the other vitamin K-dependent
proteins eluted a t higher salt. Each isolated protein was highly pure
3.30
as indicated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970) using Coomassie Blue as the stain. Each
2.89
protein was assayed for membrane binding as described previously
2
(Nelsestuen and Lim, 1977).Prothrombin, F-1, FactorX, and protein
2.48 x
c
C were quantitated by absorbance a t 280 nm using E'" of 14.1, 10.1
(Heldebrant and Mann, 1973), 12.4, and 13.7(Sugo et al., 1984),
2.06
respectively; the M, values used for each protein were 72,500 (Bajaj
c
1.65
et al., 1975), 23,400 (Lim et al., 1977), 56,000,and 55,000 (Sugo et al.,
1984), respectively.
\
L
Methods
Light Scattering-Relative light scattering intensity a t 90" from
the incident lightwas used to measure protein binding to phospholipid
vesicles (Nelsestuen and Lim, 1977). For these light scatteringmeasurements a Perkin-Elmer MPF-44A fluorescence spectrophotometer
was used. The temperature was held at 25 "C using a Lauda RM-3
circulating water bath. Unless otherwise indicated, all of the experiments were conducted in 50 mM K2PIPES, pH6.5.
Quasielastic light scatteringwas conducted as described previously
(Lim et al., 1977) using the 488-nm l i e of a Lexel, Model 95, argon
ion laser. Hydrodynamic radiireported inthis paper arethe Z
averaged values only. No corrections for the degree ofprotein packing
or surface irregularities weremade. Such uncorrected resultsare
useful for direct comparison of two samples. In all cases, proteininduced membrane binding by either T b 3 + or Ca" was fully reversible
with EDTA.
Fluorescence Intensities and Spectra Measurements-The T b 3 +
480 510 540
570 600 630 660
EMISSION h (nm)
FIG. 1. CoEDTA absorption spectrum overlap with Tb3+prothrombin emission spectrum. The prothrombin concentration
was 10 p~ and the TbC4concentration was 60 FM in a total volume
of 1.5 ml. The T b 3 + emission spectrum (the spectrum showing several
maxima) was scanned from480 to 660 nm at 15 nm/min with a 430nm cutoff filter in the emission port. The absorbance spectrum of
CoEDTA (the single broad spectrum) was obtained with a Beckman
Acta CII absorbance spectrophotometer. A 1.2 mM solution of
CoEDTA with a 1-cm pathlength was scanned from 480 to 660 nm
at a scan rate of 10 nm/min.
6224
Tb3+Probe of Ca2+-bindingSites
Equation 4 was used to calculate E from the experimental data, and
Equation 3 was used to analyze E in terms of the intermolecular
distance that determines kT.
Under the conditions of these experiments, energy transfer from
Tb3+to CoEDTA is enhanced due to diffusion. CoEDTA is a freely
diffusing ( D is about
cmz/s) acceptor while Tb3+ bound to
cm2/s), or the prothrombin-phosphoprothrombin ( D is about
lipid complex ( D is about
cmZ/s) is a freely diffusing donor with
lifetimes that range from 1.0 to 1.5 ms. Under these conditions for
spherical, uncharged donors and acceptors the second-order rate
constant for energy transfer in the rapid diffusion limit is
k,
= ~T/[COEDTA]
= 2.52
x 1021R06a-3k,,~"
s-l
(5)
where a is the distance of closest approach between the donor and
acceptor (for review, seeStryer et aL, 1982). Due to thecomplexity of
the Tb3+binding sites in this system no simple model could bemade
that described the diffusion-enhanced energy transfer. Therefore,
Equation 5 was used to get a first approximation of & for CoEDTA
quenching of Tb3+bound to thissystem. For example, if a population
of Tb3+with a 12,value of 763 s-l were 10 A (a) from the surface of
the prothrombin-membrane complex, the upper limit for the secondorder energy transfer rate constant(kZ= h/[CoEDTA]) for diffusionenhanced dipole-dipoleoenergy transfer would be 1.6 X lo5 M-' s-l
(assuming Ro was 21 A). Therefore, experimentally measured rate
constants greater than this value would suggest that the quenching
of Tb3+by CoEDTA would be due to contact quenching instead of
dipole-dipole diffusion-enhanced energy transfer. Other more complex models for energy transfer in the rapid diffusion limit (Stryer et
al., 1982) include nonuniform accessibility of the donor and receptor
on all sides. These properties would decrease the upper limit for k,
and would shift the theoretical curves to the left (see Fig. 8, below)
thus decreasing the value of a.
