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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Purification of Canine Plasma Renin by Affinity Chromatography a
a
Robert Walsh , Dean T. Mason , Melvln J. a
Tonkon & Joan Wikman-coffelt
a
a
Section of Cardiovascular Medicine, Department of Medicine , University of California, Davis School of Medicine Published online: 05 Dec 2006.
To cite this article: Robert Walsh , Dean T. Mason , Melvln J. Tonkon & Joan Wikman-coffelt (1976) Purification of Canine Plasma Renin by Affinity Chromatography, Preparative Biochemistry, 6:2-3, 177-191, DOI: 10.1080/00327487608061611 To link to this article: http://dx.doi.org/10.1080/00327487608061611
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PREPARATIVE BIOCHPlISTRY, 6(2&3), 177-191 (1976)
PURIFICATION OF CANINE PLASMA R E N I N BY AFFINITY CHROMATOGRAPHY
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Robert Walsh, Dean T. Mason, Melvin J. Tonkon and Joan Wikman-Coffelt S e c t i o n of Cardiovascular Medicine, Department of Medicine, University of C a l i f o r n i a , Davis School of Medicine
,
ABSTRACT An a f f i n i t y column f o r t h e p u r i f i c a t i o n of canine plasma ren i n was prepared using goat a n t i - r e n i n
(dog kidney) yG g l o b u l i n s .
The antiserum was prepared a g a i n s t a p u r i f i e d kidney r e n i n preparation.
The a n t i - r e n i n g l o b u l i n s were coup1 ed t o cyanogen bromide-
a c t i v a t e d Sepharose.
Using t h e a n t i - r e n i n
globulin-coupled Sepha-
rose as an imuno-adsorbant, a method w a s devised allowing p u r i f i c a t i o n of plasma r e n i n t o a 1,000-fold p u r i t y . INTRODUCTION Renin has been p a r t i a l l y p u r i f i e d from muse s u b p a x i l l a r y glands (11, and kidneys of human (2), hog ( 3 ) . b e e f , r a b b i t ( 4 1 , and dog ( 5 ) .
These procedures i n c l u d e a c i d treatment and (NH4)*
SO4 p r e c i p i t a t i o n , as w e l l as e t h a n o l e x t r a c t i o n ( 5 ) . procedures i n c l u d e Sephadex-G-100
Other
f i l t r a t i o n ( l ) , and a f f i n i t y
chromatography where analogs of t h e r e n i n s u b s t r a t e were coupled t o agarose ( 6 ) .
Radioimmunoassays have been developed t o quan-
t i t a t e sera r e n i n (7) as w e l l as a n g i o t e n s i n I (8,9,10,11,12),
a
177 Copyright 0 1976 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying. microfilmmg, and recording, or by any information storage and retrieval system, without permission in writing from the publisher
WALSH ET AL.
178 product of renin.
This study presents the first purification of
renin directly from the serum using affinity chromatography where renin was assayed by quantitating production of angiotensin I using a radioimmunoassay. MATERIALS AND METHODS
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Kidney Renin Purification.
For purification of canine kidney
renin a modified procedure of Haas et a1 (5) was used.
Six to
eight kidoeys were homogenized in 0.05 M potassium phosphate, pH
7.0, 0.001 M EDTA, 0.001 M DTT at '4 using a Naring blender. volume was brought to 18 d / g m of tissue with water. was stirred for 15 min at
The
The homogenate
centrifuged at 9,000 x g (10 mfn),
4O,
and the supernatant collected.
The pellet was resuspended in the
starting buffer, rehomogenized and the supernatants pooled.
The
superpatant was brought to pH 2.5 with 5N H2S04 and stirred €or 10 min.
The pH was adjusted to 6.5 with 5N KOH and centrifuged.
The
pellet was reextracted and the combined supernatants were brought to pH 4.5 with 5 N H2S04.
Solid (NH4)2 SO4 was added, bringing the
supernatant to 70% ( N H 4 ) *
SO4 saturation.
without stirring (4')
After 2 hours of settling
a pellet containing renin was obtained by
centrifugation at 10,000 x g (10 min).
The partially purified
renin was resuspended in 0.001 M DTT, 0.001 M EDTA and H 2 0 (1 ml/20gm of original kidney weight).
The partially purified renin was dialyzed
in 0.001 M DTT, 0.001 M EDTA and H 2 0 for 16 hours ( 4 ' ) . According to the methods of Haas, et a1 (131, the pH of the solution was brought to 7.5 with. 5N KOH.
