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Purification and Properties of Guine Pig Antithrombin III a
a
L. Heck , R. Rosenberg & H. Remold
a
a
Departments of Medicine , Harvard Medical School and Robert B. Brigham Hospital and Sidney Farber Cancer Institute , Boston, Massachusetts, 02115 Published online: 05 Dec 2006.
To cite this article: L. Heck , R. Rosenberg & H. Remold (1979) Purification and Properties of Guine Pig Antithrombin III, Preparative Biochemistry, 9:4, 359-377, DOI: 10.1080/00327487908061700 To link to this article: http://dx.doi.org/10.1080/00327487908061700
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PREPARATIVE BIOCHEMISTRY, 9(4) , 359-377 (1979)
PURIFICATION AND PROPERTIES OF GUINEA PIG ANTITHROMBIN I11
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L. Heck, R. Rosenberg, and H. Remold Departments of Medicine, Harvard Medical School and Robert B. Brigham Hospital and Sidney Farber Cancer Institute Boston, Massachusetts 02115 ABSTRACT Guinea pig antithrombin I11 has been purified from plasma by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose chromatography, isoelectric focussing, and Sephadex G-100 gel filtration chromatography.
The final product was homogeneous as
judged by sodium dodecyl sulfate disc gel electrophoresis. Purification was 202-fold with a yield of 41%. Antiproteinase activity of 4
antithrombin I11 was determined by progressive inactivation of thrombin coagulant and amidolytic activity.
Heparin cofactor activ-
ity was demonstrated by immediate inactivation of thrombin by antithrombin I11 in the presence of minute quantities of heparin. It also could be demonstrated that thrombin inactivation by antithrombin 111 occurs by formation of a bimolecular complex whose rate of formation is markedly enhanced by minute quantities o f heparin. Introduction Methods for the purification of AT 111’ from human (1-3, 6 ) , canine (4), rabbit (5) plasma have been reported by several
359 Copyrlght 0 I979 hy Marcel Dekker. Inc All Rights Keserved. Neither this work nor a n y part may he reproduced o r transmitted in any form or hy any means, electronic or mechanical, including pholocopying. microfilming. and recording. or by any information storage and retrieval s y s l e m . withoul permission in writing from the publisher.
HECK, ROSENBERG, AND REMOLD
360
investigators. Whereas a single purification step utilizing heparin-Sepharose affinity chromatography has been reported to yield a homogeneous preparation from human (6) and canine (4) sources, this has not been our experience in purifying this proteinase inhibitor from guinea pig plasma.
Four steps were necessary to purify guinea
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pig AT 111 to homogeneity. Materials and Methods Chemicals: The following chemicals were reagent grade and used without further purification: bovine fibrinogen, Fraction 1, Lot 84C-0479 from Sigma Chemical Corp., St. Louis, MO; Bz-Phe-Val-ArgpNa (S-2160) from Ortho Diagnostics, Raritan, NJ: p-nitrophenyl-pguanidinobenzoate (NPGB) and four times crystallized bovine serum albumin from ICN Pharmaceutical, Inc., Cleveland, OH; topical bovine thrombin from Parke-Davis, Detroit, MI; polybrene from Aldrich Chemical Crop., Milwaukee, WI; crude porcine intestinal heparin (Stage 14) from Inolex Crop., Park Forest, IL: DE-52 cellulose from Whatman, Inc., Clifton, NJ; Sephadex G-100 from Pharmacia, Piscataway, NJ; cellulose-phosphate (Cellex-P) from Bio-Rad, Richmond, CA; and Ampholines pH 4.0 to 6.0 from LKB, Hicksville, NY. Guinea pig plasma: Plasma was obtained by collecting cardiac blood in the presence of 3.8% trisodium citrate (1 vol citrate: 9 vol blood) with subsequent centrifugation at 2500 RPM for
30 min at 4OC.
The plasma was then recentrifuged at 10,000 RPM
for 15 min at 4°C in a Model E Sorvall. stored at -2OOC.
The frozen pool was
GUINEA PIG ANTITHROMBIN 111
36 1
Preparation of heparin-Sepharose: Crude porcine intestinal heparin, partially purified according to the method of Lindahl ( 7 ) , was insolubilized to Sepharose 4 B as described ( 8 ) . Electrophoresis: Molecular weight markers and samples were prepared as described (9).
Polypeptides were separated by a modifica-
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tion of the method of Laemmli (10) using slab gels ( 3 X 120 X 170 mm) or tube gels ( 6 mm X 150 mm). 1% Protein determination: The EZB0 of guinea pig AT I11 and bovine
thrombin was determined to be 6.9 and 19.6, respectively. Protein determination was performed by the method of Lowry using 4X crystallized bovine albumin as the reference (11). Immunoelectrophoresis was performed in agar gels as described (12). Quantitative immunodiffusion was performed by the technique of Mancini using purified AT-111 in serial dilutions as the standard antigen ( 1 3 ) . Immunization: New Zealand white rabbits were immunized with a total of 200 pg of purified AT I11 mixed with an equal volume of complete Freund's adjuvant (CFA) with multiple intradermal injections every three weeks for a total of four injections. The animals were bled after the second injection and at two week intervals. Conductivity measurements:
For conductivity determinations, a
conductivity meter, type CDC 314 (Radiometer, Copenhagen, Denmark) was used. Purification of bovine thrombin: Bovine thrombin was purified from topical thrombin by sequential DE-52 cellulose and cellulose-
362
HECK, ROSENBERG, ANTI REMOLD
phosphate chromatography (14) and stored in 0.5 M NaC1, 0.5 M sodium phosphate buffer, pH 7.0 at a concentration of 1.0 mgm/rnl at -7OOC. All experiments were done with a bovine thrombin preparation that had a specific clotting activity of 1.8 NIH units/ug and a specific amidolytic activity of 0.36 OD ( 4 0 5 nm) change/min/
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Ug.
This preparation was more than 87% active by active site ti-
tration and electrophoresed as a mixture of alpha and beta thrombin (1O:l). Measurement of thrombin coagulant activity:
Fibrinogen clotting
activity was determined (15) using Lot 3B thrombin standard (21.3 NIH units/mg) kindly provided by Dr. David Aronson, NIH. Measurement of thrombin amidolytic activity: Amidolysis of Bz-Phe-Val-Arg-pNn was measured by a modification of the method of Gdegard (16).
Three hundred p1 of substrate (0.5 mg/ml),
100 p 1 of polybrene (1 mg/ml), and 100 1.11 of Tris-HC1, O.lM, pH 8.1, were sequentially added to a 1.0 ml quartz cuvette. Thrombin in 1-10 ~1aliquots was added, mixed rapidly, and the absorbance was monitored over a 5 minute period at 405 nm (25 C). Active site titration of thrombin:
Active site titration with
NPGB was performed by the method of Chase and Shaw (17). Measurement of residual thrombin coagulant activity: Thrombin 5.4 .pg (10 NIH units) was added to 0-25 pg AT 111 in a prealbumin-
ized plastic tube and the volume adjusted to 100 p1 with O.lM, Tris-HC1, pH 8.1, and incubated at 37'C.
At 60 second intervals,
10 p1 fractions were withdrawn and added to the fibrinogen solu-
tion and the standard chrombin assay performed (15).