By solving Equation 5 for and substituting into Equation 3, one
obtains the following.
E = kz[CoEDTA]/(h[CoEDTA]
+ k)
70
=
=
1/(k+ k,[CoEDTA])
2.oop
(6)
Equation 6 relates changes in E and a with changes in the CoEDTA
concentration. This equation was used to calculate the theoretical
lines drawn below in Fig. 9. E was determined experimentally from
Equation 4 while a was estimated from the plot shown in Fig. 9. If
values determined for a were less than 10 A this would suggest that
contact quenching or other types of energy transfer are dominant
factors for the quenching experiments.
If CoEDTA does quench T b 3 + by exchange interaction (contact
quenching), involving electron cloud overlap (Wensel and Meares,
1983), then this quenching can also be described by a second-order
Measurements of the T b 3 + emission lifetimes
quenching constant (b).
in the presence ( 7 ) and absence (70) of CoEDTA can be used to
analyze this kind of quenching as well.
T
Ca2+were required for binding each prothrombin molecule. A
portion of these ions were thought to be bound to theprotein
and another portion
to thephospholipid (Nelsestuen and Lim,
1977). These measurements were conducted in a mixture of
components (protein, phospholipids, and protein-phospholipid complexes), each of which were able to bind Ca2+.Interpretation of these results required correction for metal ions
bound to sites otherthan those involved in theprothrombinmembrane complex.
Measurements of Ca2+-mediatedbinding of prothrombin to
phospholipid vesicles under conditions of very little free Ca2+
are shown in Fig. 2. Linear extrapolation of protein binding
to the theoretical maximum level of binding (dashed line in
Fig. 2 A ) indicated that about 11 Ca2+were needed for each
bound prothrombin molecule. This wouldbe a maximum
stoichiometry since it is possible that some of the metal ions
3-
L
B
\
1
2
B
(7)
(8)
Thus, by taking the ratio of Equation 7/Equation 8, one obtains the
Stern-Volmer equation as follows.
ro/r = 1
+ kq~OICoEDTA]
(9)
A plot of T ~ / Tuersus [CoEDTA] willgive a straight line with the
intercept at 1 if the quencher has equal accessibility to all of the
fluorophores (Lakowicz, 1983).
Tb3+Binding to Factor X , Protein C, and F-1 f PhospholipidEach sample was titrated with Tb3+.Terbium binding was measured
by changes in Tb3+ intensitydue to excitation a t 488 nm and due to
energy transfer from tryptophan residues (Sommerville et al., 1985).
Excitation of Th3+at 488 nm in a system including F-1 and phospholipid could not be measured because the large light scattering peak
interfered with the Tb3+ emission. For this reason lifetimes rather
than intensity measurements were used to estimate quenching. In all
of the experiments reported here Tb3+was always added to mixtures
of protein/phospholipid.
RESULTS
Metal Ion Stoichiometry for the Prothrombin-Phospholipid
Complex-Equilibrium binding studies of Ca2+ binding to
prothrombin and phospholipids indicated that about 9 +. 1
[Ca210r [Tb31/CPROTHROMBIN]
FIG. 2. Prothrombin binding to phospholipid vesicles under
conditions where Ca2+and Tb3+binding is stoichiometric. The
experiments with Ca" ( A and B ) were conducted in 50 mM Tris, pH
7.5, 0.1 M NaC1. The experiments conducted with Tb3+ (C) were in
50 mM K,PIPES, pH 6.5. All of the experiments used a total volume
of 1.5ml. The theoretical maximum binding is indicated by the
dashed horizontal line.The number of metal ions needed for binding
were determined by linear extrapolation to thetheoretical maximum
binding. The concentrations of phospholipid and prothrombin in A
were 8 and 7.25 mg/ml (0.1 mM) with 0 ( 0 ) , 3 (O),6 (O), and 8 (A)
eq of Mn2+.The concentrations of phospholipid in B were 8 mg/ml
(0)
and 14.5 mg/ml ( 0 )while the prothrombin concentration was
7.25 mg/ml. Eight equivalents of Mn2+were used in this experiment.
The concentration of phospholipid in C was 363 pg/ml (0)and 50
)
pg/ml(O) with prothrombin concentrations of 363 pg/ml (5 p ~ (0)
and 12.5 pg/ml (0.17 pM) (0).
