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Multiple forms of human factor IX in chromatography and isoelectric focusing
Vof*
TKROXBOSIS RESEARCH Printed in r;he TVnited Szates
YULTIPLE
FORMS
C H R 3 :::,A,TCSRAPtJY
OF
Red
Helsinki,
(Received
ABSTRACT
IX
I?\.
F C C iJ S I r,:C
GFID ISOELECTRIC
Hannu Finnish
HUKA?,; FACTOR
7, pp. 101-112, Pergamon Press,
Suomela
Cross
Blood
Transfusion
Service
Finland
in revised form 11.5.1975. 3.3.1975; Accepted by Editor B. Blombkk)
Factor 600-3000 genecus
IX
concentrate made from a plasma pool of persons was demonstrated to be heteroin DEAE-Sephadex and hydroxyapatite chromatography . Factor IX activity was separated into three main groups and each of these groups was shown to be heterogeneous in isoelectric focusing. Factor IX concentrate made from individual plasma samples was found to be homogeneous when the same techniques were used. Variation between individuals was demonstrated.
INTRODUCTION Differences strated.
in
The
coagulation found variants
has lation
been study
polymorphism Veltkamp
of
al.
normal
least
8
the
patients
heterogeneity
males
that in
not
et
the
confirm 101
al. there levels this
B have
been
both
immunological
four
variants
literature
demonand have
been
concerning
(21. of
Lester
variation could
at
on
reviewed
Hemophilia of
Hemophilia based
and
suggested.
for et
is
recently
in
occurrence
also
of
analysis,
Denson found
The
types
classification time
(I).
the
normal
human
concluded may of result
Factor
from
well
be
Factor in
a pcpugenetic
IX their
(31. twin
IX
1975 Inc.
stories
[.I).
In
‘1?2
Factor
sours=
7s”;)
zf
._loFy-_=ri
IX (F IX) froT pcoled
fepeataaly
fount
CEAE-Sephadex phate)
_
c:
in sevaral
nur-an plasya, sompsnents.
chromatography,
chromatography
per presents
__
F 1. aCt
This ::,a-e
aopsren:
i,n
!salci~~ phosf:-using :51. This pa-
and in isoelectric
from pooled
MATERIAL
-‘,,
in hydroxyaparite
the chromatographic
F IX originating
?.,>_i_-._ _ _ i--z
>.;r:-f;a;
and issele~tric
plasma
and four
properties
of
tndividuala.
AIND METHODS
F IX concentrates a)
The pooled
plasma:
The plasma
pool of about 3000 persons was separated
within
was cryo-supernatant
again,
separated.
and F IX concentrate
from a
plasma,
six hours of the bleeding,
thawed and the cryoprecipitate frozen
for F IX concentrate
frozen
which at -7G°C,
The supernatant
prepared
as described
was earlier
(61. bl
Individual
either
from repeated
The plasmas
The plasmas
plasma:
donations
were stored
ted. F IX concentrate
frozen.
taining
0.2 M phosphate
about 600 ml of plasma transferred
Cryoprecipitate
buffers
gently
onto a Biichner funnel
contained
as fellows:
in G.14 M phosphate-
were diluted
both 0.20 and 0.25 M phosphate-NaCl
concentrate
was not separa-
from a buffer con-
and 0.8 M NaCl, pH 6.91 was added to and mixed
II, VII, Xl were eluted
received
plasmapheresis.
&as prepared
A-50 R equilibrated
(all phosphate-NaCl
were
or after a double
from individuals
25 ml of DEAE-Sephadex NaCl
of the donors
with
for l/2 h; the gel was
and washed buffers.
with 600 mi of F IX (and Factors
50 ml of 1.0 M phosphate-?iaCi. The
5-6 mg of protein/ml
and 5-8 units of
F IX/ml. Purification
with A1(OH13
gel
was carried
Swart et al. (71 at O'C. F IX concentrate volymes
of distilled
each mg of protein by adding
water.
and mixed
a gel volume
0.3 ml Al(OH13
out as described was diluted
of 0.3 M phosphate
with three
22.1 was pipetted
for 5 min. Elution
by
for
was performed
buffer,
pH 6.0, mixing
FKLTIPLE
t'ol.;,~o.l
iiata
r?~rs
olustSS
\.*;SrS
FCClZd
3;ai;_ __ $-z
ChT-!Ifil~tCl--
z;
3:
_i
-7
_-.Y
FOR%
OF FACTOR
_,3 r
p^31
2nd
*
CT
frCzSn
prCCpS.S?_:
cr isoelectric
E’aa”y
_jl
CL
J 2 per "=.._ ct
tr.3::
IX
a”+_ivity, __
+‘_r”, +“er
T;i-_ : y r: r f x _” -
“4
fC=!,si"g.
CE.GE-Sephadex ChronatograpSv. 2C ml CEAE-Se~?a:ex gel ec,uili:ra-y 2.2 t-_n c e n L* r a te ,_,~F _, __ in 3.96 ?, zitrafe, pP 7.0, was atided c, [ 2 t .>'At T:H
13CC
ucits
,Gft;r
7.5,.
of
gent?e
F
I;()
dissclvsd
Fixirg
For
in
l/2
h
:zc
5:s
7-1 gel
:.'I3 :yss
F
citr3-j
p;lcko:2
ink=
3
and e:uted ~.+itha linear citrate g:-aziant, ,;b 7.3, ,usic' cz l_i?n = 6 113OC iiltrograd R gradient mixer. ihe protein ‘dZS ret----an L ,< _v_i ed by Cvicorcf II (LKE). The citrate measured forc
cf each fracticr
F':eter(Wiss.-Tec~n.Werkst~tter,
ty a Conductivity
Kailheim)
content
cali'bratec with a series
2,?42
buffers
made of the
wss
used
5~
the gradient. The gel was prepared
Hytiroxyapatite chromatography. to Tiselius buffer,
et al. (61 and equilibrated
pH 6.6. The mclarity
about 2 mg per milliiitre gradient
lJ/ith5 mM phosphate
of the charge
ed t 0 about 0.1 M with distilled
according
water.
soltition was adjust-
The protein
of gel. Chromatography
elut ion was done with phosphate
load was
with a linear
suffer,
pP 6.8, and
LKE equipment. Isoelectric
focusing
was carried
(110 ml) using ampholytes The sample
containing
hours against against followed
7-17 mg of protein
solution.
with regard
buffer,
was dialyzed
pH 7.0, and for two hours
The manufacturer's
for column
for twc
and sample
instructions
leading,
pump at the rate of 33 ml/h.
were allowed
to reach room temperature
with Beckman
Zeromatic
Factor
IX activity
cephalin-kaolin
and/or
Findip
measurements.
system
1-3 ml fractions
555. pH meters.