363
GUINEA PIG ANTITHROMBIN I11 Measurement of residual thrombin amidolytic activity:
Thrombin
5 ug was added to 9.4 ug of AT 111 in prealbuminized plastic tubes apd the volume adjusted to 100 ~1 with assay buffer incubated at
37°C. At 15 sec, 30 sec and 60 sec intervals, 10 p 1 fractions were withdrawn and added to the substrate solution in the standard
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amidolytic assay. Measurement of heparin-cofactor activity:
Sodium heparin, 0.1 IT,
was added to 9.4 ug of AT I11 and incubated at 25°C for 5 min and then placed in an ice bucket.
The amidolytic assay was then per-
formed as described above so that the final concentration of heparin/ assay did not exceed 0.01 U and did not interfere with the amidolysis of the tripeptide substrate by thrombin. Heparin-Sepharose affinity chromatography: All chromatography steps were performed at 4°C.
500 ml aliquots of citrated guinea
pig plasma were mixed with 75 ml of settled heparin-Sepharose and placed on a roller platform for one hour at 4°C.
The plasma-
slurry mixture was then transferred to a large scintered glass funnel and washed with four liters of 0.05 M Tris-HC1 saline, pH 7.6. After performing this step three additional times, the slurries were combined and transferred to a Lucite column ( 4 X 60 cm) and washed with 0.05 M Tris-HC1 in 0.4 M NaC1, pH 7.6, until
the OD280 was less than 0.03 unitslml.
The antithrombin contain-
ing fraction was desorbed with 0.05M Tris-HC1 in 1.OM NaC1, pH 7.6 and concentrated ten-fold by vacuum dialysis to a final protein concentration of 18.7 OD280 unitslml.
The resin could be re-
utilized at least 8-10 times for the purification of AT I11 if
HECK, ROSENBERG, AND REMOLD
364
i t was immediately r e e q u i l i b r a t e d w i t h 0.15 M N a C l i n 0.05 M T r i s -
HC1, pH 7 . 6 , and s t o r e d i n 0.1% sodium a z i d e a t 4OC.
DE-52 c e l l u l o s e i o n exchange chromatography:
The f r a c t i o n from t h e
p r e v i o u s s t e p was d i a l y z e d a g a i n s t 4 l i t e r s of 0.005 M sodium phosp h a t e b u f f e r , pH 8.1, w i t h f r e q u e n t changes u n t i l t h e c o n d u c t i v i t y
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of t h e p r o t e i n s o l u t i o n w a s e q u a l t o t h e b u f f e r .
DE-52 c e l l u l o s e
was e q u i l i b r a t e d w i t h t h i s b u f f e r and 250 m l of t h e s e t t l e d r e s i n w a s packed i n a L u c i t e column (30 X 4 cm).
The p r o t e i n s o l u t i o n
w a s allowed t o p e r c o l a t e s l o w l y t h r o u g h t h e r e s i n , which was t h e n
washed w i t h 500 m l of s t a r t i n g b u f f e r a t a flow r a t e of 75 m l / h r m a i n t a i n e d by a p e r i s t a l t i c pump.
A l i n e a r sodium c h l o r i d e g r a d i -
e n t was t h e n employed f o r f r a c t i o n a t i o n .
The r e s e r v o i r c o n t a i n e d
1000 m l of 0 . 3 M N a C l i n 0.005 M sodium phosphate b u f f e r , pH 8 . 1 , and t h e mixing chamber c o n t a i n e d 1000 m l of 0.005 M sodium phosp h a t e b u f f e r , pH 8.1.
F r a c t i o n s of 1 5 m l were c o l l e c t e d i n
p l a s t i c tubes. I s o e l e c t r i c focussing i n a sucrose density gradient:
The pool
of a c t i v e f r a c t i o n s from t h e p r e v i o u s s t e p w a s c o n c e n t r a t e d t o 5 m l by vacuum d i a l y s i s and s u b j e c t e d t o i s o e l e c t r i c f o c u s s i n g on a LKB 8102 column c o n t a i n i n g 1.5% pH 4 . 0 - 6 . 0 Ampholines i n a l i n e a r 0-47% s u c r o s e d e n s i t y g r a d i e n t .
The column w a s pre-
e l e c t r o f o c u s s e d f o r 2 1 h r a t 6 W b e f o r e a p p l i c a t i o n of t h e sample. AT I11 w a s d i a l y z e d a g a i n s t 4 l i t e r s of d o u b l e d i s t i l l e d water
a t 4 O C f o r 2 hrs.
The d e n s i t y of t h e a n t i t h r o m b i n sample was
a d j u s t e d t o t h e d e n s i t y of t h e sample e x t r a c t e d from t h e c e n t e r of t h e s u c r o s e d e n s i t y g r a d i e n t w i t h c o n c e n t r a t e d s u c r o s e
365
GUINEA PIG ANTITHROMBIN 111 solution. This resulted in a sample volume of 8-10 ml which was subjected to isoelectric focussing for 22 hr at 6 W.
The gradient
was then drained with a pump, and 4 ml aliquots were collected into tubes and the pH measured.
0.5 ml of 0.1 M Tris-HC1, pH 8.0,
was added to the fractions and functional activity studies were
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performed. Sephadex G-100 fel filtration chromatography:
The pool of active
protein fractions was exhaustively dialyzed against PBS, concentrated to 5 ml, and then applied to a Sephadex G-100 (5 X 100 cm) column.
The flow rate of 30 ml/hr was maintained with a peristal-
tic pump and 10 ml fractions were collected. Results Purification of guinea pig AT 111:
Guinea pig AT 111 was purified
by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose ion-exchange chromatography, isoelectric focussing in a sucrose density gradient, and Sephadex G-100 gel filtration chromatography.
It was homogeneous as judged by SDS-disc gel
electrophoresis. Table I outlines this purification method including quantitative values for each purification step. Heparin-Sepharose affinity chromatography exploited the affinity of AT I11 for heparin.
It resulted in a 128-fold purification
with recovery of virtually all plasma AT I11 antigenic activity as determined by quantitative immunodiffusion and 85% of the plasma functional antithrombin activity as determined by measurement of residual thrombin coagulant activity.
The second purification
step, DE-52 cellulose chromatography, is illustrated in Figure 1.
HECK, ROSENBERG, AND REMOLD
366 TABLE I
Summary of Purification of AT I11 from Guinea Pig Plasma
Step
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1. Guinea pig plasma (2000 ml)
PurifiAbsorbance Antithrombin Specific cation units (280 nm) units* activity units Recovery 90,000
270,000
3
1
100%
2. HeparinSepharose
600
230,000
383
128
85%
3. DE-52 Cellulose
324
157,000
486
162
58%
192
120,960
630
210
44%
185
112,300
607
202
41%
4 . Isoelectro-
focussing 5. Sephadex G-100
*Based on arbitrarily assigning 135 antithrombin units/ml to guinea pig plasma in the standard thrombin coagulant assay.
The upper panel depicts a typical chromatogram of AT I11 on DE-52 cellulose. This step resulted in a 162-fold purification and 58% yield of the inhibitor.