After centrifugation the
PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
179
volume was brought to 15 m1/100gm of original kidney weight, and
4 ' ) . acidified to pH 2.8 with 1N H2S04 and stirred 10 min (
The
supernatant was acidified to pH 2.8 with 1N H2S04 and stirred 10 min. After centrifuging at 20,000 x g (10 min), sufficient ethanol was added to the supernatant to make a final concentration of 10%
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ethanol and stored for 2 hours without stirring (0').
The pH of
the ethanol solution was adjusted to 4.5 with 1N NaOH and solid (NH4)2 SO4 was added to bring the supernatant to 70% (NH4)2 SO4 saturation. After stirring for 10 min and centrifuging the precipitate was resuspended in H20 (1.8 m l / l O O g m of starting kidney weight) and dialyzed overnight against 0.05 M citrate, pH 4.6 and 0.05 M Na C1. The supernatant was applied
to
a Sephadex G-100
column (1.5 x 90 cm) and the activity peak collected, lyophilized; and dialyzed against 0.05 M citrate, pH 4.6 and 0.05 M Na C1.
The
purified renin gave approximately 3.2 GU/mg protein. Preparation of Antiserum. Antiserum was produced in goats against canine kidney renin by using the Sephadex G-100 peak renin (Figure 1) which contained the renin activity.
One half milliliter
of solution (kidney renin) containing 2 mg of protein was mixed with an equal volume of Freund's Adjuvant and injected into the axilla of a goat. The first 2 injections were 14 days apart, followed by 3 injections, one every 7 days. After 5 injections the goats were bled.
The g a m globulins were purified from the
4 fractionation of the goat antiserum. Purification of Renin from Canine Serum. The goat g a m a G
antiserum by a 35% (NH4I2
SO
anti-renin (kidney) was complexed to an affinity column using the
WALSH ET AL.
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180
TUBE NUMBER
FIGURE 1 Gel f i l t r a t i o n of dog kidney r e n i n . Sample volume of 4 m l c o n t a i n i n g 60 mg p r o t e i n was a p p l i e d t o a Sephadex G 1 0 0 column (90 x 1.5 cm) and e l u t e d w i t h 0.001 M sodium acetate (pH 5 . 9 ) buff e r c o n t a i n i n g 0.1 M NaC1. The flow rate w a s 2.0 ml/cm2 p e r hour and 3 m l f r a c t i o n s were c o l l e c t e d . An a l i q u o t was t a k e n t o a s s a y f o r angiotensin I a c t i v i t y .
techniques of March e t a1 ( 1 4 ) , e x c e p t t h e c o u p l i n g w a s c a r r i e d o u t
a t pH 7.0 i n s t e a d o f pH 9.4 ( 1 6 ) .
Sepharose 48 beads were washed
e x t e n s i v e l y w i t h d i s t i l l e d water, followed by e q u i l i b r i u m o f t h e beads i n 2 M sodium c a r b o n a t e g i v i n g a volume c o n s i s t i n g o f e q u a l volumes o f packed beads and c a r b o n a t e b u f f e r .
The m i x t u r e , cyanogen
bromide in a c e t o n i t r i l e , (2g cyanogen bromide p e r m l of a c e t o n i t r i l e ) ,
was s t i r r e d v i g o r o u s l y u s i n g approximately 1 / 2 0 of t h e volume of t h e bead c a r b o n a t e s l u r r y .
S t i r r i n g continued for 2 minutes.
The
PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
181
slurry was then transferred to a Buchner funnel where it was washed with 5-10 volumes of 0.1 M sodium bicarbonate and then water.
For
thess experiments 0.2 M sodium citrate pH 7.0 was used at the last washing step and at the subsequent coupling step.
The washed acti-
vated beads were then transferred to a plastic bottle in which 1
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volume of 0 . 2 M sodium citrate pH 7.0 containing the amino-ligand was added. Coupling was performed at '4
for 24 hours during which
time the bottle was gently agitated. After coupling, the beads were washed with 20 volumes each of 0.1 Y sodium acetate, pH 4.0, 0.1 M sodium bicarbonate, pH 9.5, 0.01 M Tris-HC1, pH 6.8.
All three
washes contained 0.3 M KC1. Acrylic plastic columns (Pharmacia Kl5/90, 1.5 x 90 cm) were poured with variable bed volumes of the coupled beads. a l s o used).
(Columns of smaller dimensions, 0.9 x 30 cm, were
Prior to first using a prepared globulin-Sepharose
column, it was washed with 2-3 bed volumes of elution buffer (0.2
M acetic acid, 0.3 M KCl), followed by at least 20 bead volumes of loading buffer (0.01 M Tris-HC1, pH 6.8, 0.3 M KC1).
The globulh-
coupled Sepharose column could be reused several times.