One-stage
the study
with an
and the pH was measured
using haemophilia
strate was used throughout
WPZ
and focused
for 72-69 hours at 560 V at 3'C. The column was emptied LK6 peristaltic
8100-l
pH 3-5, 4-6 and 5-7, 2 ml of each.
5 mM phosphate
ampholyte
out with an LK6 column
assay with the
6 plasma
as the sub-
(9).
RESULTS Pooled Figures
plasma
Factor
IX.
1 and 2 demonstrate
the hetercgeneity
of the F IX con-
Ji;i”cF,3---
c
2)
-5’
There there three
nenrs. t
(Fi-
_,krag,hy
he+arogene;us. U..,&
:s 3
the concentration absarbed
at
=
I;(
a,~!--~i~it
-3:n
’
ccnponents,
3
which
components
c,rpg-
3~~earzd
(Fig. I]
chrzrcat~graphy
were eluted
and the main component
0.100 and G.115 M, and the third component
betwaen
,*
sever‘3
was critirq1. ^_i.i, F IX activity
K and the first
0.08 and '3.100 Fi. The second
5::
eaok
gE,&E-Sephadex
In
cf citrate
L1.08
_., Y appears:
was
was between eluted
between
0.115
and 0.130 P-1. A steeper Several
gradient
fractions
concentrations eluted
was needed
with
F IX activities
of 0.100
at 0.175
- 0.240
- 0.175
M. Secondly
molarity
(Fig. 21.
were eluted at phosphate
M, and finally
0.24 - 0.26 M. The elution hydroxyapatite
in hydroxyapatite
a double
peak
was
cne peak in the range
depended
on the lot of
gel used.
The F IX activities chromatography
in the above-mentioned
hydroxyapatite
were pooled
and designated
pools I, II and III.
These pools were subjected
to isoelectric
focusing
(Figs. 3a
and 351. Isoelectric
focusing.
Pool I resolved
in isoelectric
with the following
focusing
pIs and parts
into three subcomponents
calculated
in percentages:
p1 3.98, 30%; p1 4.16, 61% and p1 4.50, 9%. Pooi
II seemed
to be even more heterogeneous.
nents were separated,
three of them constituted
although
cent, and four small subcomponents pIs and parts
in percentages
Seven subcompo-
together
25 per cent. The
were p1 3.85, 24%; p1 4.12, 6.5%;pI
4 .42, 22%; p1 5.35, 5.5%; p1 5.80, 7% and p1 Pool III consisted 4.10,
75 per
of four subcomponents:
4.5%.
6.C4,
p1 3.85,
15.5%; p1
16%; p1 4.36, 48% and p1 4.83, 20.5%.
Individual
Factor
Donor No. 1. nations
6540 ml of plasma
during
about
F IX concentrate A1(OH13
IX
was prepared
gel adsorption.
to hydroxyapatite activity
four years.
was found
was collected
from several
do-
The plasma was stored at -25'C.
with DEAE-Sephadex
Part of the A1(OH13eluate
gel chromatography;
followed
was subjected
one peak containing
(Fig. 41. After focusing
by
this material,
F IX a
N-ELTIPLE FOR%
63~11
OF FACTOR
10 20 Fraction NP
30
IOj
IX
40
FIG. 1 DEAE-Sephadex chromatography of pooled Factor IX from about 600 persons. F IX concentrate containing about 1000 units of F IX was adjusted in 0.08 M citrate buffer, pH 7.0, and absorbed with 20 ml of DEAE-Sephadex equilibrated in the same buffer. The gel was then transferred to an empty column and the column was eluted with a linear citrate gradient formed by 250 ml each of 0.08 M and 0.15 M buffers, pk 7.0. Fractions (12.5 ml1 were Factor IX activity -x collected at a flow rate of 30 ml/h -xx adsorbance at 280 nm. Phosphate
200 50 100 150 Fraction NO FIG. 2 gel chromatography of pooled F IX (from about 0
Hydroxyapatite 3000 persons). gel and 5 ml of the F IX concentrate was adsorbed with Al(CH) eluant was diluted to 15 ml with water. T?i s sample contained 200 mg of protein and was adsorbed in the c01umr~ (2 x 10 cm1 and eluted with a linear phosphate gradient formed by 300 ml each of 0.1 M and 0.5 M phosphate buffers, pH 6.8. The column was run at +4'C with a flow rate of 4 ml/h and 190 fractions of about 2 ml were collected. The pools which were further run in isoelectric focusing are indicated by arrows on the abscissa. Pool I contains fractions 40-99 (122 ml), Pool II fractions loo-135 (61 ml1 and Pool III fractions 136-161: (57 ml!. x absorbance at 280 nm. Factor IX x -
Dooi
c
L.L2
;c1;;;< L.90
L.12 hi
10
5.90 6.04
20
Fraction
Fig.
3a.
Pool
I,
Isoelectric in
the
LO
NO
FIG.
hydroxyapatite
30
FIG.