Tb3' Probe of Ca2+-binding
Sites
6225
the presence of MnZ+were not helpful since T b 3 + and Mn2+
compete for the sites involved in stabilizing the prothrombin
conformation and T b 3 + displaced all Mn2+from the protein
(data notshown). The results indicated that a totalof 11Tb3+
were required for membrane binding of prothrombin. Experiments at two different protein, phospholipid, and protein/
phospholipid ratios showed that the stoichiometry rem,ained
unchanged.
Equilibrium Binding of Prothrombin to Phospholipid Vesicles-If the models proposing protein association with anionic
phospholipids through Ca2+ions are correct, the affinity of
the protein for the phospholipid would be greater with lanthanide ions rather than
Ca2+ions. The basis for this prediction is the affinity of lanthanide ions for protein ligands which
is 100- to 1000-fold greater (see Sommerville et al., 1985 and/
or Furie et al., 1979) than thatfor Caz+.
The Tb3+binding experiments had to be carried out in a
manner different than thatused previously for Ca2+(Nelsestuen and Lim, 1977). Normally, prothrombin and the phospholipids are equilibrated with Ca2+ separately andthen
P + PL + nCa + P - PL - Ca
(10) mixed. A similar approach with lanthanidelprotein mixtures
Kd = [P][PL][Ca]"/[P - PL -Can]
resulted in prothrombin-phospholipid binding that was only
about 60% of the theoretical value.2 This level of membrane
where P is protein and PLis phospholipid.
Due to the several equilibria contributing to theoverall Kd binding is similar to that reported by Rhee et al. (1982). It
(Nelsestuen and Lim, 1977), it was difficult to calculate the appeared that T b 3 + caused some irreversible aggregation (or
actual free Ca2+concentration a t high protein and phospho- inactivation) of free prothrombin which prevented membrane
lipid concentrations and estimateits contribution to the total binding.
A second problem, vesicle fusion, resulted when lanthanides
Ca2+ added. However, if free Ca2+ were significant in the
were mixed with acidic phospholipid-containing vesicles as
experiments shown in Fig. 2, the observed stoichiometry
illustrated inFig. 4. Two populations of vesicles wereprepared
should be sensitive to a change in the phospholipid or procontaining NBD-PE and LRBS-PE. Fusion of these vesicles
thrombin concentration (see Equation 10). Despite altering
results in energy transfer from NBD-PE to LRBS-PE with
the phospholipid and
prothrombinlphospholipid ratio by almost
quenching of the NBD-PE signal and enhancement of the
a factor of two, the estimated number of Ca2' required for
LRBS-PE signal. Fig. 4A shows a 50150 mixture of the two
quantitative binding of prothrombin to thephospholipid vespopulation of vesicles with no protein or Tb3+while Fig. 4B
icles remained unchanged (Fig. 2B). A number of experiments
shows the same mixture of vesicles with 33 p~ TbCl,. Fig. 4,
of this type were carried out. There were nodetectable changes
C andD,shows the same population of vesicles in thepresence
in the Ca" stoichiometry due to a 2-fold alteration of the
of prothrombin and prothrombin plus 6 eq of Tb3+, respecconcentration of an individual component. The titrations in
tively. The prothrombin bound to the vesicles apparently
Fig. 2 appeared to represent actual bound metal ions.
F-1binding to membranes was investigated using Tb3+(Fig. prevented vesicle fusion and the membrane binding appar3). Linear extrapolationof protein binding to thequantitative ently prevented the irreversible protein aggregation.
The results of binding experiments for prothrombin/phoslevel showed that about 8 Tb3+ions were required for full Fpholipid mixtures are shown in Fig. 5. A different protein1binding (Fig. 3). About 6 Tb3+can bindto F-1 in theabsence
of phospholipid (Sommerville et al., 1985). Consequently, a phospholipid sample was used for each experimental mea2 mM CaCl, or 12 eq of TbC13 were added
larger number of Tb3+ ions were required for membrane surement. Either
to the protein-lipid mixture. The solid lines are theoretical
binding. Again, the results inFig. 3 showed that theminimum
curves forprotein-membrane binding with the Kd values
number of metal ions needed for membrane binding was 8.
indicated.
For CaC1, this modified experimental approach
Titration of prothrombin-phospholipid binding with Tb3+
indicated
protein-membrane
dissociation constants of 0.3 to
is shown in Fig. 2C. In this case, experiments carried out in
0.5 pM which are similar to those reported previously (Nelsestuen and Broderius, 1977). The data with Tb3' were not
as satisfying since the data points did not follow the shape
for a single dissociation constant. An upper limit for the Kd
r
- - -- - - appeared to be 0.1 p~ while other portions of the curve
suggested a Kd as low as 0.01 PM.
