Preparative Biochemistry
ISSN: 0032-7484 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/lpbb19
Purification of Cathepsin D by Ah-Sepharose Affinity Chromatography A. Linde & B. Persliden To cite this article: A. Linde & B. Persliden (1978) Purification of Cathepsin D by AhSepharose Affinity Chromatography, Preparative Biochemistry, 8:4, 231-240, DOI: 10.1080/00327487808065523 To link to this article: http://dx.doi.org/10.1080/00327487808065523
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PREPARATIVE BIOCHEMISTRY, 8(4), 231-240 (1978)
PURIFICATION OF CATHEPSIN D BY AH-SEPHAROSE AFFINITY CHROMATOGRAPHY A. L i n d e and B. P e r s l i d e n
L a b o r a t o r y o f O r a l B i o l o g y , Department o f H i s t o l o g y , U n i v e r s i t y o f Goteborg, Fack, S-400 33, GUTEBORG, Sweden ABSTRACT A r a p i d and r e l i a b l e method f o r c o u p l i n g t h e p r o t e a s e i n -
h i b i t o r p e p s t a t i n t o AH-Sepharose 48 was developed. The m a t r i x prepared was used t o p u r i f y c a t h e p s i n D from r a t l i v e r . The enzyme was e l u t e d i n one f r a c t i o n and proved t o be p u r e b y g e l e l e c t r o p h o r e s i s , two t y p e s of i o n exchange chromatography, m o l e c u l a r s i e v e chromatography, and i m m u n o l o g i c a l l y homogenous b y immunoelectrophoresis. T h i s method i s more r a p i d and g i v e s a h i g h e r y i e l d t b a n p r e v i o u s techniques. The p o s s i b i l i t y t o use t h i s t e c h n i q u e f o r t h e p u r i f i c a t i o n o f o t h e r enzymes i n h i b i t a b l e by p e p s t a t i n s h o u l d be considered. INTRODUCTION The lysosomal p r o t e a s e c a t h e p s i n 0 (EC 3.4.23.5)
has drawn
c o n s i d e r a b l e a t t e n t i o n due t o i t s p o s s i b l e r o l e i n t h e degrad a t i o n o f p r o t e o g l y c a n s i n v a r i o u s t i s s u e s . Thus, t h e enzyme has been c o n s i d e r e d t o be t h e f a c t o r r e s p o n s i b l e f o r t h i s p r o c e s s
231 Copyright 0 1978 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, microfilming, and recording, or by any information storage and retriwal system, without permission in writing from the publisher.
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232
i n c a r t i l a g e ( 4 ) and a t t h e m i n e r a l i z a t i o n f r o n t i n b i o l o g i c a l c a l c i f i c a t i o n (8). I n o u r s t u d i e s o f b i o l o g i c a l c a l c i f i c a t i o n c a t h e p s i n D was p u r i f i e d from r a t l i v e r b y a method m o d i f i e d a f t e r
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B a r r e t t ( 1 ) and used t o produce s p e c i e s - s p e c i f i c a n t i b o d i e s f o r immunohistochemical l o c a l i z a t i o n o f t h e enzyme ( 9 ) . T h i s p r e p a r a t i o n method f o r c a t h e p s i n D i s r a t h e r l a b o r i o u s and g i v e s low enzyme y i e l d s . O c c a s i o n a l l y , t h e c a t h e p s i n D prepared w i t h i s o e l e c t r i c f o c u s i n g as l a s t s t e p was impure, g i v i n g two p r e c i p i t a t i o n l i n e s when t e s t e d by immunoelectrophoresis a g a i n s t a n t i b o d i e s o b t a i n e d from t h e same preparation. T h i s communication p r e s e n t s a t e c h n i q u e by which t h e a c i d p r o t e a s e i n h i b i t o r p e p s t a t i n i s bound t o an agarose m a t r i x f o r a f f i n i t y chromatography. By t h i s method c a t h e p s i n D has been r a p i d l y p u r i f i e d w i t h a high y i e l d . The method w i l l be o f v a l u e f o r t h e p u r i f i c a t i o n of o t h e r enzymes i n h i b i t a b l e b y p e p s t a t i n , e.g.
p e p s i n and r e n i n .
EXPERIMENTAL Chemi ca 1s AH-Sepharose 48, Sephacryl S-200 S u p e r f i n e and Sephadex 6-100 were purchased from Pharmacia F i n e Chemicals, Uppsala, Sweden. DEAE-cellulose (Whatman DE52) and CM-cellulose (Whatman CM52) were o b t a i n e d from W . & B a l s t o n e ( M o d i f i e d C e l l u l o s e ) Ltd.,
S p r i n g f i e l d M i l l , Maidstone, Kent,
England. C e l i t e was from Kebo AB, Stockholm, Sweden. The p r o t e a s e i n h i b i t o r p e p s t a t i n (5, 7, 13, 14), a p r o d u c t f r o m Streptomyces
urgenteoZus v a r . toyonakensis was k i n d l y s u p p l i e d b y D r . H. Umezawa, I n s t . o f M i c r o b i a l Chemistry, Tokyo, Japan. A l l o t h e r reagents were of ana l y t i c a l grade.