3a focusing
gel chromatogram middle Pool II
of
Pools
described and at the
30
I-111 in
obtained Fig.
bottom
Relative F IX activities of different Fig. 3b. fractions calculated from activity distribution Each bar represent one isoelectric peak and its aoscissa is determined by the isoelectric point pT 2 respectively.
2.
Pool
in
the
ijn the
top,
III.
isoelectric in Fig. 3a. position on (the actual
the
l-01 .;,50.1
X-VLTIPLE
0
20
FOR%
OF FACTOR
40 60 Fraction NQ FIG.
107
IS
80
100
4
Hydroxyapatite gel chromatography of donor P.o 1. The column (2 x IO cm) was run at +4’C with a flo;, IO ml/h. 12 ml of F IX concentrate The sample wzs adsorption diluted witi24 ml of water. It contair,ed protein. The phosphate gradient was formed by 0.1 buffers, pH 6.0. 106 fractions of about 4 mi were to isoelectric Fractions 38-45 I32 ml1 were subjected
-
F IX
activity
-
x -
x absorbance
at
280
rate of after A!(Ck!13 106 mg of V and 0.5 M collected.
focusing.
nm.
Isoelectric focusing of fractions 38-45 obtained in hydroxyapatite chromatography of donor Ko I,
Fraction NQ
Si?ile
i
-
.-.__
:;c.
33nzr ‘“,j
I
ps3<
-
2.
-.
-
-
--
_ -25
r-=r: _
3:35--3
‘ys LEA,E-Sepka”sx
t
Conors N0 .
3
3
and
,?lasmas
4.
repea
lad and
isoelectric
pH
each
one
respectively. was
_
4.55
in
they
achieved;the
b
* , i ,?. , -.i;,l-i
doucle
were pis
rbtained.
?.:o.
(Fig.
or
mixed peak
mixed
together
4.13
and
of
4 NB~E
A part
of
cis a
4.42
5). conor
purifisd them
with
was
together. with
;~a+
This
focusing
single
were
25r:-a:egr33i;z
alas-apheresis
donor
alone a
3
:y-es”e;
:.:a5
an?
:
:+.,a~
adsorpticn.
as
5).
isceJ.e,ctris
cf
either
focused
::i,. ~~3s--~~+,~“~s-:s
peal:
from
Al(GHI3
k’hen
68
._*ri -_,
donations
focusing
alone,
c_s,i,_3
-.j-,
3
;A, single
at
CEAE-Sephadex
them
;;?I.
focuseri
ano
__,_a:ri
c-
3 3
a:p.SC?-h”i AC__
in hydrc;..yapa:iza material
8‘
run
When 4.15
(Fig.
run
and
separation
in
4.43
between
7).
4.55
T
I
i,
g24 f /
i
FIG. (Fig. i?jo.
(Fig. 4
61 2.
The
71
purified
Isoelectric sample
Isoelectric by
was
6
FIG. focusing
of
the
purified
by
DEAE-Sephadex
focusing
of
the
DEA E-Sephadex
and
Al(OH13.
F
IX
mixture
7
concentrate and
of
of
donor
Al(OH13.
samples
3
and
i-01 .;,?;0.1
present
The
3 pool
WLTIPLE
froc
matzgr.aphy. There elution 7-q / Y
patterns
3ctivity
apatite nen:s.
both
suggest The
focusing,
steps.
of pooled
cept that pooled ly improbable
from enzymatic F IX
was
ortained further
partly
showed
were
used during
chrobut the
splitting.
compo-
in hy-
the purification
treated
degrada-
samples
and
in the same way, ex-
focusing
Similarly,
during
heterogeneity
plasma,
F IX forms appe3ring
and isoelectric
least
focusing.
as the individual
plasma were
at
in hydrcxy-
homogeneity
plasma was cryo supernatant
observed
intc se-
that some proteolytic
However,
in
overlapping
and in isoelectris
that the multiple
chromatographies
activity
hydroxyapatite
showed
studied
possible
tion may have occurred.
IX
main cc?ponents,
but contained
inhibitors
It is therefore
the samples
ani
grocps
I-III)
chromatography
?; o enzyme
nziin
(Pool
The four individuals
drcxyapatite
Factcr
it is possi'sle *:y~t there are three
chromatography
iO9
IS
can Se sep3rated
DEAE-Seohadex
are three distinct
peaks.
in isoelectric
that
number of persons by
veral components
OF FACTOR
derrznstrate
results
a zrsat
FORYS
it is highboth in
could derive
no indication
the purification
merely
of activated
steps and isoelectric
focusing. It seems that hydroxyapatite for the separation
chromatography
of components
of pooled
is very suitable
F IX. Although
the
flow rate of the gel is very slow, F IX seems
to be very sta-
bile in these runs, and the expectable
were obtained
from the chromatographic
runs.
yields
(to be published).
shown by Hjerten
that for serum proteins
tion
isoelectric
between
the
points
order of their emergence with
phosphate
gradient
and the phenomenon electric
patterns
ween OEAE-Sephadex
(101.
derived
This seems
clearly
that there
is
no
proteins
correla-
and the
columns
eluted
to apply also to
from a comparison
from Pools
chromatography
3 study of this point
the
from hydroxyapatite
emerges
It can be assumed
of
there
It has been
F IX
of the iso-
I-III.
is a better and isoelectric
relationship focusing,
betand
is in progress.
In isoelectric focusing relatively few fractions were collected from each of the runs. Therefore, the accuracy of the
is_=e;~c-“‘.-
__
‘_C_
;I:.
_J
t--.-
“‘-e
-1
-?-
.‘&_
,.L2
c+r
;I7
+
lZ*2L)
-
suggest
units,
such
and
:-.:_
either
as
the
sialic
The
iscelectric
5.04,
but
the
tric
points
of
F IX
cf
individuals
and
within
with
the
F
relatively
true
hydrate
existence
CT
‘=“‘“;” __i
3
_i;h-=
L
2.35
__
-I-,
3z,rzx1-3_L_
of
pooled
cc,mponents
.1/+
- _ _
I -
_
==-
____srzs
__
,_L_ _,_airaz.