Dog sera
(100 ml) was passed through the anti-renin globulin Sepharose complexing the serum renin with goat XC anti-renin (dog kidney) for 2 hours at room temperature. After applying the dog sera the renin-
globulin Sepharose column was washed with 0.01 M Tris pH 6.8, 0.30
M KC1 until absorption was below 0.01 absorbance units (280 mp) (15).
Renin was eluted from anti-renin globulin-Sepharose with 0.2
M acetic acid pH 2. 8.
Two milliliter volume fractions were col-
lected and monitored for absorbance and pH.
Elution of protein was
WALSH ET AL.
182
completed w i t h 2 bed volumes of b u f f e r (0.2 M a c e t i c a c i d , pH 2 . 8 ) . The p u r i f i e d serum r e n i n w a s l y o p h i l i z e d , and d i a l y z e d a g a i n s t c i t r a t e - p h o s p h a t e b u f f e r pH 4.4. Ouchterlony Double-Diffusion.
For immunodiffusion p l a t e s , 10%
agarose w a s prepared i n 0.05 M Tris HC1, pH 6.8, 0 . 3 M K C 1 , and 0.01%
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thimersol. I
The mixture was h e a t e d t o b o i l i n g and cooled t o about
45OC and then poured (10 m l / P e t r i d i s h ) .
I n c u b a t i o n c o n d i t i o n s were
as t h o s e d e s c r i b e d e a r l i e r (17). Renin A c t i v i t y Assav.
The radioimmunoassay o f Schwarz-Efann
was used except t h a t r e n i n w a s assayed a t pH 6.8 u s i n g a Tris a c e t a t e buffer.
P u r i f i e d r e n i n was assayed by adding an a l i q u o t of t h e
prepared r e n i n t o 1 ad of dog serum and a n a l y z i n g t h e r e n i n a c t i v i t y over and above t h a t p r e s e n t i n t h e normal dog serum.
The a c t i v i t y
of r e n i n i n dog serum of t h e hemorrhaged dog u $ e d . i n t h e s e experiments
was approximately 6 ng a n g i o t e n s i n I / m l plasma, hour-', GUfml.
-4 o r 6.0 x 10
The r e n i n a c t i v i t y of normal dogs sera was between 1 and 2 w
ng of a n g i o t e n s i n I/ml plasma, hour-'. Bioassay of Renin.
Kidney r e n i n was i n f u s e d i n p a r t i a l l y
a n a e s t h e s l z e d dogs f o r one hour a t a n i n c r e a s i n g i n f u s i o n r a t e of
0.005 t o 0 . 1 GU. kg".
dn-'
t o a s s a y t h e p r e s s o r response i n dogs
t o p u r i f i e d renin. RESULTS AND DISCUSSION Renin was p u r i f i e d from dog kidney and chromatographed on Sephadex G-100 (Figure 1). The peak corresponding t o r e n i n a c t i v -
ity having an approximate 42,000 molecular weight a c c o r d i n g t o Sephadex g e l f i l t r a t i o n (Figure 2) was used f o r producing a n t i -
PLASMA RENIN BY AFFINITY C K R O M A T O W H Y
183
99
88 W
77 66
J
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w
55 44
33
't
RENIN' (42,000) I
1
1
\ I
I
1
1
1
1
,
3 4 5 678910 MOLECULAR WEIGHT ( x I O - ~ ) I
2
FIGURE 2 Sephadex g e l f i l t r a t i o n w a s c a r r i e d o u t as d e s c r i b e d i n F i g u r e 1. S t a n d a r d s were chromatographed and p l o t t e d on semi-log p a p e r t o approximate t h e m o l e c u l a r w e i g h t of r e n i n .
renin i n goats.
The p u r i f i e d p r o t e i n was d e f i n e d as r e n i n by as-
s a y i n g a c t i v i t y and q u a n t i t a t i n g a n g i o t e n s i n I p r o d u c t i o n , (peak of h i g h e s t a c t i v i t y , 157 ug a n g i o t e n s i n I/mg p r o t e i n , h o u r
-1
).
Normal dog plasma gave a n immune r e a c t i o n a g a i n s t g o a t a n t i - r e n i n , whereas human plasma gave no r e a c t i o n ( F i g u r e 3 ) .
I n a double d i f -
f u s i o n a s s a y t h e dog plasma gave p r e c i p i t i n bands which f u s e d w i t h p u r i f i e d dog kidney r e n i n when r e a c t e d a g a i n s t g o a t a n t i - r e n i n (Figure 4 ) .
(dog)
The amount o f plasma r e n i n i n c r e a s e d , b a s e d on
a n g i o t e n s i n I p r o d u c t i o n , when t h e dog w a s s e v e r e l y hemorrhaged. When dog plasma was r e a c t e d w i t h g o a t a n t i - r e n i n and t h e a n t i g e n - a n t i b o d y complex removed by c e n t r i f u g a t i o n , t h e r e m a i n i n g plasma had no r e n i n a c t i v i t y .