Two protein peaks elute after initia-
tion of the salt gradient. The first peak, which elutes between 0.06-0.1M added NaC1, contained all of the antithrombin activity as determined by functional inhibition of thrombin and quantitative immunodiffusion. The second peak, which elutes between 0.13-0.16M added NaC1, contained no functional AT I11 activity. Two cathodal bands and three anodal bands, which did not bind to thrombin, were noted on SDS-disc gel electrophoresis. When fractions containing AT I11 were subjected to preparative isoelectric focussing, maxi-
GUINEA PIG ANTITHROMBIN I11
36 7
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1 DEAE CELLULOSE CffROMATOGRAFffY pH 8.I
I
TUBE NUMBER FIGURE 1 Upper panel: Chromatography of AT 111 on DE-52 cellulose. DE-52 cellulose was packed into a column ( 4 X 60 cm) and 600 units from step 2 were applied and a linear salt gradient was initiated with the reservoir containing 1000 ml of 0 . 3 M NaCl in 0.005 M sodium phosphate, pH 8.1 and the mixing chamber containing 1000 ml of 0.005 M sodium phosphate buffer, pH 8.1. Fractions of 15 m l were collected into plastic tubes. Lower panel: Isoelectric focussing was performed in a 440 ml glass column in a 0-47% sucrose density gradient containing 1.5% pH 4-6 Ampholines. The column was prefocussed for 21 hr at 6 W. After application of the sample, the antithrombin was electrofocussed for 22 hr at 6 W and drained with a pump and 4 ml aliquots were collected into plastic tubes.
mum AT 111 activity was demonstrated in fractions with an isoelectric point of 5.15 (Figure 1, lower panel).
Greater than 90% of
the functional and antigenic AT I11 activity was found to exist
368
HECK, ROSENBERG, AND REMOLD
over a narrow 0.2 pH range (pH 5.0-5.2).
This s t e p resulted i n a
210-fold p u r i f i c a t i o n and a 44% y i e l d of t h e i n h i b i t o r .
Despite
exhaustive d i a l y s i s , t h e presence of Ampholines w i t h AT 111 w a s detected.
When t h i s mixture w a s chromatographed on Sephadex G-100,
complete e l i m i n a t i o n of t h e Ampholines with minimal l o s s of AT 111
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was observed.
SDS-disc g e l e l e c t r o p h o r e s i s revealed
one homo-
geneous band a t an apparent molecular weight of 64,000 ( F i g u r e 2 ) .
FIGURE 2
The SDS-disc g e l e l e c t o p h o r e t i c p a t t e r n u s i n g t h e Laemmli system obtained with 50 ug of p u r i f i e d AT 111.
369
GUINEA PIG ANTITHROMBIN I11 Effect of pH on stability of inhibitor at 37OC for 24 hr:
The pH
stability of AT I11 was determined by incubation of the inhibitor in buffers ranging from pH 2-12 for 24 hr at 37°C under sterile conditions (5).
A t p H 2 the inhibitor activity was 10% of
freshly thawed AT 111. At pH 6 , 80%; pH 7-8,
95%; pH 9 , 84%, and
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at pH 12, 35% of control activity. Effect of temperature at 56OC on inhibitor stability: The temperature stability of AT I11 incubated at 56OC for varying times in 0.1M
NaC1, 0.05M Tris-HC1, pH 7.5 was determined (5).
After a
10 minute incubation, 50% o f the original activity was detected. No activity was found after a 30 min incubation. Inhibition of thrombin coagulant activity by antithrombin:
Fig-
ure 3 demonstrates inhibition of the coagulant activity of thrombin by AT 111.
At approximate equimolar concentrations of thrombin and
antithrombin, the thrombin time is increased from 15 to 30 seconds. As the molar concentration of antithrombin to thrombin is increased to approximately 2:l and 5:1, the thrombin time is dramatically increased to 88 sec and 300 sec respectively. Inhibition of thrombin amidolysis activity by AT I11 and evidence for heparin cofactor activity:
This assay measured the amount of
p-nitrophenol released from the chromogenic substrate, Bz-Phe-ValArg-pNa, by thrombin in the presence of AT 111. When thrombin and AT I11 were mixed at equimolar ratios and aliquots removed at specific intervals, a progressive decrease in the amount of released p-nitrophenol was observed (Figure 4).
When a minute
amount of heparin was preincubated with AT I11 and then mixed
HECK, ROSENBERG, AND REMOLD
3 70
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F
cL lo
60
OJJg 1 k
'240
3b0
360
INCUBA TION TIME OF JHROMBIN Wl T t i AN TI THROMBINfl 6ecJ
FIGURE 3 Inhibition of the coagulant activity of thrombin AT 111. The graph depicts the straight line relationship between the log of the thrombin time (ordinate) and the thrombin-antithrombin incubation time (abscissa). Each point represents the mean obtained for four separate incubation mixtures k SEM.
with thrombin, instantaneous inhibition of thrombin was noted. Thrombin preincubated with the same amount of heparin in the absence of AT TI1 and then mixed with the substrate exhibited full activity. SDS-disc gel electrophoresis analysis of thrombin-AT I11 complexes: Thrombin and AT 111 were incubated together for varying times at 37'C
and the reaction products analyzed by
SDS-disc gel electrophoresis to determine complex formation of thrombin with AT 111.
When thrombin and AT 111 were in-
cubated at equimolar concentrations for 10 sec, 60 see, and 300 sec
GUINEA PIG ANTITHROMBIN I11
371
MEASUREMENT OF RESIDUAL THROMBIN BY AMlDOLYSlS OF BzPhe-Val-Arg-pNa 6-2160)
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10
ThrondnnWeporin
Li
0
I
I
I
I
Ttromlmnt Anlithrmbn t Heporin I
I
30
60 120 I80 240 300 INCUBATION TIME OF THROMBIN WITH ANTITHROMBINlDf? Heparin&x 15
FIGURE 4 Inhibition of the amidolytic activity o f thrombin by AT I11 in the presence or absence of heparin. The graph depicts the progressive inhibition of thrombin by an equimolar concentration of antithrombin and the instantaneous inhibition of thrombin by the mixture of antithrombin and heparin. Each point represents the SEM. mean obtained for four separate incubation mixtures
*
at 37"C, gradual formation of two complexes at apparent molecular weights of 7 8 , 0 0 0 and 8 9 , 0 0 0 and a dimunition over time in both the alpha and beta thrombin can be observed (Figure 5
-
Lanes G, H, I).
In contrast, the pattern observed after incubating thrombin with heparin-pretreated AT I11 resulted in instantaneous formation of both complexes with apparent molecular weights of 7 8 , 0 0 0 and
89,000 (Lanes D, E, F).
AT I11 migrated as a single band in this
system with an apparent molecular weight of 6 4 , 0 0 0 (Lane C ) . Thrombin was a mixture of alpha thrombin ( 3 4 , 0 0 0 ) and beta thrombin (21,000 - Lane B).
I n addition, modification of AT I11
occurred as manifested by formation of a new band at an apparent
HECK, ROSENBERG, ANTI REMOLD
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372
FIGURE 5 Effect of incubating thrombin with and without heparin-pretreated AT I11 as examined by slab 10% sodium dodecyl sulfate disc gel electrophoresis. Antithrombin (* heparin) was mixed with an equimolar concentration of thrombin and the reaction terminated after 10 sec, 60 sec, and 300 sec. The gel represents A , molecular weight markers; B, thrombin 15 ug, D-F, heparin pretreated antithrombin added to thrombin: D. 10 sec; E, 60 sec; F, 300 sec; G-I, antithrombin added to thrombin; G , 10 sec; H, 60 sec; and I, 300 sec. molecular weight of 56,000 suggesting an initial cleavage by thrombin of the native inhibitor. Production and demonstration of a monospecific antibody to AT 111: Immunoelectrophoresis w a s performed to determine the specificity of antisera produced in rabbits immunized with purified AT 111.
When electrophoresed guinea pig plasma was reacted with rabbit antisera to guinea pig AT 111, one precipitin arc in the alpha-2 globulin zone was observed (Figure 6 ) .