_ -.-. _=
csficiency
F
IX
of
focus
vary
T’: -- = rsceatics
;i;bliSk_ad
F
(11).
Correspondingly,
IX
_’ C.35
were these
between
between
other
studied
has
been
shown
carbohydrate of
human
due
5.1
d3ts
fcund
on
_
cV arc
tkS
fit
4.5.
pIs
of
4. ‘3
those
3.e;
f;oelec-
the between
values
IX.
The
to
phosphate
nature
of
amino
acids
Either
of
these
two
be
a
-
4 .ES,
obtained
(121
in
sialic
acid
the
and
cr
polypeptide
reasons
may
be
a is
most
glycoproteins
content
of
carbo-
and
galactose
!or
the
difference
In
chain
the
same
of
the
to
eizn
the
hetercgeneity
residues1 in
glycoprctein
variation
especially
of
to content
F
either
moieties,
content
i
FlZSTt?.
IX
generally
-
bjith
limits
high
probably
the
human
pcoled
Scvine
is
well
the
__-_-
acid.
main
CGrI-elatE!S
_
milltinle:)
ooints
This
the
icr
--“-=“-q
-‘31<
-__,
___er
;=” Lnl ‘ts
2.33
might
__
s,--_”
.-_
‘J”_,x.
-.m=qnr’
_
cause
in the
(13,14,?5,‘lE1. of
the
observed
heterogeneity. The plasma and
became
es
of
apparent
therefore
rather may
indications
the
laborious be
to
for
the
individuai The
More these
simple
F
of IX
samples reason
polymorphism
resolve.
needed
heterogeneity
during
ways.
genetic
the
and
methods
in
heterogeneity
would of
the
require
detect
pooled
precesses,
processed
the
this
in
purification
were for
F IX
ing
the
sane,
of family
F
studi-
var iants
the
studies.
ACKNOWLEDGEMENTS The
skil-Jl
Mrs
Maila
due
to
kind
Dr.
interest
technical !L’ummelin G.
is
Myllyla, and
assistance
of
gratefully
acknowledged
MD and
criticism.
Dr.
Miss
Ulla
Pirkko
Hamberg,
Hiitunen and
thanks
PhO
fcr
IX
and are their
are
?-cl.
w_*LTIPLE
-, ,x0.1
FOR_%
OF
FACTOR
i 11
IX
REFE.RExCES 1.
?;EAL, li.R., TAYLOE, D.T. Jr., CEDERBATJ?~ Detection of genetic variants of H.R.: immunosorbenx technique. Brit.J.Haemat.
2.
DENSOS, k.k-;.E.: Editorial: Diath.Haemorrh. Thrombos. B.
3.
LESTER, LexreLs normal -2h:16S,
, X.1. and ROBERTS, haemophilia B wir;h an 35:63, 1973. L
>loLecular s-ariants (Stuttg.) 3:317,
R.H., ELSTON, R.C. and GRMA>l, J.B.: Factors IX and X in of blood clotting Possible genetic pal)-morphisms. men: 19;3.
a
of
haemophilia 19;3.
i'ariations in population of Xmer.J.Hum.Genet.
G.
VEI,TCX?IP, J.J., YUYO, O., MOTULSKY, A.G. and FRaSER, G.R.: Blood coagulation Factors I, II, V, 17.1, VIII, IX, X, XI and XII in twins. Human Heredity &?:103, 1972.
5.
SUO>IELA, H. : Preparation of IVth Int.Cong.Thromb.Haemostasis,
6.
SUOFIELA, treatment
e I.
H. and NYLLYLA, of haemophilia
a
tlighly purified human Factor p. l%O, Vienna 1973.
G.: A 5'actor IX concentrate B. Duodecim 1971. -87:31,
for
IX.
the
SWART, A.C.W., BLOYS-VAN TRESLONG-DE GROOT, KLAASSEN, B.H.>l., C.H.F. and HEMKER, H.C.: The adsorption of blood coagulation Factors II, VII, IX and X from human plasma to aluminium hydroxide. Thromb.Diath.Haemorrh. 3:!190, 1972.
8.
TISELIUS, A., phy on calcium 1956.
9.
HARDISTY, R.N. and MACPHERSON, Janet C.: VIII (Antihaemophilic globulin) assay and and capillary plasma. Thromb.Diath.Iiaemorrh.
HJERTEN, S. and LEVIS, 0.: Protein chromatographosphate columns. Arch.Biochem.Biophys. %:132,
A one-stage its
Factor use on venous 1:213,1962.
3.: Calcium 10 * HJERTEN, phosphate chromatography of normal human serum and of electrophoretically isolated serum proteins. Biochim.Biophys.Acta 31~216, 1959. 11.
SUDHISH, C. and PECHET, L.: The separation of clotting factors. Purification of human Factor IX by isoelectric focusing. II. Biochim.Biophys.Acta 328:456, 1973.
12.
FUJIKAWA, K., DAVIE, E.W.: IX (Christmas
13.
MOSHER, gen and
14,
SIEFRING, G.E. Jr. and CASTELLISO, F.J.: The role of sialic acid in the determination of distinct properties of the isozymes 1974. of rabbi3 plasminogen, J.Bil)l.Chem. 2&9:7742,
THONPSON, Isolation Factor).
D.F. and fibrin.
A.R., LEGAZ, ?I.E., NEYER, R.G. and characterization of bovine Biochemistry 1973. -12:4938,
BLOUT, E.R.: J.Biol.Chem.
Heterogeneity of 248:6896, 1973.
bovine
and Factor
fibrino-
15.
16.
NELSESTL'ES, G.L. aglycoprothrombin,
and
SIJTTIE, J.V.: Properties Biochem.Biophys.Res.Commun.
or^ asialo 3:198,
and 1971.