5
Tb3+ hasbeen shown to cause prothrombin to aggregate to
' 1.1
0
the apparentlevel of trimers (Nelsestuen et al., 1981).Binding
2
I
of such aggregates to the vesicle could alter the apparent
1.o
affinity calculations (Fig. 5A). However, the hydrodynamic
0
4
8
12
16
20
properties of the particles formed with calcium or terbium
pCIs]/p]
and saturating prothrombinwere indistinguishable (Fig. 5B).
FIG. 3. Tb3+-mediated membrane binding of F-1 to phos- Binding of aggregates of prothrombin to themembrane should
pholipid vesicles. The theoretical maximum binding is indicated by cause substantial changes in this parameterregardless cf the
the dashed horizontal line. The concentrations of F-1 and phospho- orientation of the second sphere of proteins (30-100 A de-
are binding at sites not involved in forming a productive
protein-membrane complex.However, control experiments
indicated that thisproblem was not a significant factor.
Mn2+ions stabilize the membrane-binding conformation of
prothrombin but Mn2+ions do not supportmembrane binding
(Nelsestuen et al., 1976). The number of Ca2+ions needed for
membrane binding should be less in the presence of Mn2+.
This was found to be the case and theresults of a number of
titrations showed that 5 Ca2+ions were rcquired per prothrombin molecule at quantitative binding (e.g. Fig. 2 A ) . The maximum apparent error, determined
from a number of titrations,
appeared to be 5 5 1. If Mn2+ions could compete for Ca2+binding sites involved in the membrane binding, increased
Mn2+should increase the requirements for Ca2' in this titration. This did not occur and the Ca2+/prothrombin ratio at
quantitative prothrombin binding was the same in the presence of 3,6, or 8 eq of Mn2+(Fig. 2 A ) .
The experiments in Fig. 2 measure a single overall equilibrium which could be abbreviated
l,2m
"
b
lipid were 50 pg/ml (2.1 p ~ and
) 200 pg/ml, respectively, in a total
volume of 1.5 ml. Two sets of data collected with different preparations of F-1 and phospholipids are shown.
' L. E. Sommerville, R. M. Resnick, D. D. Thomas, and G . L.
Nelsestuen, unpublished data.
6226
Tb3+Probe of Ca2+-bindingSites
D
0
10
20
30
[PL]
I
500 560 500 560 500 560
!
IO
,
40
50
200
(pg/ml)
2501
I
560
EMISSION h ( n m )
FIG.4. Measurement of Tb3+-induced aggregation/fusionof
phospholipid vesicles f prothrombin. These emission spectra
were scanned from 500 to 590 nm at 30 nm/min with excitation a t
460 nm. The total phospholipid concentration in each experiment
was 218 pg/ml with half of the phospholipid vesicles comprised of
10% LRBS-PE, 20% PS, 70% PC and the other half comprised of
10% NBD-PE, 20% PS, 70% PC in a total volume of 1.5 ml. Upon
fusion or aggregation of these vesicles, energy transfer from NBDPE to LRBS-PE would occur causing a quenching of the NBD-PE
signal and an enhancement of the LRBS-PE signal. The results
shown' are for vesicles alone ( A ) , vesicles plus 33 pM TbC13 (B),
vesicles plus 218 pgjml prothrombin ( C ) ,and vesicles plus 218 pg/ml
(3 p ~prothrombin
)
with 10 eq of TbCL. Similar resultswere obtained
if F-1was substituted for prothrombin.
pending on the horizontal or vertical addition of another layer
of protein molecules). It appeared that thetwo metals bound
prothrombin to themembrane with a similar geometry.
Tb3+Binding to Protein C and FactorX-Metal ion binding
to protein C and factorX was examined by Tb3+emission due
to direct excitation of Tb3+ at 488 nm and due to energy
transfer from tryptophan residues (Fig. 6 ) . Irradiation a t 488
nm excites all Tb3+in the solution equally but the emission
signal is dominated by thoseTb3+ with the fewest water
ligands. Irradiation a t 295 nm selects those Tb3+ which are
bound near a tryptophan donor. Emission intensities from
488-nm exrjitation of T b 3 + in thepresence of factor X showed
a linear increase up to about 7 Tb3+/factor X (Fig. 6A). After
8 T b 3 + had been added, a precipitate formed which precluded
further accurate measurements of T b 3 + binding to factor X.