A d d i t i o n of dog k i d n e y r e n i n re-
s t o r e d t h e plasma r e n i n a c t i v i t y .
WALSH ET A.L.
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184
FIGURE 3 Immuno double diffusion pattern o f (1 and 3 ) dog plasma ( 2 and 4) human plasma. Center well contains antiserum against dog kidney renin (ab). Fifty microliters of serum and antiserum was added to each well.
An affinity column for the purification of canine serum renin was prepared using goat anti-renin (dog kidney) yG globulins. The antiserum was prepared against a purified kidney renin preparation. The anti-renin globulins were coupled to cyanogen bromideactivated Sepharose. Dog plasma was then applied to the immunoadsorbant Sepharose. The eluant containing the antigen fused with dog plasma (Figure 5) and purified canine renal renin (Figure 6 ) in
185
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PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
FIGURE 4 Ouchterlony p a t t e r n of ( 1 and 3) dog plasma (2 and 4) dog kidney r e n i n (25 u l ) , a n d (Ab) goat anti-kidney r e n i n (dog). F i f t y m i c r o l i t e r s of plasma and a n t i s e r u m were added t o r e s p e c t i v e wells.
an Ouchterlony r e a c t i o n incubated w i t h goat anti-(dog)
kidney r e n i n .
The s p e c i f i c a c t i v i t y of t h e e l u t e d plasma r e n i n was 20 ng angiotensin I / m l ,
hour
-1
Imp p r o t e i n beginning w i t h dog plasma a t 0.02
ng a n g i o t e n s i n Ilmg plasma p r o t e i n , hour
-1
.
( I n o t h e r r e p o r t s an
a f f i n i t y column, a s d e s c r i b e d h e r e , w a s used t o p u r i f y i n s u l i n from serum ( 1 8 ) ) .
I f t h e sample of plasma a p p l i e d t o a globulin-coupled
WALSH ET AL.
186
0.9
0.8
1
0.7
c
5 0.6
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W
#m
0.4
0.3 m 4
0.2 0.I
TUBE NUMBER
FIGURE 5 A f f i n i t y chromatography o f c a n i n e plasma r e n i n i n a n t i - r e n i n 25 m l o f serum was added t o t h e column (30 x globulin-Sepharose. 0.9 cm) as d e s c r i b e d in Methods. (Columns o f o t h e r dimensions were used f o r r e n i n p u r i f i c a t i o n u s i n g l a r g e r serum volumes).
Sepharose column was c o m p a r a t i v e l y small (10 m l s serum a p p l i e d t o a 1 . 5 x 90 cm column) v i r t u a l l y a l l o f t h e r e n i n a c t i v i t y c o u l d b e e x t r a c t e d from t h e serum.
P a s s a g e o f 100 mls o f serum t h r o u g h t h e
90 cm column r e s u l t e d in a t least a 30% r e c o v e r y o f r e n i n a c t i v i t y based on r a d i o i m u n o a s s a y r e s u l t s .
I n comparison a k i d n e y e x t r a c t
s o l u t i o n was p r e p a r e d c o n s i s t i n g o f t h e s u p e f n a t a n t from t h e f i r s t a c i d i f i c a t i o n s t e p i n t h e scheme f o r kidney r e n i n p u r i f i c a t i o n ; t h i s was d i l u t e d t o where t h e r e n i n a c t i v i t y approximated t h a t of
187
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PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
FIGURE 6
Double diffusion assay of (1 and 3) dog plasma, and ( 2 and 4 ) antigen from dog serum eluted from an affinity column; all were incubated with goat anti- (dog) kidney renin.
native dog serum ( 2 ng/ml/hr angiotensin I formation).
Under these
conditions, recovery of renin activity from either normal plasma or kidney, using an affinity column, was approximately equal. The technique described here gives a simple purification of serum renin by affinity chromatography such that the specific activity of renin in plasma could be increased 1,000 fold in a one step purification.
WALSH ET AL.
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188
FIGURE 7 Ouchterlony assay of (1 and 3) dog kidney renin (2 and 4 ) antigen from dog plasma eluted from an affinity column and incubated in the double diffusion plate with goat anti-renin (dog kidney).
ABBREVIATIONS EDTA: (ethylenedinitri1o)-tetracetic acid disodium salt DTT: dithiothreitol GU Units: A Goldblatt unit of renin is based on the pressor response of normal intact conscious dogs to the injection of renin (5) Saturated (NH4)*SO4 (Biochemistry Handbook)
PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
189
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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Purification of Canine Plasma Renin by Affinity Chromatography a
a
Robert Walsh , Dean T. Mason , Melvln J. a
Tonkon & Joan Wikman-coffelt
a
a
Section of Cardiovascular Medicine, Department of Medicine , University of California, Davis School of Medicine Published online: 05 Dec 2006.