By Ouchterlony analysis,
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GUINEA PIG ANTITHROMBIN I11
373
FIGURE 6
Immunoelectrophoresis of guinea pig plasma to demonstrate a monospecific antisera. The center wells contain the electrophoresed guinea pig plasma and the troughs contain the antisera; trough A contains rabbit antisera to guinea pig antithrombin and trough B contains rabbit antisera to guinea pig serum.
a strong line of identity was observed between purified AT I11 and citrated guinea pig plasma reacted with the monospecific antisera.
No species crossreactivity was noted between this
antisera and citrated plasma from mouse, rat, rabbit, goat, bovine, monkey, and human sources. The plasma concentration was determined by quantitative immunodiffusion to be 310 2 18 Ug/rnl. No difference was noted in plasma concentration between outbred (Hartley) and the inbred (Strain 2 and 13) guinea pigs. Discussion The four step purification procedure of guinea pig AT I11 described in this communication reproducibly yielded a highly active AT I11 that was free of any contaminants as judged by SDS-disc gel electrophoresis. Furthermore, we investigated the inhibitor's capacity to inactivate purified bovine thrombin.
If AT I11 is
374
HECK, ROSENBERG, ANTI REMOLD
added to thrombin at approximately a 1:l molar stoichiometry, there is a linear decline in the enzyme's coagulant activity. The slope o f the line, reflecting loss of thrombin coagulant activity, is
proportional to the AT I11 concentration. To demonstrate that this single molecular species possessed
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both antithrombin and heparin cofactor activity, we examined the kinetics of thrombin inhibition by AT I11 in the presence and absence of heparin.
When thrombin and AT I11 are mixed at a
1:l molar ratio, there is a progressive decline in the enzyme's amidolytic activity over a period of time.
In contrast, if throm-
bin is incubated with heparin-pretreated AT 111, its enzymatic activity is inhibited instantaneously. The mechanism of inhibition appears to be the formation of a bimolecular complex between AT 111 and thrombin in a 1:l molar ratio. When the reaction products were analyzed by SDS-disc gel electrophoresis, the gradual disappearance of both thrombin and AT I11 and the gradual formation of two complexes with apparent molecular weights 89,000 and 78,000 could be observed. When heparin was added to the AT 111 and then reacted with thrombin, complex formation was observed within 10 seconds of incubation. The observations demonstrate that AT I11 neutralizes the activity of thrombin by formation of a 1:l inhibitor-enyzme complex and that heparin functions to dramatically accelerate the rate of complex formation. As can be seen in Figure 5 , incubation of thrombin with AT I11 in both the presence and absence of heparin did not result in complete conversion of both thrombin forms to higher
molecular
GUINEA PIG ANTITHROMBIN 111 weight thrombin-AT 111 complexes.
375 T h i s can b e s t b e e x p l a i n e d by
t h e p r e s e n c e of small amounts of i n a c t i v e thrombin and AT I11 i n t h i s r e a c t i o n m i x t u r e which c o u l d n o t i n t e r a c t t o form b i m o l e c u l a r complexes. The i n h i b i t o r we have i s o l a t e d from g u i n e a p i g plasma e x h i b i t s
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many s i m i l a r p h y s i c a l p r o p e r t i e s t o AT 111 from o t h e r s p e c i e s .
The
m o l e c u l a r weight of o u r p u r i f i e d i n h i b i t o r i s 64,000, which i s i n c l o s e agreement w i t h t h o s e r e p o r t e d f o r human plasma, 62,300 ( 1 ) and 63,700 ( 2 ) ; and r a b b i t plasma 68,000 ( 5 ) .
On t h e o t h e r hand,
c a n i n e AT I11 h a s a m o l e c u l a r weight of 78,000 ( 4 ) .
Guinea p i g
AT I11 e x i s t s as a s i n g l e p o l y p e p t i d e c h a i n a f t e r r e d u c t i o n w i t h
B-mercaptoethanol which i s s i m i l a r t o AT I11 from o t h e r s p e c i e s (1, 4 , 5 ) .
The i s o e l e c t r i c p o i n t of g u i n e a p i g AT I11 w a s d e t e r -
mined t o b e 5.15, which i s s i m i l a r t o t h a t r e p o r t e d f o r human AT 111 ( 5 . 1 2 ) (1). The pH s t a b i l i t y r a n g e was d e t e r m i n e d t o be
maximal a t pH 7-8, which i s n a r r o w e r t h a n t h a t r e p o r t e d f o r r a b b i t AT 111 of pH 6.5-8.5
The t e m p e r a t u r e s t a b i l i t y a t 56OC of
(5).
o u r i n h i b i t o r c h a r a c t e r i z e s i t a s more l a b i l e t h a n t h a t o f r a b b i t AT I11 ( 5 ) .
The f i n a l y i e l d of AT I11 w i t h h i g h s p e c i f i c a c t i v i t y w a s h i g h e r t h a n t h a t r e p o r t e d f o r o t h e r p r e p a r a t i o n s of AT 111 and t h e p r e s e n t method c o u l d b e u t i l i z e d f o r l a r g e s c a l e p r e p a r a t i o n of t h i s i n h i b i t o r .
P u r i f i e d AT 111 s h o u l d b e a v a l u a b l e
r e a g e n t t o i n v e s t i g a t e t h e r o l e of p r o t e i n a s e s i n t h e r e g u l a t i o n of c e l l u l a r f u n c t i o n s . ' A b b r e v i a t i o n s : AT 111 ( a n t i t h r o m b i n 111, a n t i t h r o m b i n - h e p a r i n cofactor). SDP - sodium d o d e c y l s u l f a t e .
376
HECK, ROSENBERG, AND REMOLD Acknowledgments To D r . David L i u f o r h e l p f u l s u g g e s t i o n s i n the p r e p a r a t i o n
of t h i s m a n u s c r i p t .
T h i s work w a s s u p p o r t e d by G r a n t s RR05669 and A112110 and a Grant-in-Aid
from t h e American Heart A s s o c i a t i o n .
HGR,
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r e c i p i e n t of a n E s t a b l i s h e d I n v e s t i g a t o r s h i p from t h e American Heart A s s o c i a t i o n . REFERENCES 1. R.D.
Rosenberg and P.S. Damus, J. B i o l . Chem. w : 6 4 9 0 - 6 5 0 5 ,
1973.
2.
U . Abi l dgaard. Scand. J. C l i n . Lab. I n v e s t . 2 : 1 9 0 - 1 9 5 ,
3.
E. T h a l e r and G. Schmer. B r i t . J. Haelratol. 1 : 2 3 3 - 2 4 3 ,
1967.
1975. 4.
P . Damus, andG. Wa lla c e . Biochem. Biophys. Res. Comun.
61:1147-1153, 5.
1974.
E. T. Yin, S. Wessler, S .
246~3694-3792, 6.
and P. S t o l l . J . B i o l . Cliem.
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I. Dani s hefs ky, A . Zweben and B. Slomiany. J . B i o l . Chem. 2 5 3 ~ 3 2 - 3 7 , 1978.
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U. L i n d a h l , L. A. C i f o n e l l i , B. L i n d a h l and L . Roden. J . B i o l . Chem. =:2817-2820,
8.
1965.
P. Damus and R. Rosenberg, i n "Methods i n Enzymology" Vol.
XLV, P a r t B , L . Lorand, e d . , Academic P r e s s , New York, 1976, pp. 653-669. 9.
L . Heck, E . Remold and H . Remold. Biochem. Biophys. Res.