Tb3+emission intensity due to energy transfer from tryptophan residues showed a lag for the first4 Tb3+followed by an
increase up to 8 Tb3+where the precipitate formed.
Terbium binding to protein C is shown in Fig. 6B.Emission
intensity due to excitation at 488 nm increased steadily for
the first 10 T b 3 + added and then plateaued. These results
showed that theadded Tb3+were binding to theprotein with
high affinity. Emission intensity due to energy transfer from
tryptophan residues again showed a delay for the first4 T b 3 +
followed bya rapid increase for the next6 Tb3'. After addition
of about 10 T b 3 + there was no additional increase in the Tb3+
emission due to energy transfer. The final emission intensity
of Tb3+ bound to protein C was about 10-fold greater than
that observed for either factor X or prothrombin (Sommerville
et al., 1985). No Tb3+-inducedprotein precipitation was observed with protein C. These resultsshowed that theincrease
in Tb3+ emission due to energy transfer from tryptophan
'""If
170
150
0
A
6
[PROTHROMBIN]/[PL]
(w/w)
FIG. 5. Prothrombin binding to 20% PS, 80% P C vesicles
mediated by Tb3+and Ca2+. The experiments shown in A were
conducted with a constant prothrombin/phospholipid ratio of 0.5 and
0,
.
and
,A) or with 2 mM CaClz (0 and A).
with 12 eq of TbCL (
The different symbols represent experiments with separate preparations of prothrombin andphospholipids.The solid lines are theoretical
prothrombin-membrane binding curves for the indicated Kd values
and a maximum binding of 1.2 g of prothrombin/g of PL (Nelsestuen
and Broderius, 1977a). E shows the hydrodynamic radius of prothrombin-vesicle complexes generated by calcium).( or terbium (0).
The phospholipid concentration was 200 pg/ml in a total volume of
1.0 ml. The measurements conducted with TbC13(0)involved adding
prothrombin to thephospholipid solution followed bythe addition of
12 eq of TbCL. Fifteen to 20 min were allowed before making the
measurements. PL, phospholipid.
residues was due to sequential filling of sites which have very
little tryptophanenergy transfer to Tb3+followed bythe filling
of sites which have a larger amount of energy transfer rather
than to equilibrium binding of Tb3+ as suggested previously
(Johnson et al., 1983). Initial filling of sites with little energy
transfer was similar to theproperties observed for prothrombin (Sommerville et al., 1985).
Tb3+-mediatedmembrane binding of protein C and factor
X was also attempted. However, Tb3+-inducedprecipitation
of factor X precluded such studies. Protein C did not precipitate with Tb3+ butthe light scattering intensitieswere always
higher than theoretical values for quantitative protein Cmembrane binding. This indicated that there was reversible
aggregation of vesicles byprotein C plus Tb3+.Consequently,
these comparisons could not be completed.
Lifetimes of Emission from Tb3+Bound to Prothrombin and
Prothrombin plus Phospholipid-A representative sample of
T b 3 + emission decay for Tb3+ bound to prothrombin in the
presence of phospholipid is shown in Fig. 7A. Table I summarizes the Tb3+emission lifetimes obtained with 1-12 T b 3 +
Sites Tb3+ Probeof Ca.2+-binding
6227
TABLEI
emission lifetimes with prothrombin
rt:phospholipid
The lifetimes were fit to l(t) = Al(exp) - t / +~Az(exp)
~
t/T2
&. Pro, prothrombin; PL, phospholipid.
Tb3'
-
Sample
+ Pro
+ Pro
5 T b + Pro
6 T b + Pro
8 Tb + Pro
10 Tb + Pro
12 Tb + Pro
1T b + Pro + PL
3 T b + Pro + PL
5 T b + Pro + PL
6 Tb + Pro + PL
8 T b + Pro + PL
10 Tb + Pro + PL
1Tb
3 Tb
12 Tb -k Pro
+ PL
AI
72
Az
m
1.24
0.043
0.011
1.56
0.056
0.011
0.604
0.011
0.011
0.13
1.63
0.941
0.061
1.56
0.090
0.011
0.900
0.030
0.0083
0.14
1.54
0.797
0.082
0.714
0.098
1.47
0.21
0.0087
0.595
0.12
0.0086
0.28
1.36
0.150 0.0028 1.29
0.076
0.012
0.390 0.016
1.46
0.088
0.011
0.467 0.086 0.012
0.21
1.33
0.451 0.039
1.31
0.11
0.011
0.447 0.099
0.0082
0.26
1.33
0.422
0.19
1.34 0.50
0.382
0.22
0.0087
0.54
1.28
71
m
A2
+
(7)'
ms
1.24
1.40
1.41
1.39
1.27
1.23
1.13
1.25
1.30
1.08
1.08
1.09
0.0088 1.09
1.02
[TbC13]/[PROTEIN]
FIG.6. Tb3+binding to protein C and factor X. The concentration of factor X ( A ) and protein C ( B ) in these experiments was
10 FM in a total volume of 1.5 ml. The intensities were measured as
described under "Methods." Excitation a t 488 (0,right axis) and 295
(0,left axis) are given for each protein.