To cite this article: Robert Walsh , Dean T. Mason , Melvln J. Tonkon & Joan Wikman-coffelt (1976) Purification of Canine Plasma Renin by Affinity Chromatography, Preparative Biochemistry, 6:2-3, 177-191, DOI: 10.1080/00327487608061611 To link to this article: http://dx.doi.org/10.1080/00327487608061611
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PREPARATIVE BIOCHPlISTRY, 6(2&3), 177-191 (1976)
PURIFICATION OF CANINE PLASMA R E N I N BY AFFINITY CHROMATOGRAPHY
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Robert Walsh, Dean T. Mason, Melvin J. Tonkon and Joan Wikman-Coffelt S e c t i o n of Cardiovascular Medicine, Department of Medicine, University of C a l i f o r n i a , Davis School of Medicine
,
ABSTRACT An a f f i n i t y column f o r t h e p u r i f i c a t i o n of canine plasma ren i n was prepared using goat a n t i - r e n i n
(dog kidney) yG g l o b u l i n s .
The antiserum was prepared a g a i n s t a p u r i f i e d kidney r e n i n preparation.
The a n t i - r e n i n g l o b u l i n s were coup1 ed t o cyanogen bromide-
a c t i v a t e d Sepharose.
Using t h e a n t i - r e n i n
globulin-coupled Sepha-
rose as an imuno-adsorbant, a method w a s devised allowing p u r i f i c a t i o n of plasma r e n i n t o a 1,000-fold p u r i t y . INTRODUCTION Renin has been p a r t i a l l y p u r i f i e d from muse s u b p a x i l l a r y glands (11, and kidneys of human (2), hog ( 3 ) . b e e f , r a b b i t ( 4 1 , and dog ( 5 ) .
These procedures i n c l u d e a c i d treatment and (NH4)*
SO4 p r e c i p i t a t i o n , as w e l l as e t h a n o l e x t r a c t i o n ( 5 ) . procedures i n c l u d e Sephadex-G-100
Other
f i l t r a t i o n ( l ) , and a f f i n i t y
chromatography where analogs of t h e r e n i n s u b s t r a t e were coupled t o agarose ( 6 ) .
Radioimmunoassays have been developed t o quan-
t i t a t e sera r e n i n (7) as w e l l as a n g i o t e n s i n I (8,9,10,11,12),
a
177 Copyright 0 1976 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying. microfilmmg, and recording, or by any information storage and retrieval system, without permission in writing from the publisher
WALSH ET AL.
178 product of renin.
This study presents the first purification of
renin directly from the serum using affinity chromatography where renin was assayed by quantitating production of angiotensin I using a radioimmunoassay. MATERIALS AND METHODS
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Kidney Renin Purification.
For purification of canine kidney
renin a modified procedure of Haas et a1 (5) was used.
Six to
eight kidoeys were homogenized in 0.05 M potassium phosphate, pH
7.0, 0.001 M EDTA, 0.001 M DTT at '4 using a Naring blender. volume was brought to 18 d / g m of tissue with water. was stirred for 15 min at
The
The homogenate
centrifuged at 9,000 x g (10 mfn),
4O,
and the supernatant collected.
The pellet was resuspended in the
starting buffer, rehomogenized and the supernatants pooled.
The
superpatant was brought to pH 2.5 with 5N H2S04 and stirred €or 10 min.
The pH was adjusted to 6.5 with 5N KOH and centrifuged.
The
pellet was reextracted and the combined supernatants were brought to pH 4.5 with 5 N H2S04.
Solid (NH4)2 SO4 was added, bringing the
supernatant to 70% ( N H 4 ) *
SO4 saturation.
without stirring (4')
After 2 hours of settling
a pellet containing renin was obtained by
centrifugation at 10,000 x g (10 min).
The partially purified
renin was resuspended in 0.001 M DTT, 0.001 M EDTA and H 2 0 (1 ml/20gm of original kidney weight).
The partially purified renin was dialyzed
in 0.001 M DTT, 0.001 M EDTA and H 2 0 for 16 hours ( 4 ' ) . According to the methods of Haas, et a1 (131, the pH of the solution was brought to 7.5 with. 5N KOH.
After centrifugation the
PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
179
volume was brought to 15 m1/100gm of original kidney weight, and
4 ' ) . acidified to pH 2.8 with 1N H2S04 and stirred 10 min (
The
supernatant was acidified to pH 2.8 with 1N H2S04 and stirred 10 min. After centrifuging at 20,000 x g (10 min), sufficient ethanol was added to the supernatant to make a final concentration of 10%
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ethanol and stored for 2 hours without stirring (0').