Comun. g : 1 5 7 6 - 1 5 8 3 ,
1978.
G U I N E A PIG ANTITHROMBIN I11
377
10.
U . K . Laemmli, N a t u r e . K : 6 8 0 - 6 8 5 ,
11.
0. H . Lowry, N . J . Rosebrough, A . L . F a r r
J . B i o l . Chem. =:265-275,
1970. and R . J . R a n d a l L
1951.
C . Wllliams , i n "Methods i n Immunology and Immunochemis t r y "
12
Volume 111, C . W i l l i a m s and
M. C h a s e , e d s , Academic Press,
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New York, 1971, p p . 237-273.
13.
14
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G . M a n c i n i , A . D . Carbonara and J . F. Heremans. Immuno-
c h e m i s t r y . 2:235-242,
1965.
D . J . Baughman and D.
F . Waugh. J . B i o l . Chem. =:5252-5260,
1967. 15.
R . L . Lundblad, H. S . Kingdon and K . G . Mann. i n "Methods
i n Enzymology" Vol XLV, P a r t B , L . Lorand, e d , Academic P r e s s , N e w York, 1976, p p . 653-669. 16.
0 . R . d d e g a r d , M . L i e and U . A b i l d g a a r d . Thrombosis R e s e a r c h 6:287-294,
17.
1975.
T . Chase and E. Shaw. i n "Methods i n Enzymology'' Vol. X I X , L . Lorand and G. Pearlman, e d s , Academic P r e s s , New York,
1970, p p . 20-27.
Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Purification and Properties of Guine Pig Antithrombin III a
a
L. Heck , R. Rosenberg & H. Remold
a
a
Departments of Medicine , Harvard Medical School and Robert B. Brigham Hospital and Sidney Farber Cancer Institute , Boston, Massachusetts, 02115 Published online: 05 Dec 2006.
To cite this article: L. Heck , R. Rosenberg & H. Remold (1979) Purification and Properties of Guine Pig Antithrombin III, Preparative Biochemistry, 9:4, 359-377, DOI: 10.1080/00327487908061700 To link to this article: http://dx.doi.org/10.1080/00327487908061700
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PREPARATIVE BIOCHEMISTRY, 9(4) , 359-377 (1979)
PURIFICATION AND PROPERTIES OF GUINEA PIG ANTITHROMBIN I11
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L. Heck, R. Rosenberg, and H. Remold Departments of Medicine, Harvard Medical School and Robert B. Brigham Hospital and Sidney Farber Cancer Institute Boston, Massachusetts 02115 ABSTRACT Guinea pig antithrombin I11 has been purified from plasma by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose chromatography, isoelectric focussing, and Sephadex G-100 gel filtration chromatography.
The final product was homogeneous as
judged by sodium dodecyl sulfate disc gel electrophoresis. Purification was 202-fold with a yield of 41%. Antiproteinase activity of 4
antithrombin I11 was determined by progressive inactivation of thrombin coagulant and amidolytic activity.
Heparin cofactor activ-
ity was demonstrated by immediate inactivation of thrombin by antithrombin I11 in the presence of minute quantities of heparin. It also could be demonstrated that thrombin inactivation by antithrombin 111 occurs by formation of a bimolecular complex whose rate of formation is markedly enhanced by minute quantities o f heparin. Introduction Methods for the purification of AT 111’ from human (1-3, 6 ) , canine (4), rabbit (5) plasma have been reported by several
359 Copyrlght 0 I979 hy Marcel Dekker. Inc All Rights Keserved. Neither this work nor a n y part may he reproduced o r transmitted in any form or hy any means, electronic or mechanical, including pholocopying. microfilming. and recording. or by any information storage and retrieval s y s l e m . withoul permission in writing from the publisher.
HECK, ROSENBERG, AND REMOLD
360
investigators. Whereas a single purification step utilizing heparin-Sepharose affinity chromatography has been reported to yield a homogeneous preparation from human (6) and canine (4) sources, this has not been our experience in purifying this proteinase inhibitor from guinea pig plasma.
Four steps were necessary to purify guinea
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pig AT 111 to homogeneity. Materials and Methods Chemicals: The following chemicals were reagent grade and used without further purification: bovine fibrinogen, Fraction 1, Lot 84C-0479 from Sigma Chemical Corp., St. Louis, MO; Bz-Phe-Val-ArgpNa (S-2160) from Ortho Diagnostics, Raritan, NJ: p-nitrophenyl-pguanidinobenzoate (NPGB) and four times crystallized bovine serum albumin from ICN Pharmaceutical, Inc., Cleveland, OH; topical bovine thrombin from Parke-Davis, Detroit, MI; polybrene from Aldrich Chemical Crop., Milwaukee, WI; crude porcine intestinal heparin (Stage 14) from Inolex Crop., Park Forest, IL: DE-52 cellulose from Whatman, Inc., Clifton, NJ; Sephadex G-100 from Pharmacia, Piscataway, NJ; cellulose-phosphate (Cellex-P) from Bio-Rad, Richmond, CA; and Ampholines pH 4.0 to 6.0 from LKB, Hicksville, NY. Guinea pig plasma: Plasma was obtained by collecting cardiac blood in the presence of 3.8% trisodium citrate (1 vol citrate: 9 vol blood) with subsequent centrifugation at 2500 RPM for
30 min at 4OC.
The plasma was then recentrifuged at 10,000 RPM
for 15 min at 4°C in a Model E Sorvall. stored at -2OOC.
The frozen pool was
GUINEA PIG ANTITHROMBIN 111
36 1
Preparation of heparin-Sepharose: Crude porcine intestinal heparin, partially purified according to the method of Lindahl ( 7 ) , was insolubilized to Sepharose 4 B as described ( 8 ) . Electrophoresis: Molecular weight markers and samples were prepared as described (9).
Polypeptides were separated by a modifica-
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tion of the method of Laemmli (10) using slab gels ( 3 X 120 X 170 mm) or tube gels ( 6 mm X 150 mm). 1% Protein determination: The EZB0 of guinea pig AT I11 and bovine
thrombin was determined to be 6.9 and 19.6, respectively. Protein determination was performed by the method of Lowry using 4X crystallized bovine albumin as the reference (11). Immunoelectrophoresis was performed in agar gels as described (12). Quantitative immunodiffusion was performed by the technique of Mancini using purified AT-111 in serial dilutions as the standard antigen ( 1 3 ) . Immunization: New Zealand white rabbits were immunized with a total of 200 pg of purified AT I11 mixed with an equal volume of complete Freund's adjuvant (CFA) with multiple intradermal injections every three weeks for a total of four injections. The animals were bled after the second injection and at two week intervals. Conductivity measurements:
For conductivity determinations, a
conductivity meter, type CDC 314 (Radiometer, Copenhagen, Denmark) was used. Purification of bovine thrombin: Bovine thrombin was purified from topical thrombin by sequential DE-52 cellulose and cellulose-
362
HECK, ROSENBERG, ANTI REMOLD
phosphate chromatography (14) and stored in 0.5 M NaC1, 0.5 M sodium phosphate buffer, pH 7.0 at a concentration of 1.0 mgm/rnl at -7OOC. All experiments were done with a bovine thrombin preparation that had a specific clotting activity of 1.8 NIH units/ug and a specific amidolytic activity of 0.36 OD ( 4 0 5 nm) change/min/
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Ug.