1.2
m^
>
t
v)
0,
1.0
Sample
3
x
v
2
+ Pro
5 Tb + Pro
6 Tb + Pro
3 Tb
w
I-
E
TABLE
I1
CoEDTA quenching of Tb3+bound to prothrombin f phospholipids
All of the experiments contained 0.3 mM CoEDTA except those
with higher concentrations. The latter all contained 6 Tb3' ions
without (upper part) and with (lower part) phospholipid. Pro, prothrombin; PL, phospholipid.
0.8
m
0
L
0.6
-3
0.4
+
+
+
+
+
+
+
0 2 4 6 510
0.2
0
I
0
2
4
6
8
1
0
2
TIME (msec)
4
6
8
1
+0.6 mM COEDTA
+1.2 mM COEDTA
8 T b Pro
10 Tb Pro
12 T b Pro
1Tb Pro PL
3 Tb Pro PL
5 Tb Pro PL
6 T b Pro PL
+0.6 mM CoEDTA
+1.2 mM CoEDTA
8 T b Pro PL
10 Tb Pro + PL
12 Tb Pro PL
0
FIG.7. Tb3+emission decays f CoEDTA. These decays were
collected with 6 eq ofTb3' bound to 5 p M prothrombin in thepresence
of 363 pg/ml phospholipid with no CoEDTA (A) and in the presence
of 0.6 mM CoEDTA ( B )in a total volume of 1.0 ml. The insets show
the residuals ([exp-fit]/[exp]"') for each decay. The datawere fit best
by a two-exponential fitting routine. The best fit was determined by
visual inspection of the residuals and thefit overlap with the data.
+
+
+
+
+
+
+
+
+
A,"
12'
m
ms
0.287
0.034
0.866
0.019
0.010
0.495
0.367
0.061
1.17
0.058
0.011
0.758
0.292
0.036
1.10
0.052
0.011
0.769
0.279 0.12 1.01
0.177 0.091 0.775
0.302 0.082 1.15
0.303 0.084 1.14
0.262 0.092 1.07
0.259 0.052 0.714
0.320 0.046 0.912
0.303 0.097 0.960
0.334 0.053 1.04
0.250 0.18 0.906
0.201 0.12 0.778
1.11
0.348 0.13
0.322 0.354 1.11
0.341 0.179 1.12
~1~
Azo
41"
(T)*
ms
0.10
0.059
0.14
0.15
0.18
0.011
0.023
0.094
0.057
0.16
0.086
0.14
0.51
0.245
0.0080
0.0080
0.0082
0.0088
0.0085
0.011
0.011
0.011
0.011
0.0079
0.0078
0.0079
0.0088
0.0088
0.560
0.412
0.837
0.839
0.797
0.338
0.517
0.626
0.670
0.559
0.442
0.743
0.787
0.791
CoEDTA was exchange-inert. Prothrombin showed similar
Ca2+-or Tb3+-dependentmembrane-binding properties in the
presence or absence of CoEDTA; so this component did not
bound to prothrombin in the absence and presence of phos- appear to undergo exchange (data not shown).
The T b 3 + emission lifetimes for Tb3+bound to prothrombin
pholipid. Most of the Tb3' emission decays could be fit better
by a two-exponential fit (Table I), but for comparison with in the presence and absence of phospholipid, plus CoEDTA,
the quenching data, shown in Table 11, the averaged lifetimes are summarized in Table 11. In all cases the Tb3+ emission
decays could be fit better by a two-exponential function (Fig.