The pH of
the ethanol solution was adjusted to 4.5 with 1N NaOH and solid (NH4)2 SO4 was added to bring the supernatant to 70% (NH4)2 SO4 saturation. After stirring for 10 min and centrifuging the precipitate was resuspended in H20 (1.8 m l / l O O g m of starting kidney weight) and dialyzed overnight against 0.05 M citrate, pH 4.6 and 0.05 M Na C1. The supernatant was applied
to
a Sephadex G-100
column (1.5 x 90 cm) and the activity peak collected, lyophilized; and dialyzed against 0.05 M citrate, pH 4.6 and 0.05 M Na C1.
The
purified renin gave approximately 3.2 GU/mg protein. Preparation of Antiserum. Antiserum was produced in goats against canine kidney renin by using the Sephadex G-100 peak renin (Figure 1) which contained the renin activity.
One half milliliter
of solution (kidney renin) containing 2 mg of protein was mixed with an equal volume of Freund's Adjuvant and injected into the axilla of a goat. The first 2 injections were 14 days apart, followed by 3 injections, one every 7 days. After 5 injections the goats were bled.
The g a m globulins were purified from the
4 fractionation of the goat antiserum. Purification of Renin from Canine Serum. The goat g a m a G
antiserum by a 35% (NH4I2
SO
anti-renin (kidney) was complexed to an affinity column using the
WALSH ET AL.
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180
TUBE NUMBER
FIGURE 1 Gel f i l t r a t i o n of dog kidney r e n i n . Sample volume of 4 m l c o n t a i n i n g 60 mg p r o t e i n was a p p l i e d t o a Sephadex G 1 0 0 column (90 x 1.5 cm) and e l u t e d w i t h 0.001 M sodium acetate (pH 5 . 9 ) buff e r c o n t a i n i n g 0.1 M NaC1. The flow rate w a s 2.0 ml/cm2 p e r hour and 3 m l f r a c t i o n s were c o l l e c t e d . An a l i q u o t was t a k e n t o a s s a y f o r angiotensin I a c t i v i t y .
techniques of March e t a1 ( 1 4 ) , e x c e p t t h e c o u p l i n g w a s c a r r i e d o u t
a t pH 7.0 i n s t e a d o f pH 9.4 ( 1 6 ) .
Sepharose 48 beads were washed
e x t e n s i v e l y w i t h d i s t i l l e d water, followed by e q u i l i b r i u m o f t h e beads i n 2 M sodium c a r b o n a t e g i v i n g a volume c o n s i s t i n g o f e q u a l volumes o f packed beads and c a r b o n a t e b u f f e r .
The m i x t u r e , cyanogen
bromide in a c e t o n i t r i l e , (2g cyanogen bromide p e r m l of a c e t o n i t r i l e ) ,
was s t i r r e d v i g o r o u s l y u s i n g approximately 1 / 2 0 of t h e volume of t h e bead c a r b o n a t e s l u r r y .
S t i r r i n g continued for 2 minutes.
The
PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
181
slurry was then transferred to a Buchner funnel where it was washed with 5-10 volumes of 0.1 M sodium bicarbonate and then water.
For
thess experiments 0.2 M sodium citrate pH 7.0 was used at the last washing step and at the subsequent coupling step.
The washed acti-
vated beads were then transferred to a plastic bottle in which 1
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volume of 0 . 2 M sodium citrate pH 7.0 containing the amino-ligand was added. Coupling was performed at '4
for 24 hours during which
time the bottle was gently agitated. After coupling, the beads were washed with 20 volumes each of 0.1 Y sodium acetate, pH 4.0, 0.1 M sodium bicarbonate, pH 9.5, 0.01 M Tris-HC1, pH 6.8.
All three
washes contained 0.3 M KC1. Acrylic plastic columns (Pharmacia Kl5/90, 1.5 x 90 cm) were poured with variable bed volumes of the coupled beads. a l s o used).
(Columns of smaller dimensions, 0.9 x 30 cm, were
Prior to first using a prepared globulin-Sepharose
column, it was washed with 2-3 bed volumes of elution buffer (0.2
M acetic acid, 0.3 M KCl), followed by at least 20 bead volumes of loading buffer (0.01 M Tris-HC1, pH 6.8, 0.3 M KC1).
The globulh-
coupled Sepharose column could be reused several times.