This preparation was more than 87% active by active site ti-
tration and electrophoresed as a mixture of alpha and beta thrombin (1O:l). Measurement of thrombin coagulant activity:
Fibrinogen clotting
activity was determined (15) using Lot 3B thrombin standard (21.3 NIH units/mg) kindly provided by Dr. David Aronson, NIH. Measurement of thrombin amidolytic activity: Amidolysis of Bz-Phe-Val-Arg-pNn was measured by a modification of the method of Gdegard (16).
Three hundred p1 of substrate (0.5 mg/ml),
100 p 1 of polybrene (1 mg/ml), and 100 1.11 of Tris-HC1, O.lM, pH 8.1, were sequentially added to a 1.0 ml quartz cuvette. Thrombin in 1-10 ~1aliquots was added, mixed rapidly, and the absorbance was monitored over a 5 minute period at 405 nm (25 C). Active site titration of thrombin:
Active site titration with
NPGB was performed by the method of Chase and Shaw (17). Measurement of residual thrombin coagulant activity: Thrombin 5.4 .pg (10 NIH units) was added to 0-25 pg AT 111 in a prealbumin-
ized plastic tube and the volume adjusted to 100 p1 with O.lM, Tris-HC1, pH 8.1, and incubated at 37'C.
At 60 second intervals,
10 p1 fractions were withdrawn and added to the fibrinogen solu-
tion and the standard chrombin assay performed (15).
363
GUINEA PIG ANTITHROMBIN I11 Measurement of residual thrombin amidolytic activity:
Thrombin
5 ug was added to 9.4 ug of AT 111 in prealbuminized plastic tubes apd the volume adjusted to 100 ~1 with assay buffer incubated at
37°C. At 15 sec, 30 sec and 60 sec intervals, 10 p 1 fractions were withdrawn and added to the substrate solution in the standard
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amidolytic assay. Measurement of heparin-cofactor activity:
Sodium heparin, 0.1 IT,
was added to 9.4 ug of AT I11 and incubated at 25°C for 5 min and then placed in an ice bucket.
The amidolytic assay was then per-
formed as described above so that the final concentration of heparin/ assay did not exceed 0.01 U and did not interfere with the amidolysis of the tripeptide substrate by thrombin. Heparin-Sepharose affinity chromatography: All chromatography steps were performed at 4°C.
500 ml aliquots of citrated guinea
pig plasma were mixed with 75 ml of settled heparin-Sepharose and placed on a roller platform for one hour at 4°C.
The plasma-
slurry mixture was then transferred to a large scintered glass funnel and washed with four liters of 0.05 M Tris-HC1 saline, pH 7.6. After performing this step three additional times, the slurries were combined and transferred to a Lucite column ( 4 X 60 cm) and washed with 0.05 M Tris-HC1 in 0.4 M NaC1, pH 7.6, until
the OD280 was less than 0.03 unitslml.
The antithrombin contain-
ing fraction was desorbed with 0.05M Tris-HC1 in 1.OM NaC1, pH 7.6 and concentrated ten-fold by vacuum dialysis to a final protein concentration of 18.7 OD280 unitslml.
The resin could be re-
utilized at least 8-10 times for the purification of AT I11 if
HECK, ROSENBERG, AND REMOLD
364
i t was immediately r e e q u i l i b r a t e d w i t h 0.15 M N a C l i n 0.05 M T r i s -
HC1, pH 7 . 6 , and s t o r e d i n 0.1% sodium a z i d e a t 4OC.
DE-52 c e l l u l o s e i o n exchange chromatography:
The f r a c t i o n from t h e
p r e v i o u s s t e p was d i a l y z e d a g a i n s t 4 l i t e r s of 0.005 M sodium phosp h a t e b u f f e r , pH 8.1, w i t h f r e q u e n t changes u n t i l t h e c o n d u c t i v i t y
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of t h e p r o t e i n s o l u t i o n w a s e q u a l t o t h e b u f f e r .
DE-52 c e l l u l o s e
was e q u i l i b r a t e d w i t h t h i s b u f f e r and 250 m l of t h e s e t t l e d r e s i n w a s packed i n a L u c i t e column (30 X 4 cm).
The p r o t e i n s o l u t i o n
w a s allowed t o p e r c o l a t e s l o w l y t h r o u g h t h e r e s i n , which was t h e n
washed w i t h 500 m l of s t a r t i n g b u f f e r a t a flow r a t e of 75 m l / h r m a i n t a i n e d by a p e r i s t a l t i c pump.
A l i n e a r sodium c h l o r i d e g r a d i -
e n t was t h e n employed f o r f r a c t i o n a t i o n .
The r e s e r v o i r c o n t a i n e d
1000 m l of 0 . 3 M N a C l i n 0.005 M sodium phosphate b u f f e r , pH 8 . 1 , and t h e mixing chamber c o n t a i n e d 1000 m l of 0.005 M sodium phosp h a t e b u f f e r , pH 8.1.
F r a c t i o n s of 1 5 m l were c o l l e c t e d i n
p l a s t i c tubes. I s o e l e c t r i c focussing i n a sucrose density gradient:
The pool
of a c t i v e f r a c t i o n s from t h e p r e v i o u s s t e p w a s c o n c e n t r a t e d t o 5 m l by vacuum d i a l y s i s and s u b j e c t e d t o i s o e l e c t r i c f o c u s s i n g on a LKB 8102 column c o n t a i n i n g 1.5% pH 4 . 0 - 6 . 0 Ampholines i n a l i n e a r 0-47% s u c r o s e d e n s i t y g r a d i e n t .
The column w a s pre-
e l e c t r o f o c u s s e d f o r 2 1 h r a t 6 W b e f o r e a p p l i c a t i o n of t h e sample. AT I11 w a s d i a l y z e d a g a i n s t 4 l i t e r s of d o u b l e d i s t i l l e d water
a t 4 O C f o r 2 hrs.
The d e n s i t y of t h e a n t i t h r o m b i n sample was
a d j u s t e d t o t h e d e n s i t y of t h e sample e x t r a c t e d from t h e c e n t e r of t h e s u c r o s e d e n s i t y g r a d i e n t w i t h c o n c e n t r a t e d s u c r o s e
365
GUINEA PIG ANTITHROMBIN 111 solution. This resulted in a sample volume of 8-10 ml which was subjected to isoelectric focussing for 22 hr at 6 W.
The gradient
was then drained with a pump, and 4 ml aliquots were collected into tubes and the pH measured.
0.5 ml of 0.1 M Tris-HC1, pH 8.0,
was added to the fractions and functional activity studies were
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performed. Sephadex G-100 fel filtration chromatography:
The pool of active
protein fractions was exhaustively dialyzed against PBS, concentrated to 5 ml, and then applied to a Sephadex G-100 (5 X 100 cm) column.
The flow rate of 30 ml/hr was maintained with a peristal-
tic pump and 10 ml fractions were collected. Results Purification of guinea pig AT 111:
Guinea pig AT 111 was purified
by sequential heparin-Sepharose affinity chromatography, DE-52 cellulose ion-exchange chromatography, isoelectric focussing in a sucrose density gradient, and Sephadex G-100 gel filtration chromatography.
It was homogeneous as judged by SDS-disc gel
electrophoresis. Table I outlines this purification method including quantitative values for each purification step. Heparin-Sepharose affinity chromatography exploited the affinity of AT I11 for heparin.
It resulted in a 128-fold purification
with recovery of virtually all plasma AT I11 antigenic activity as determined by quantitative immunodiffusion and 85% of the plasma functional antithrombin activity as determined by measurement of residual thrombin coagulant activity.
The second purification
step, DE-52 cellulose chromatography, is illustrated in Figure 1.