RYDEX, L.: Human ceruloplasmin as a polymorphic glycoprotein. Analysis by chromatography on hydrosyapatitc. Int.J.Protein 1971. Research 111:131,
TKROXBOSIS RESEARCH Printed in r;he TVnited Szates
YULTIPLE
FORMS
C H R 3 :::,A,TCSRAPtJY
OF
Red
Helsinki,
(Received
ABSTRACT
IX
I?\.
F C C iJ S I r,:C
GFID ISOELECTRIC
Hannu Finnish
HUKA?,; FACTOR
7, pp. 101-112, Pergamon Press,
Suomela
Cross
Blood
Transfusion
Service
Finland
in revised form 11.5.1975. 3.3.1975; Accepted by Editor B. Blombkk)
Factor 600-3000 genecus
IX
concentrate made from a plasma pool of persons was demonstrated to be heteroin DEAE-Sephadex and hydroxyapatite chromatography . Factor IX activity was separated into three main groups and each of these groups was shown to be heterogeneous in isoelectric focusing. Factor IX concentrate made from individual plasma samples was found to be homogeneous when the same techniques were used. Variation between individuals was demonstrated.
INTRODUCTION Differences strated.
in
The
coagulation found variants
has lation
been study
polymorphism Veltkamp
of
al.
normal
least
8
the
patients
heterogeneity
males
that in
not
et
the
confirm 101
al. there levels this
B have
been
both
immunological
four
variants
literature
demonand have
been
concerning
(21. of
Lester
variation could
at
on
reviewed
Hemophilia of
Hemophilia based
and
suggested.
for et
is
recently
in
occurrence
also
of
analysis,
Denson found
The
types
classification time
(I).
the
normal
human
concluded may of result
Factor
from
well
be
Factor in
a pcpugenetic
IX their
(31. twin
IX
1975 Inc.
stories
[.I).
In
‘1?2
Factor
sours=
7s”;)
zf
._loFy-_=ri
IX (F IX) froT pcoled
fepeataaly
fount
CEAE-Sephadex phate)
_
c:
in sevaral
nur-an plasya, sompsnents.
chromatography,
chromatography
per presents
__
F 1. aCt
This ::,a-e
aopsren:
i,n
!salci~~ phosf:-using :51. This pa-
and in isoelectric
from pooled
MATERIAL
-‘,,
in hydroxyaparite
the chromatographic
F IX originating
?.,>_i_-._ _ _ i--z
>.;r:-f;a;
and issele~tric
plasma
and four
properties
of
tndividuala.
AIND METHODS
F IX concentrates a)
The pooled
plasma:
The plasma
pool of about 3000 persons was separated
within
was cryo-supernatant
again,
separated.
and F IX concentrate
from a
plasma,
six hours of the bleeding,
thawed and the cryoprecipitate frozen
for F IX concentrate
frozen
which at -7G°C,
The supernatant
prepared
as described
was earlier
(61. bl
Individual
either
from repeated
The plasmas
The plasmas
plasma:
donations
were stored
ted. F IX concentrate
frozen.
taining
0.2 M phosphate
about 600 ml of plasma transferred
Cryoprecipitate
buffers
gently
onto a Biichner funnel
contained
as fellows:
in G.14 M phosphate-
were diluted
both 0.20 and 0.25 M phosphate-NaCl
concentrate
was not separa-
from a buffer con-
and 0.8 M NaCl, pH 6.91 was added to and mixed
II, VII, Xl were eluted
received
plasmapheresis.
&as prepared
A-50 R equilibrated
(all phosphate-NaCl
were
or after a double
from individuals
25 ml of DEAE-Sephadex NaCl
of the donors
with
for l/2 h; the gel was
and washed buffers.
with 600 mi of F IX (and Factors
50 ml of 1.0 M phosphate-?iaCi. The
5-6 mg of protein/ml
and 5-8 units of
F IX/ml. Purification
with A1(OH13
gel
was carried
Swart et al. (71 at O'C. F IX concentrate volymes
of distilled
each mg of protein by adding
water.
and mixed
a gel volume
0.3 ml Al(OH13
out as described was diluted
of 0.3 M phosphate
with three
22.1 was pipetted
for 5 min. Elution
by
for
was performed
buffer,
pH 6.0, mixing
FKLTIPLE
t'ol.;,~o.l
iiata
r?~rs
olustSS
\.*;SrS
FCClZd
3;ai;_ __ $-z
ChT-!Ifil~tCl--
z;
3:
_i
-7
_-.Y
FOR%
OF FACTOR
_,3 r
p^31
2nd
*
CT
frCzSn
prCCpS.S?_:
cr isoelectric
E’aa”y
_jl
CL
J 2 per "=.._ ct
tr.3::
IX
a”+_ivity, __
+‘_r”, +“er
T;i-_ : y r: r f x _” -
“4
fC=!,si"g.
CE.GE-Sephadex ChronatograpSv. 2C ml CEAE-Se~?a:ex gel ec,uili:ra-y 2.2 t-_n c e n L* r a te ,_,~F _, __ in 3.96 ?, zitrafe, pP 7.0, was atided c, [ 2 t .>'At T:H
13CC
ucits
,Gft;r
7.5,.
of
gent?e
F
I;()
dissclvsd
Fixirg
For
in
l/2
h
:zc
5:s
7-1 gel
:.'I3 :yss
F
citr3-j
p;lcko:2
ink=
3
and e:uted ~.+itha linear citrate g:-aziant, ,;b 7.3, ,usic' cz l_i?n = 6 113OC iiltrograd R gradient mixer. ihe protein ‘dZS ret----an L ,< _v_i ed by Cvicorcf II (LKE). The citrate measured forc
cf each fracticr
F':eter(Wiss.-Tec~n.Werkst~tter,
ty a Conductivity
Kailheim)
content
cali'bratec with a series
2,?42
buffers
made of the
wss
used
5~
the gradient. The gel was prepared
Hytiroxyapatite chromatography. to Tiselius buffer,
et al. (61 and equilibrated
pH 6.6. The mclarity
about 2 mg per milliiitre gradient
lJ/ith5 mM phosphate
of the charge
ed t 0 about 0.1 M with distilled
according
water.