Dog sera
(100 ml) was passed through the anti-renin globulin Sepharose complexing the serum renin with goat XC anti-renin (dog kidney) for 2 hours at room temperature. After applying the dog sera the renin-
globulin Sepharose column was washed with 0.01 M Tris pH 6.8, 0.30
M KC1 until absorption was below 0.01 absorbance units (280 mp) (15).
Renin was eluted from anti-renin globulin-Sepharose with 0.2
M acetic acid pH 2. 8.
Two milliliter volume fractions were col-
lected and monitored for absorbance and pH.
Elution of protein was
WALSH ET AL.
182
completed w i t h 2 bed volumes of b u f f e r (0.2 M a c e t i c a c i d , pH 2 . 8 ) . The p u r i f i e d serum r e n i n w a s l y o p h i l i z e d , and d i a l y z e d a g a i n s t c i t r a t e - p h o s p h a t e b u f f e r pH 4.4. Ouchterlony Double-Diffusion.
For immunodiffusion p l a t e s , 10%
agarose w a s prepared i n 0.05 M Tris HC1, pH 6.8, 0 . 3 M K C 1 , and 0.01%
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thimersol. I
The mixture was h e a t e d t o b o i l i n g and cooled t o about
45OC and then poured (10 m l / P e t r i d i s h ) .
I n c u b a t i o n c o n d i t i o n s were
as t h o s e d e s c r i b e d e a r l i e r (17). Renin A c t i v i t y Assav.
The radioimmunoassay o f Schwarz-Efann
was used except t h a t r e n i n w a s assayed a t pH 6.8 u s i n g a Tris a c e t a t e buffer.
P u r i f i e d r e n i n was assayed by adding an a l i q u o t of t h e
prepared r e n i n t o 1 ad of dog serum and a n a l y z i n g t h e r e n i n a c t i v i t y over and above t h a t p r e s e n t i n t h e normal dog serum.
The a c t i v i t y
of r e n i n i n dog serum of t h e hemorrhaged dog u $ e d . i n t h e s e experiments
was approximately 6 ng a n g i o t e n s i n I / m l plasma, hour-', GUfml.
-4 o r 6.0 x 10
The r e n i n a c t i v i t y of normal dogs sera was between 1 and 2 w
ng of a n g i o t e n s i n I/ml plasma, hour-'. Bioassay of Renin.
Kidney r e n i n was i n f u s e d i n p a r t i a l l y
a n a e s t h e s l z e d dogs f o r one hour a t a n i n c r e a s i n g i n f u s i o n r a t e of
0.005 t o 0 . 1 GU. kg".
dn-'
t o a s s a y t h e p r e s s o r response i n dogs
t o p u r i f i e d renin. RESULTS AND DISCUSSION Renin was p u r i f i e d from dog kidney and chromatographed on Sephadex G-100 (Figure 1). The peak corresponding t o r e n i n a c t i v -
ity having an approximate 42,000 molecular weight a c c o r d i n g t o Sephadex g e l f i l t r a t i o n (Figure 2) was used f o r producing a n t i -
PLASMA RENIN BY AFFINITY C K R O M A T O W H Y
183
99
88 W
77 66
J
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w
55 44
33
't
RENIN' (42,000) I
1
1
\ I
I
1
1
1
1
,
3 4 5 678910 MOLECULAR WEIGHT ( x I O - ~ ) I
2
FIGURE 2 Sephadex g e l f i l t r a t i o n w a s c a r r i e d o u t as d e s c r i b e d i n F i g u r e 1. S t a n d a r d s were chromatographed and p l o t t e d on semi-log p a p e r t o approximate t h e m o l e c u l a r w e i g h t of r e n i n .
renin i n goats.
The p u r i f i e d p r o t e i n was d e f i n e d as r e n i n by as-
s a y i n g a c t i v i t y and q u a n t i t a t i n g a n g i o t e n s i n I p r o d u c t i o n , (peak of h i g h e s t a c t i v i t y , 157 ug a n g i o t e n s i n I/mg p r o t e i n , h o u r
-1
).
Normal dog plasma gave a n immune r e a c t i o n a g a i n s t g o a t a n t i - r e n i n , whereas human plasma gave no r e a c t i o n ( F i g u r e 3 ) .
I n a double d i f -
f u s i o n a s s a y t h e dog plasma gave p r e c i p i t i n bands which f u s e d w i t h p u r i f i e d dog kidney r e n i n when r e a c t e d a g a i n s t g o a t a n t i - r e n i n (Figure 4 ) .
(dog)
The amount o f plasma r e n i n i n c r e a s e d , b a s e d on
a n g i o t e n s i n I p r o d u c t i o n , when t h e dog w a s s e v e r e l y hemorrhaged. When dog plasma was r e a c t e d w i t h g o a t a n t i - r e n i n and t h e a n t i g e n - a n t i b o d y complex removed by c e n t r i f u g a t i o n , t h e r e m a i n i n g plasma had no r e n i n a c t i v i t y .