HECK, ROSENBERG, AND REMOLD
366 TABLE I
Summary of Purification of AT I11 from Guinea Pig Plasma
Step
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1. Guinea pig plasma (2000 ml)
PurifiAbsorbance Antithrombin Specific cation units (280 nm) units* activity units Recovery 90,000
270,000
3
1
100%
2. HeparinSepharose
600
230,000
383
128
85%
3. DE-52 Cellulose
324
157,000
486
162
58%
192
120,960
630
210
44%
185
112,300
607
202
41%
4 . Isoelectro-
focussing 5. Sephadex G-100
*Based on arbitrarily assigning 135 antithrombin units/ml to guinea pig plasma in the standard thrombin coagulant assay.
The upper panel depicts a typical chromatogram of AT I11 on DE-52 cellulose. This step resulted in a 162-fold purification and 58% yield of the inhibitor.
Two protein peaks elute after initia-
tion of the salt gradient. The first peak, which elutes between 0.06-0.1M added NaC1, contained all of the antithrombin activity as determined by functional inhibition of thrombin and quantitative immunodiffusion. The second peak, which elutes between 0.13-0.16M added NaC1, contained no functional AT I11 activity. Two cathodal bands and three anodal bands, which did not bind to thrombin, were noted on SDS-disc gel electrophoresis. When fractions containing AT I11 were subjected to preparative isoelectric focussing, maxi-
GUINEA PIG ANTITHROMBIN I11
36 7
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1 DEAE CELLULOSE CffROMATOGRAFffY pH 8.I
I
TUBE NUMBER FIGURE 1 Upper panel: Chromatography of AT 111 on DE-52 cellulose. DE-52 cellulose was packed into a column ( 4 X 60 cm) and 600 units from step 2 were applied and a linear salt gradient was initiated with the reservoir containing 1000 ml of 0 . 3 M NaCl in 0.005 M sodium phosphate, pH 8.1 and the mixing chamber containing 1000 ml of 0.005 M sodium phosphate buffer, pH 8.1. Fractions of 15 m l were collected into plastic tubes. Lower panel: Isoelectric focussing was performed in a 440 ml glass column in a 0-47% sucrose density gradient containing 1.5% pH 4-6 Ampholines. The column was prefocussed for 21 hr at 6 W. After application of the sample, the antithrombin was electrofocussed for 22 hr at 6 W and drained with a pump and 4 ml aliquots were collected into plastic tubes.
mum AT 111 activity was demonstrated in fractions with an isoelectric point of 5.15 (Figure 1, lower panel).
Greater than 90% of
the functional and antigenic AT I11 activity was found to exist
368
HECK, ROSENBERG, AND REMOLD
over a narrow 0.2 pH range (pH 5.0-5.2).
This s t e p resulted i n a
210-fold p u r i f i c a t i o n and a 44% y i e l d of t h e i n h i b i t o r .
Despite
exhaustive d i a l y s i s , t h e presence of Ampholines w i t h AT 111 w a s detected.
When t h i s mixture w a s chromatographed on Sephadex G-100,
complete e l i m i n a t i o n of t h e Ampholines with minimal l o s s of AT 111
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was observed.
SDS-disc g e l e l e c t r o p h o r e s i s revealed
one homo-
geneous band a t an apparent molecular weight of 64,000 ( F i g u r e 2 ) .
FIGURE 2
The SDS-disc g e l e l e c t o p h o r e t i c p a t t e r n u s i n g t h e Laemmli system obtained with 50 ug of p u r i f i e d AT 111.
369
GUINEA PIG ANTITHROMBIN I11 Effect of pH on stability of inhibitor at 37OC for 24 hr:
The pH
stability of AT I11 was determined by incubation of the inhibitor in buffers ranging from pH 2-12 for 24 hr at 37°C under sterile conditions (5).
A t p H 2 the inhibitor activity was 10% of
freshly thawed AT 111. At pH 6 , 80%; pH 7-8,
95%; pH 9 , 84%, and
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at pH 12, 35% of control activity. Effect of temperature at 56OC on inhibitor stability: The temperature stability of AT I11 incubated at 56OC for varying times in 0.1M
NaC1, 0.05M Tris-HC1, pH 7.5 was determined (5).
After a
10 minute incubation, 50% o f the original activity was detected. No activity was found after a 30 min incubation. Inhibition of thrombin coagulant activity by antithrombin:
Fig-
ure 3 demonstrates inhibition of the coagulant activity of thrombin by AT 111.
At approximate equimolar concentrations of thrombin and
antithrombin, the thrombin time is increased from 15 to 30 seconds. As the molar concentration of antithrombin to thrombin is increased to approximately 2:l and 5:1, the thrombin time is dramatically increased to 88 sec and 300 sec respectively. Inhibition of thrombin amidolysis activity by AT I11 and evidence for heparin cofactor activity:
This assay measured the amount of
p-nitrophenol released from the chromogenic substrate, Bz-Phe-ValArg-pNa, by thrombin in the presence of AT 111. When thrombin and AT I11 were mixed at equimolar ratios and aliquots removed at specific intervals, a progressive decrease in the amount of released p-nitrophenol was observed (Figure 4).
When a minute
amount of heparin was preincubated with AT I11 and then mixed
HECK, ROSENBERG, AND REMOLD
3 70
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F
cL lo
60
OJJg 1 k
'240
3b0
360
INCUBA TION TIME OF JHROMBIN Wl T t i AN TI THROMBINfl 6ecJ
FIGURE 3 Inhibition of the coagulant activity of thrombin AT 111. The graph depicts the straight line relationship between the log of the thrombin time (ordinate) and the thrombin-antithrombin incubation time (abscissa). Each point represents the mean obtained for four separate incubation mixtures k SEM.
with thrombin, instantaneous inhibition of thrombin was noted. Thrombin preincubated with the same amount of heparin in the absence of AT TI1 and then mixed with the substrate exhibited full activity. SDS-disc gel electrophoresis analysis of thrombin-AT I11 complexes: Thrombin and AT 111 were incubated together for varying times at 37'C
and the reaction products analyzed by
SDS-disc gel electrophoresis to determine complex formation of thrombin with AT 111.
When thrombin and AT 111 were in-
cubated at equimolar concentrations for 10 sec, 60 see, and 300 sec
GUINEA PIG ANTITHROMBIN I11
371
MEASUREMENT OF RESIDUAL THROMBIN BY AMlDOLYSlS OF BzPhe-Val-Arg-pNa 6-2160)
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10
ThrondnnWeporin
Li
0
I
I
I
I
Ttromlmnt Anlithrmbn t Heporin I
I
30
60 120 I80 240 300 INCUBATION TIME OF THROMBIN WITH ANTITHROMBINlDf? Heparin&x 15
FIGURE 4 Inhibition of the amidolytic activity o f thrombin by AT I11 in the presence or absence of heparin. The graph depicts the progressive inhibition of thrombin by an equimolar concentration of antithrombin and the instantaneous inhibition of thrombin by the mixture of antithrombin and heparin. Each point represents the SEM. mean obtained for four separate incubation mixtures
*
at 37"C, gradual formation of two complexes at apparent molecular weights of 7 8 , 0 0 0 and 8 9 , 0 0 0 and a dimunition over time in both the alpha and beta thrombin can be observed (Figure 5
-
Lanes G, H, I).
In contrast, the pattern observed after incubating thrombin with heparin-pretreated AT I11 resulted in instantaneous formation of both complexes with apparent molecular weights of 7 8 , 0 0 0 and
89,000 (Lanes D, E, F).