soltition was adjust-
The protein
of gel. Chromatography
elut ion was done with phosphate
load was
with a linear
suffer,
pP 6.8, and
LKE equipment. Isoelectric
focusing
was carried
(110 ml) using ampholytes The sample
containing
hours against against followed
7-17 mg of protein
solution.
with regard
buffer,
was dialyzed
pH 7.0, and for two hours
The manufacturer's
for column
for twc
and sample
instructions
leading,
pump at the rate of 33 ml/h.
were allowed
to reach room temperature
with Beckman
Zeromatic
Factor
IX activity
cephalin-kaolin
and/or
Findip
measurements.
system
1-3 ml fractions
555. pH meters.
One-stage
the study
with an
and the pH was measured
using haemophilia
strate was used throughout
WPZ
and focused
for 72-69 hours at 560 V at 3'C. The column was emptied LK6 peristaltic
8100-l
pH 3-5, 4-6 and 5-7, 2 ml of each.
5 mM phosphate
ampholyte
out with an LK6 column
assay with the
6 plasma
as the sub-
(9).
RESULTS Pooled Figures
plasma
Factor
IX.
1 and 2 demonstrate
the hetercgeneity
of the F IX con-
Ji;i”cF,3---
c
2)
-5’
There there three
nenrs. t
(Fi-
_,krag,hy
he+arogene;us. U..,&
:s 3
the concentration absarbed
at
=
I;(
a,~!--~i~it
-3:n
’
ccnponents,
3
which
components
c,rpg-
3~~earzd
(Fig. I]
chrzrcat~graphy
were eluted
and the main component
0.100 and G.115 M, and the third component
betwaen
,*
sever‘3
was critirq1. ^_i.i, F IX activity
K and the first
0.08 and '3.100 Fi. The second
5::
eaok
gE,&E-Sephadex
In
cf citrate
L1.08
_., Y appears:
was
was between eluted
between
0.115
and 0.130 P-1. A steeper Several
gradient
fractions
concentrations eluted
was needed
with
F IX activities
of 0.100
at 0.175
- 0.240
- 0.175
M. Secondly
molarity
(Fig. 21.
were eluted at phosphate
M, and finally
0.24 - 0.26 M. The elution hydroxyapatite
in hydroxyapatite
a double
peak
was
cne peak in the range
depended
on the lot of
gel used.
The F IX activities chromatography
in the above-mentioned
hydroxyapatite
were pooled
and designated
pools I, II and III.
These pools were subjected
to isoelectric
focusing
(Figs. 3a
and 351. Isoelectric
focusing.
Pool I resolved
in isoelectric
with the following
focusing
pIs and parts
into three subcomponents
calculated
in percentages:
p1 3.98, 30%; p1 4.16, 61% and p1 4.50, 9%. Pooi
II seemed
to be even more heterogeneous.
nents were separated,
three of them constituted
although
cent, and four small subcomponents pIs and parts
in percentages
Seven subcompo-
together
25 per cent. The
were p1 3.85, 24%; p1 4.12, 6.5%;pI
4 .42, 22%; p1 5.35, 5.5%; p1 5.80, 7% and p1 Pool III consisted 4.10,
75 per
of four subcomponents:
4.5%.
6.C4,
p1 3.85,
15.5%; p1
16%; p1 4.36, 48% and p1 4.83, 20.5%.
Individual
Factor
Donor No. 1. nations
6540 ml of plasma
during
about
F IX concentrate A1(OH13
IX
was prepared
gel adsorption.
to hydroxyapatite activity
four years.
was found
was collected
from several
do-
The plasma was stored at -25'C.
with DEAE-Sephadex
Part of the A1(OH13eluate
gel chromatography;
followed
was subjected
one peak containing
(Fig. 41. After focusing
by
this material,
F IX a
N-ELTIPLE FOR%
63~11
OF FACTOR
10 20 Fraction NP
30
IOj
IX
40
FIG. 1 DEAE-Sephadex chromatography of pooled Factor IX from about 600 persons. F IX concentrate containing about 1000 units of F IX was adjusted in 0.08 M citrate buffer, pH 7.0, and absorbed with 20 ml of DEAE-Sephadex equilibrated in the same buffer. The gel was then transferred to an empty column and the column was eluted with a linear citrate gradient formed by 250 ml each of 0.08 M and 0.15 M buffers, pk 7.0. Fractions (12.5 ml1 were Factor IX activity -x collected at a flow rate of 30 ml/h -xx adsorbance at 280 nm. Phosphate
200 50 100 150 Fraction NO FIG. 2 gel chromatography of pooled F IX (from about 0
Hydroxyapatite 3000 persons). gel and 5 ml of the F IX concentrate was adsorbed with Al(CH) eluant was diluted to 15 ml with water. T?i s sample contained 200 mg of protein and was adsorbed in the c01umr~ (2 x 10 cm1 and eluted with a linear phosphate gradient formed by 300 ml each of 0.1 M and 0.5 M phosphate buffers, pH 6.8. The column was run at +4'C with a flow rate of 4 ml/h and 190 fractions of about 2 ml were collected. The pools which were further run in isoelectric focusing are indicated by arrows on the abscissa. Pool I contains fractions 40-99 (122 ml), Pool II fractions loo-135 (61 ml1 and Pool III fractions 136-161: (57 ml!. x absorbance at 280 nm. Factor IX x -
Dooi
c
L.L2
;c1;;;< L.90
L.12 hi
10
5.90 6.04
20
Fraction
Fig.
3a.
Pool
I,
Isoelectric in
the
LO
NO
FIG.
hydroxyapatite
30
FIG.