(7) (Table I) were used. The error associated with the ( T )
values in repetitive determination was less than *lo%. This 7B shows a typical decay). However, initial attempts to anacontrasted with the observed differences in the lifetimes for lyze the quenching data from T~ values alone was not very
satisfactory since T~ represents only a fraction (usually about
T b 3 + bound to free prothrombin and for T b 3 + bound to prothrombin in the presence of phospholipid (about 30% differ- %) of the total Tb3+ in the complex and could vary slightly
ence at 5-6 Tb3+/prothrombin). Therefore, it appeared that a depending on theexact fitting parameters. Thisincreased the
population of the T b 3 + ions bound to theprothrombin-mem- variability in quenching constants. The averaged lifetimes
brane complex were in a different environment from the Tb3+ ((7))were more consistent and were therefore used to analyze
the CoEDTA quenching data.
bound to prothrombin alone.
For diffusion-enhanced energy transfer the lifetime of the
CoEDTA Quenching of Tb3+ Bound to Prothrombin in the
donor in the presence of the acceptor is shown calculated in
Presence and Absence of Phospholipid-Terbium-mediated
Equation 11.
binding of prothrombin to phospholipid vesicles in the presT = ( ~ ~ [ C O E D T+
A ]( l / ~ , J ) - l
(11)
ence and absence of CoEDTA was carried out to assure that
Tb3+Probe of Ca2'-binding Sites
6228
By substituting the ( T ) values from Table I1 and Table I for
T and 7 0 in Equation 11, an apparent second-order rate constant for energy transfer (k2)could be calculated. These values
ranged from 7.2 X lo6 M" s-' with 1 Tb3+ ion bound to
prothrombin in the presence of phospholipids to 9.5 X lo5 M"
s" with 12 Tb3+ions bound to prothrombin in the presence
of phospholipids. These values are alllarger than what would
be expected if the energy transfer was due to diffusionenhanced dipole-dipole energy transfer (see "Methods").
The kz values for 6 Tb3+ions bound to prothrombin alone
or in the presence of phospholipids were used to calculate E
(Equation 6 ) for quenching by 0.3,0.6, and 1.2 mM CoEDTA.
These values, shown in Fig. 8, are compared to theoretical
curves calculated for 0.3,0.6, and 1.2 mM CoEDTA quenching
of Tb3+.From these results the distance of closest approach
for either system was estimated to be between 4 and 6
CoEDTA andTbEDTA, based on x-ray crystallographic
data, have a distance of closest approach of about 7 A. A
closest approach of 4 to 6 A indicatedthat contact quenching
of Tb3+ by CoEDTA was probably the dominant factor in
these experiments. The results indicatedtpat approach of the
fluorophore and quencher was within 10 A or less. They also
suggested that therewere no buried Tb3+for either prothrombin alone or prothrombin plus phospholipid.
The ( T ) values from Table I and Table I1 were analyzed
using the Stern-Volmer equation (Equation 9 under "Methods"), where 70 is ( T ) from Table I, and 7 is (7) from Table
11. A Stern-Volmer plot for CoEDTA quenching of 6 Tb3+
bound to prothrombin in the presence and absence of phospholipid is shown in Fig. 9A. The downward curvature shown
in Fig. 9A indicated that therewas a heterogeneous population
of Tb3+ with different accessibilities to CoEDTA quenching.
The slope of the curve (Fig. 9A) from zero to 0.3 mM CoEDTA
was used to estimate an apparent quenching constant (kq,
Equation 9) for various Tb3+/protein ratios. These apparent
kq values were plotted as a functionof Tb3+/protein ratio (Fig.
9B). There was about a 10-fold change in the apparent kq
between 1 Tb3' and 12 Tb3+bound to prothrombin in either
the absence or presence of phospholipid. These quenching
studies indicatedthat even though there were no buried Tb3+,
there was a population of Tb3+that become more protected
from the quencher due to prothrombin aggregation or to
membrane-binding.
:w
1
0
0.3
0.6
[CoEDTA]
0.9
1.2
(mM)
i
A.
0
0
2
4
6
8
1
0
1
2
a 6 )
FIG. 8. CoEDTA quenching of Tb3+ analyzed according to
models for diffusion-enhanced energy transfer.
The solid lines
are theoretical curves calculated from Equations 4 and 6 for quencher
concentrations of 0.3 mM (left line), 0.6 mM (center line),and 1.2 mM
(right line). The six data points shown are for 6 eq of Tb3+bound to
prothrombin in the absence (0)and presence (0)of phospholipid
and at0.3, 0.6, and 1.2 mM CoEDTA, respectively.