AT I11 migrated as a single band in this
system with an apparent molecular weight of 6 4 , 0 0 0 (Lane C ) . Thrombin was a mixture of alpha thrombin ( 3 4 , 0 0 0 ) and beta thrombin (21,000 - Lane B).
I n addition, modification of AT I11
occurred as manifested by formation of a new band at an apparent
HECK, ROSENBERG, ANTI REMOLD
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372
FIGURE 5 Effect of incubating thrombin with and without heparin-pretreated AT I11 as examined by slab 10% sodium dodecyl sulfate disc gel electrophoresis. Antithrombin (* heparin) was mixed with an equimolar concentration of thrombin and the reaction terminated after 10 sec, 60 sec, and 300 sec. The gel represents A , molecular weight markers; B, thrombin 15 ug, D-F, heparin pretreated antithrombin added to thrombin: D. 10 sec; E, 60 sec; F, 300 sec; G-I, antithrombin added to thrombin; G , 10 sec; H, 60 sec; and I, 300 sec. molecular weight of 56,000 suggesting an initial cleavage by thrombin of the native inhibitor. Production and demonstration of a monospecific antibody to AT 111: Immunoelectrophoresis w a s performed to determine the specificity of antisera produced in rabbits immunized with purified AT 111.
When electrophoresed guinea pig plasma was reacted with rabbit antisera to guinea pig AT 111, one precipitin arc in the alpha-2 globulin zone was observed (Figure 6 ) .
By Ouchterlony analysis,
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GUINEA PIG ANTITHROMBIN I11
373
FIGURE 6
Immunoelectrophoresis of guinea pig plasma to demonstrate a monospecific antisera. The center wells contain the electrophoresed guinea pig plasma and the troughs contain the antisera; trough A contains rabbit antisera to guinea pig antithrombin and trough B contains rabbit antisera to guinea pig serum.
a strong line of identity was observed between purified AT I11 and citrated guinea pig plasma reacted with the monospecific antisera.
No species crossreactivity was noted between this
antisera and citrated plasma from mouse, rat, rabbit, goat, bovine, monkey, and human sources. The plasma concentration was determined by quantitative immunodiffusion to be 310 2 18 Ug/rnl. No difference was noted in plasma concentration between outbred (Hartley) and the inbred (Strain 2 and 13) guinea pigs. Discussion The four step purification procedure of guinea pig AT I11 described in this communication reproducibly yielded a highly active AT I11 that was free of any contaminants as judged by SDS-disc gel electrophoresis. Furthermore, we investigated the inhibitor's capacity to inactivate purified bovine thrombin.
If AT I11 is
374
HECK, ROSENBERG, ANTI REMOLD
added to thrombin at approximately a 1:l molar stoichiometry, there is a linear decline in the enzyme's coagulant activity. The slope o f the line, reflecting loss of thrombin coagulant activity, is
proportional to the AT I11 concentration. To demonstrate that this single molecular species possessed
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both antithrombin and heparin cofactor activity, we examined the kinetics of thrombin inhibition by AT I11 in the presence and absence of heparin.
When thrombin and AT I11 are mixed at a
1:l molar ratio, there is a progressive decline in the enzyme's amidolytic activity over a period of time.
In contrast, if throm-
bin is incubated with heparin-pretreated AT 111, its enzymatic activity is inhibited instantaneously. The mechanism of inhibition appears to be the formation of a bimolecular complex between AT 111 and thrombin in a 1:l molar ratio. When the reaction products were analyzed by SDS-disc gel electrophoresis, the gradual disappearance of both thrombin and AT I11 and the gradual formation of two complexes with apparent molecular weights 89,000 and 78,000 could be observed. When heparin was added to the AT 111 and then reacted with thrombin, complex formation was observed within 10 seconds of incubation. The observations demonstrate that AT I11 neutralizes the activity of thrombin by formation of a 1:l inhibitor-enyzme complex and that heparin functions to dramatically accelerate the rate of complex formation. As can be seen in Figure 5 , incubation of thrombin with AT I11 in both the presence and absence of heparin did not result in complete conversion of both thrombin forms to higher
molecular
GUINEA PIG ANTITHROMBIN 111 weight thrombin-AT 111 complexes.
375 T h i s can b e s t b e e x p l a i n e d by
t h e p r e s e n c e of small amounts of i n a c t i v e thrombin and AT I11 i n t h i s r e a c t i o n m i x t u r e which c o u l d n o t i n t e r a c t t o form b i m o l e c u l a r complexes. The i n h i b i t o r we have i s o l a t e d from g u i n e a p i g plasma e x h i b i t s
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many s i m i l a r p h y s i c a l p r o p e r t i e s t o AT 111 from o t h e r s p e c i e s .
The
m o l e c u l a r weight of o u r p u r i f i e d i n h i b i t o r i s 64,000, which i s i n c l o s e agreement w i t h t h o s e r e p o r t e d f o r human plasma, 62,300 ( 1 ) and 63,700 ( 2 ) ; and r a b b i t plasma 68,000 ( 5 ) .
On t h e o t h e r hand,
c a n i n e AT I11 h a s a m o l e c u l a r weight of 78,000 ( 4 ) .
Guinea p i g
AT I11 e x i s t s as a s i n g l e p o l y p e p t i d e c h a i n a f t e r r e d u c t i o n w i t h
B-mercaptoethanol which i s s i m i l a r t o AT I11 from o t h e r s p e c i e s (1, 4 , 5 ) .
The i s o e l e c t r i c p o i n t of g u i n e a p i g AT I11 w a s d e t e r -
mined t o b e 5.15, which i s s i m i l a r t o t h a t r e p o r t e d f o r human AT 111 ( 5 . 1 2 ) (1). The pH s t a b i l i t y r a n g e was d e t e r m i n e d t o be
maximal a t pH 7-8, which i s n a r r o w e r t h a n t h a t r e p o r t e d f o r r a b b i t AT 111 of pH 6.5-8.5
The t e m p e r a t u r e s t a b i l i t y a t 56OC of
(5).
o u r i n h i b i t o r c h a r a c t e r i z e s i t a s more l a b i l e t h a n t h a t o f r a b b i t AT I11 ( 5 ) .
The f i n a l y i e l d of AT I11 w i t h h i g h s p e c i f i c a c t i v i t y w a s h i g h e r t h a n t h a t r e p o r t e d f o r o t h e r p r e p a r a t i o n s of AT 111 and t h e p r e s e n t method c o u l d b e u t i l i z e d f o r l a r g e s c a l e p r e p a r a t i o n of t h i s i n h i b i t o r .
P u r i f i e d AT 111 s h o u l d b e a v a l u a b l e
r e a g e n t t o i n v e s t i g a t e t h e r o l e of p r o t e i n a s e s i n t h e r e g u l a t i o n of c e l l u l a r f u n c t i o n s . ' A b b r e v i a t i o n s : AT 111 ( a n t i t h r o m b i n 111, a n t i t h r o m b i n - h e p a r i n cofactor). SDP - sodium d o d e c y l s u l f a t e .
376
HECK, ROSENBERG, AND REMOLD Acknowledgments To D r . David L i u f o r h e l p f u l s u g g e s t i o n s i n the p r e p a r a t i o n
of t h i s m a n u s c r i p t .
T h i s work w a s s u p p o r t e d by G r a n t s RR05669 and A112110 and a Grant-in-Aid
from t h e American Heart A s s o c i a t i o n .
HGR,
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