3a focusing
gel chromatogram middle Pool II
of
Pools
described and at the
30
I-111 in
obtained Fig.
bottom
Relative F IX activities of different Fig. 3b. fractions calculated from activity distribution Each bar represent one isoelectric peak and its aoscissa is determined by the isoelectric point pT 2 respectively.
2.
Pool
in
the
ijn the
top,
III.
isoelectric in Fig. 3a. position on (the actual
the
l-01 .;,50.1
X-VLTIPLE
0
20
FOR%
OF FACTOR
40 60 Fraction NQ FIG.
107
IS
80
100
4
Hydroxyapatite gel chromatography of donor P.o 1. The column (2 x IO cm) was run at +4’C with a flo;, IO ml/h. 12 ml of F IX concentrate The sample wzs adsorption diluted witi24 ml of water. It contair,ed protein. The phosphate gradient was formed by 0.1 buffers, pH 6.0. 106 fractions of about 4 mi were to isoelectric Fractions 38-45 I32 ml1 were subjected
-
F IX
activity
-
x -
x absorbance
at
280
rate of after A!(Ck!13 106 mg of V and 0.5 M collected.
focusing.
nm.
Isoelectric focusing of fractions 38-45 obtained in hydroxyapatite chromatography of donor Ko I,
Fraction NQ
Si?ile
i
-
.-.__
:;c.
33nzr ‘“,j
I
ps3<
-
2.
-.
-
-
--
_ -25
r-=r: _
3:35--3
‘ys LEA,E-Sepka”sx
t
Conors N0 .
3
3
and
,?lasmas
4.
repea
lad and
isoelectric
pH
each
one
respectively. was
_
4.55
in
they
achieved;the
b
* , i ,?. , -.i;,l-i
doucle
were pis
rbtained.
?.:o.
(Fig.
or
mixed peak
mixed
together
4.13
and
of
4 NB~E
A part
of
cis a
4.42
5). conor
purifisd them
with
was
together. with
;~a+
This
focusing
single
were
25r:-a:egr33i;z
alas-apheresis
donor
alone a
3
:y-es”e;
:.:a5
an?
:
:+.,a~
adsorpticn.
as
5).
isceJ.e,ctris
cf
either
focused
::i,. ~~3s--~~+,~“~s-:s
peal:
from
Al(GHI3
k’hen
68
._*ri -_,
donations
focusing
alone,
c_s,i,_3
-.j-,
3
;A, single
at
CEAE-Sephadex
them
;;?I.
focuseri
ano
__,_a:ri
c-
3 3
a:p.SC?-h”i AC__
in hydrc;..yapa:iza material
8‘
run
When 4.15
(Fig.
run
and
separation
in
4.43
between
7).
4.55
T
I
i,
g24 f /
i
FIG. (Fig. i?jo.
(Fig. 4
61 2.
The
71
purified
Isoelectric sample
Isoelectric by
was
6
FIG. focusing
of
the
purified
by
DEAE-Sephadex
focusing
of
the
DEA E-Sephadex
and
Al(OH13.
F
IX
mixture
7
concentrate and
of
of
donor
Al(OH13.
samples
3
and
i-01 .;,?;0.1
present
The
3 pool
WLTIPLE
froc
matzgr.aphy. There elution 7-q / Y
patterns
3ctivity
apatite nen:s.
both
suggest The
focusing,
steps.
of pooled
cept that pooled ly improbable
from enzymatic F IX
was
ortained further
partly
showed
were
used during
chrobut the
splitting.
compo-
in hy-
the purification
treated
degrada-
samples
and
in the same way, ex-
focusing
Similarly,
during
heterogeneity
plasma,
F IX forms appe3ring
and isoelectric
least
focusing.
as the individual
plasma were
at
in hydrcxy-
homogeneity
plasma was cryo supernatant
observed
intc se-
that some proteolytic
However,
in
overlapping
and in isoelectris
that the multiple
chromatographies
activity
hydroxyapatite
showed
studied
possible
tion may have occurred.
IX
main cc?ponents,
but contained
inhibitors
It is therefore
the samples
ani
grocps
I-III)
chromatography
?; o enzyme
nziin
(Pool
The four individuals
drcxyapatite
Factcr
it is possi'sle *:y~t there are three
chromatography
iO9
IS
can Se sep3rated
DEAE-Seohadex
are three distinct
peaks.
in isoelectric
that
number of persons by
veral components
OF FACTOR
derrznstrate
results
a zrsat
FORYS
it is highboth in
could derive
no indication
the purification
merely
of activated
steps and isoelectric
focusing. It seems that hydroxyapatite for the separation
chromatography
of components
of pooled
is very suitable
F IX. Although
the
flow rate of the gel is very slow, F IX seems
to be very sta-
bile in these runs, and the expectable
were obtained
from the chromatographic
runs.
yields
(to be published).
shown by Hjerten
that for serum proteins
tion
isoelectric
between
the
points
order of their emergence with
phosphate
gradient
and the phenomenon electric
patterns
ween OEAE-Sephadex
(101.
derived
This seems
clearly
that there
is
no
proteins
correla-
and the
columns
eluted
to apply also to
from a comparison
from Pools
chromatography
3 study of this point
the
from hydroxyapatite
emerges
It can be assumed
of
there
It has been
F IX
of the iso-
I-III.
is a better and isoelectric
relationship focusing,
betand
is in progress.
In isoelectric focusing relatively few fractions were collected from each of the runs. Therefore, the accuracy of the
is_=e;~c-“‘.-
__
‘_C_
;I:.
_J
t--.-
“‘-e
-1
-?-
.‘&_
,.L2
c+r
;I7
+
lZ*2L)
-
suggest
units,
such
and
:-.:_
either
as
the
sialic
The
iscelectric
5.04,
but
the
tric
points
of
F IX
cf
individuals
and
within
with
the
F
relatively
true
hydrate
existence
CT
‘=“‘“;” __i
3
_i;h-=
L
2.35
__
-I-,
3z,rzx1-3_L_
of
pooled
cc,mponents
.1/+
- _ _
I -
_
==-
____srzs
__
,_L_ _,_airaz.