-
00
3.0
6.0
3.0
[TbCId/PROTHROMBIN
12
3
FIG. 9. Contactquenchingof
Tb3+ by CoEDTA. A shows
results for CoEDTA quenching of 6 eq of Tb3+bound to prothrombin
in the absence (0)and presence (0)of phospholipid. T,, and T were
taken from Tables I and 11, respectively. The slope of the line between
0 and 0.3 mM CoEDTA was used to determine the apparent secondorder quenching constant (&). These quenching constants for 1to 12
eq of Tb3+bounds to prothrombin in the presence (0)and absence
(0)of phospholipid are shown in B.
DISCUSSION
Stoichiometric titrations of protein-membrane binding and
control experiments showed that a minimum of 8 metal ions
mediated the membrane binding of prothrombin (Fig. 2, A
and B , 3 Mn2+ 5 Ca2+;or Fig. 3B, 8 Tb3+ions for F-1). The
metal ions were distributed into at least two major populations. The first population of metal ions stabilizes a membrane-binding conformation of prothrombin or F-1 (3 Mn2+,
3 Tb3+,or 6 Ca" are needed for this stabilization; Nelsestuen
et al., 1981). A detailed analysis of this binding process as well
as a subdivision of these metal ions into different classes has
been presented (e.g. see Sommerville et al., 1985 and references therein). The second population was necessary for the
prothrombin-membrane complex and required 5 Tb3+ or 5
Ca2+ions (Fig. 2, A and B).
The totalnumber of Tb3' ions that can bind to F-1 or the
prothrombin-membrane complex was the same as that observed for Ca2+.Three Tb3+ bind cause
and the conformational
change in F-1. The next 5 metal ions bind and mediate the
membrane binding (Figs. 2C and 3B) to give a minimum
required stoichiometry of 8. An additional 3 Tb3+ can bind
to
the protein to account for the number of Tb3' (about 10-11)
bound to theprotein-membrane complexes. For prothrombin,
the Tb3+ stoichiometry required for membrane binding was
about 11, suggesting that all sites on the protein were filled
by the time membrane binding was complete.
With stoichiometric amounts of T b 3 + the prothrombinmembrane complex showedproperties of an equilibrium binding system (Fig. 5A) although the binding appeared to be
heterogeneous. The binding was saturable (data not shown),
and the hydrodynamic radius of the prothrombin-membrane
complex (Fig.5B) was the same in thepresence of either Tb3+
or Ca2+. A difference between the Tb3+-mediated and the
Ca2+-mediatedbinding was the Kd (Fig. 5A). This was expected if metal ions were involved in bridging between the
protein and phospholipid. However, the available evidence
+
Tb3+Probe of Ca2+-bindingSites
showed that the magnitude of the difference in Kd was small
compared to differences in Ca'' and T b 3 + affinities for protein
ligands. Therefore, the results are not conclusive and cannot
definitively differentiate a protein-membrane interactionmediated by metal ions andone that isnot. Comparison of these
results with different vitamin K-dependent proteins might
shed light onthis matter but artifacts
associated with the use
of Tb3+precluded work with factor X or protein C.
Measurements of emission lifetimes for T b 3 + bound to
prothrombin in the presence and absence of phospholipid
showed differences in the environments of T b 3 + bound to
prothrombin alone versus prothrombin bound to a membrane
(Table I). This difference was most apparent at Tb3+/protein
ratios of 5 or 6 to 1. Detailed interpretation of these differences was not possible. However, the observations contrasted
with those of Rhee et al. (1982), who saw no Eu3+ spectral
changes associated with Eu3+-mediatedmembrane binding of
prothrombin.
The results of this study showed that terbium can support
equilibrium binding of prothrombin to phospholipid vesicles
in a manner that appeared similar to calcium. All of the ions
bound to the complex were at least partially protected from
collisional quenching by CoEDTA, but no buried Tb3+ ions
were detected. This suggested that the prothrombin-membrane complex is relatively open and accessible. A relatively
open, accessible membrane complex is also supported by
iodide quenching constants of F-1 intrinsic fluorescence of
free or phospholipid-bound F-1, where no differences were
detected.' The results indicated a truly peripheralbinding for
this protein-membrane complex.
Acknowledgments-We would like to thank Rick Leder for his
assistance in measuring the Tb3+ emission lifetimes, Dr. Victor
Bloomfield for the use of his laser light scattering system, and Dr.
David Knoll for his assistance in thosemeasurements. We would also
like to thankDr. Mark Pusey for preparing sulfated Sephadex G-50.
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