A d d i t i o n of dog k i d n e y r e n i n re-
s t o r e d t h e plasma r e n i n a c t i v i t y .
WALSH ET A.L.
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184
FIGURE 3 Immuno double diffusion pattern o f (1 and 3 ) dog plasma ( 2 and 4) human plasma. Center well contains antiserum against dog kidney renin (ab). Fifty microliters of serum and antiserum was added to each well.
An affinity column for the purification of canine serum renin was prepared using goat anti-renin (dog kidney) yG globulins. The antiserum was prepared against a purified kidney renin preparation. The anti-renin globulins were coupled to cyanogen bromideactivated Sepharose. Dog plasma was then applied to the immunoadsorbant Sepharose. The eluant containing the antigen fused with dog plasma (Figure 5) and purified canine renal renin (Figure 6 ) in
185
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PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
FIGURE 4 Ouchterlony p a t t e r n of ( 1 and 3) dog plasma (2 and 4) dog kidney r e n i n (25 u l ) , a n d (Ab) goat anti-kidney r e n i n (dog). F i f t y m i c r o l i t e r s of plasma and a n t i s e r u m were added t o r e s p e c t i v e wells.
an Ouchterlony r e a c t i o n incubated w i t h goat anti-(dog)
kidney r e n i n .
The s p e c i f i c a c t i v i t y of t h e e l u t e d plasma r e n i n was 20 ng angiotensin I / m l ,
hour
-1
Imp p r o t e i n beginning w i t h dog plasma a t 0.02
ng a n g i o t e n s i n Ilmg plasma p r o t e i n , hour
-1
.
( I n o t h e r r e p o r t s an
a f f i n i t y column, a s d e s c r i b e d h e r e , w a s used t o p u r i f y i n s u l i n from serum ( 1 8 ) ) .
I f t h e sample of plasma a p p l i e d t o a globulin-coupled
WALSH ET AL.
186
0.9
0.8
1
0.7
c
5 0.6
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W
#m
0.4
0.3 m 4
0.2 0.I
TUBE NUMBER
FIGURE 5 A f f i n i t y chromatography o f c a n i n e plasma r e n i n i n a n t i - r e n i n 25 m l o f serum was added t o t h e column (30 x globulin-Sepharose. 0.9 cm) as d e s c r i b e d in Methods. (Columns o f o t h e r dimensions were used f o r r e n i n p u r i f i c a t i o n u s i n g l a r g e r serum volumes).
Sepharose column was c o m p a r a t i v e l y small (10 m l s serum a p p l i e d t o a 1 . 5 x 90 cm column) v i r t u a l l y a l l o f t h e r e n i n a c t i v i t y c o u l d b e e x t r a c t e d from t h e serum.
P a s s a g e o f 100 mls o f serum t h r o u g h t h e
90 cm column r e s u l t e d in a t least a 30% r e c o v e r y o f r e n i n a c t i v i t y based on r a d i o i m u n o a s s a y r e s u l t s .
I n comparison a k i d n e y e x t r a c t
s o l u t i o n was p r e p a r e d c o n s i s t i n g o f t h e s u p e f n a t a n t from t h e f i r s t a c i d i f i c a t i o n s t e p i n t h e scheme f o r kidney r e n i n p u r i f i c a t i o n ; t h i s was d i l u t e d t o where t h e r e n i n a c t i v i t y approximated t h a t of
187
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PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
FIGURE 6
Double diffusion assay of (1 and 3) dog plasma, and ( 2 and 4 ) antigen from dog serum eluted from an affinity column; all were incubated with goat anti- (dog) kidney renin.
native dog serum ( 2 ng/ml/hr angiotensin I formation).
Under these
conditions, recovery of renin activity from either normal plasma or kidney, using an affinity column, was approximately equal. The technique described here gives a simple purification of serum renin by affinity chromatography such that the specific activity of renin in plasma could be increased 1,000 fold in a one step purification.
WALSH ET AL.
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188
FIGURE 7 Ouchterlony assay of (1 and 3) dog kidney renin (2 and 4 ) antigen from dog plasma eluted from an affinity column and incubated in the double diffusion plate with goat anti-renin (dog kidney).
ABBREVIATIONS EDTA: (ethylenedinitri1o)-tetracetic acid disodium salt DTT: dithiothreitol GU Units: A Goldblatt unit of renin is based on the pressor response of normal intact conscious dogs to the injection of renin (5) Saturated (NH4)*SO4 (Biochemistry Handbook)
PLASMA RENIN BY AFFINITY CHROMATOGRAPHY
189
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