_ -.-. _=
csficiency
F
IX
of
focus
vary
T’: -- = rsceatics
;i;bliSk_ad
F
(11).
Correspondingly,
IX
_’ C.35
were these
between
between
other
studied
has
been
shown
carbohydrate of
human
due
5.1
d3ts
fcund
on
_
cV arc
tkS
fit
4.5.
pIs
of
4. ‘3
those
3.e;
f;oelec-
the between
values
IX.
The
to
phosphate
nature
of
amino
acids
Either
of
these
two
be
a
-
4 .ES,
obtained
(121
in
sialic
acid
the
and
cr
polypeptide
reasons
may
be
a is
most
glycoproteins
content
of
carbo-
and
galactose
!or
the
difference
In
chain
the
same
of
the
to
eizn
the
hetercgeneity
residues1 in
glycoprctein
variation
especially
of
to content
F
either
moieties,
content
i
FlZSTt?.
IX
generally
-
bjith
limits
high
probably
the
human
pcoled
Scvine
is
well
the
__-_-
acid.
main
CGrI-elatE!S
_
milltinle:)
ooints
This
the
icr
--“-=“-q
-‘31<
-__,
___er
;=” Lnl ‘ts
2.33
might
__
s,--_”
.-_
‘J”_,x.
-.m=qnr’
_
cause
in the
(13,14,?5,‘lE1. of
the
observed
heterogeneity. The plasma and
became
es
of
apparent
therefore
rather may
indications
the
laborious be
to
for
the
individuai The
More these
simple
F
of IX
samples reason
polymorphism
resolve.
needed
heterogeneity
during
ways.
genetic
the
and
methods
in
heterogeneity
would of
the
require
detect
pooled
precesses,
processed
the
this
in
purification
were for
F IX
ing
the
sane,
of family
F
studi-
var iants
the
studies.
ACKNOWLEDGEMENTS The
skil-Jl
Mrs
Maila
due
to
kind
Dr.
interest
technical !L’ummelin G.
is
Myllyla, and
assistance
of
gratefully
acknowledged
MD and
criticism.
Dr.
Miss
Ulla
Pirkko
Hamberg,
Hiitunen and
thanks
PhO
fcr
IX
and are their
are
?-cl.
w_*LTIPLE
-, ,x0.1
FOR_%
OF
FACTOR
i 11
IX
REFE.RExCES 1.
?;EAL, li.R., TAYLOE, D.T. Jr., CEDERBATJ?~ Detection of genetic variants of H.R.: immunosorbenx technique. Brit.J.Haemat.
2.
DENSOS, k.k-;.E.: Editorial: Diath.Haemorrh. Thrombos. B.
3.
LESTER, LexreLs normal -2h:16S,
, X.1. and ROBERTS, haemophilia B wir;h an 35:63, 1973. L
>loLecular s-ariants (Stuttg.) 3:317,
R.H., ELSTON, R.C. and GRMA>l, J.B.: Factors IX and X in of blood clotting Possible genetic pal)-morphisms. men: 19;3.
a
of
haemophilia 19;3.
i'ariations in population of Xmer.J.Hum.Genet.
G.
VEI,TCX?IP, J.J., YUYO, O., MOTULSKY, A.G. and FRaSER, G.R.: Blood coagulation Factors I, II, V, 17.1, VIII, IX, X, XI and XII in twins. Human Heredity &?:103, 1972.
5.
SUO>IELA, H. : Preparation of IVth Int.Cong.Thromb.Haemostasis,
6.
SUOFIELA, treatment
e I.
H. and NYLLYLA, of haemophilia
a
tlighly purified human Factor p. l%O, Vienna 1973.
G.: A 5'actor IX concentrate B. Duodecim 1971. -87:31,
for
IX.
the
SWART, A.C.W., BLOYS-VAN TRESLONG-DE GROOT, KLAASSEN, B.H.>l., C.H.F. and HEMKER, H.C.: The adsorption of blood coagulation Factors II, VII, IX and X from human plasma to aluminium hydroxide. Thromb.Diath.Haemorrh. 3:!190, 1972.
8.
TISELIUS, A., phy on calcium 1956.
9.
HARDISTY, R.N. and MACPHERSON, Janet C.: VIII (Antihaemophilic globulin) assay and and capillary plasma. Thromb.Diath.Iiaemorrh.
HJERTEN, S. and LEVIS, 0.: Protein chromatographosphate columns. Arch.Biochem.Biophys. %:132,
A one-stage its
Factor use on venous 1:213,1962.
3.: Calcium 10 * HJERTEN, phosphate chromatography of normal human serum and of electrophoretically isolated serum proteins. Biochim.Biophys.Acta 31~216, 1959. 11.
SUDHISH, C. and PECHET, L.: The separation of clotting factors. Purification of human Factor IX by isoelectric focusing. II. Biochim.Biophys.Acta 328:456, 1973.
12.
FUJIKAWA, K., DAVIE, E.W.: IX (Christmas
13.
MOSHER, gen and
14,
SIEFRING, G.E. Jr. and CASTELLISO, F.J.: The role of sialic acid in the determination of distinct properties of the isozymes 1974. of rabbi3 plasminogen, J.Bil)l.Chem. 2&9:7742,
THONPSON, Isolation Factor).
D.F. and fibrin.
A.R., LEGAZ, ?I.E., NEYER, R.G. and characterization of bovine Biochemistry 1973. -12:4938,
BLOUT, E.R.: J.Biol.Chem.
Heterogeneity of 248:6896, 1973.
bovine
and Factor
fibrino-
15.
16.
NELSESTL'ES, G.L. aglycoprothrombin,
and
SIJTTIE, J.V.: Properties Biochem.Biophys.Res.Commun.
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