R
PURIFICATION OF CATHEPSIN D
233
Tissue Preparation
A crude enzyme p r e p a r a t i o n was prepared t r o m r a t l i v e r ( 9 ) . Pooled l i v e r , 2.5 kg, was homogenized i n 5.0 L 1% NaCl c o n t a i n i n g 2% ( b y v o l . )
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n-butanol i n a S o r v a l l m n i - m i x e r . A t t e r d i s s o l v i n g 110 g CaCI2 i n t h e t o t a l homogenate, 750 m l 1 M Na2HP04
. 12 H20 were added.
of pH t o n e u t r a l i t y w i t h 2 M t r i s - H C 1 buffer,
A t t e r adjustment
pH 9 . 0 , t h e suspension was
c l e a r e d by c e n t r i f u g a t i o n . The s u p e r n a t a n t was a d j u s t e d t o p n 3.6 b y a
5 M f o r m i c a c i d b u f f e r , pH 3.0,
and i n c u b a t e d o v e r n i g h t a t 37OC f o r auto-
l y s i s . A f t e r c o o l i n g t o 2OC, 0.9 volumes o f -12OC acetone were added. The p r e c i p i t a t e was removed by c e n t r i f u g a t i o n and t h e same amount of acetone was added t o t h e supernatant. A f t e r t h e a d d i t i o n o f C e l i t e ( 2 g / l ) t h e p r e c i p i t a t e was c o l l e c t e d by f i l t r a t i o n and d i a l y z e d f o r 24 h a g a i n s t
50 mM EUTA i n 50 mM t r i s - H C 1 b u f f e r , pH 8.0.
F u r t h e r d i a l y s i s was per-
formed a g a i n s t 2 mM t r i s - H C 1 b u f f e r ,
u n t i l t h e pH i n t h e d i a l y s i s
pH 9.0,
n
bag was above 7.5 and t h e c o n d u c t i v i t y below 1.0 mmho/cmL. The C e l i t e was f i n a l l y removed by f i l t r a t i o n . The p r e p a r a t i o n o b t a i n e d was termed c r u d e c a t h e p s i n D and was f u r t h e r p u r i f i e d on t h e a f f i n i t y columns. P r i o r t o use, t h e m a t e r i a l was e x t e n s i v e l y d i a l y z e d a g a i n s t 0.1 M c i t r a t e - H C 1 b u f f e r , pH 3.0,
c o n t a i n i n g 0.5 M NaCI.
A f f i n i t y Matrix Preparation Two l i t r e s o f 0.5 M NaCl were added i n a l i q u o t s o f 200 m l t o 10 g o f AH-Sepharose 48 f o r washing and r e s w e l l i n g . P e p s t a t i n , 50 mg, was d i s s o l v e d i n 250 m l 50% e t h a n o l and added t o t h e AH-Sepharose t o g e t h e r w i t h 0.4 g o f l-ethyl-3-(3-dimethyl-amino-propyl) c a r b o d i i m i d e h y d r o c h l o r i d e (EDC).
The pH was a d j u s t e d t o 5.5 w i t h 0.05 M NaOH. The
m i x t u r e was i n c u b a t e d on a shaking w a t e r b a t h f o r 48 h a t 37OC. A f t e r c o u p l i n g , b l o c k i n g of excess amino groups was pertormed b y i n c u b a t i n g
234
LINDE AND PERSLIDEN
the m a t r i x i n 1 M a c e t i c a c i d f o r 4 h. A f t e r blocking, the AH-Sepharose was washed w i t h 500 m l p o r t i o n s of 50%, 402, 30%, 20% and 10% ethanol and f i n a l l y washed w i t h t h e e q u i l i b r a t i o n b u f f e r , 0.1
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b u f f e r , pH 3.0,
containing 0.5
M citrate-HL1
M NaC1.
I n p r e l i m i n a r y experiments, various parameters were studied: the r e a c t i o n time f o r coupling p e p s t a t i n t o AH-Sepharose 46, temperature dependence of the r e a c t i o n and the e f f e c t s o f using d i f f e r e n t concentrations o f pepstatin, EDC and ethanol i n t h e i n c u b a t i o n m i x t u r e . The method described was, however, found t o be optimal. A f f in i t y Chromatography
The crude cathepsin D preparation was a p p l i e d t o a column of the AH-Sepharose coupled p e p s t a t i n . A f t e r extensive washing w i t h t h e e q u i l i b r a t i o n b u f f e r , the enzyme was e l u t e d i n one homogenous peak w i t h 0.1
M NaHC03, pH 7.0, c o n t a i n i n g 0.5 M NaCl (Figure 1 ) . F r a c t i o n s were c o l l e c t e d and assayed f o r p r o t e i n by absorhance a t
280 nm. Cathepsin D a c t i v i t y i n 25 111 samples from each f r a c t i o n was assayed ( 9 ) . The sample was d i l u t e d t o 200 111 w i t h 0.1 pH 3.0,
M Na-citrate buffer
and 300 111 2% a c i d denatured haemoglobin i n the same b u f f e r
were added as substrate. A f t e r i n c u b a t i o n f o r I h a t 37OC and t e r m i n a t i n d the r e a c t i o n by adding 1 m l 5.2% t r i c h l o r o a c e t i c acid, t h e p r e c i p i t a t e was discarded and the supernatant was assayed a t 280 nm. The y i e l d from a r e p r e s e n t a t i v e p u r i f i c a t i o n i s given i n Table I . There i s no simple method f g r r o u t i n e l y assaying p e p s t a t i n
,
q u a n t i t a t i v e l y , so i n order t o c a l c u l a t e the capacity o f t h e p e p s t a t i n Sepharose m a t r i x , 40 m l o f the crude enzyme p r e p a r a t i o n were a p p l i e d
PURIFICATION OF CATHEPSIN D
0
235
1.0.
1
a)
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cu
a
0.5.
A
'I
B
I
elution volume(m1) FIGURE 1 A f f i n i t y chromatography on AH-Sepharose 48. 135 n i l o f a crude enzyme p r e p a r a t i o n were a p p l i e d t o a column (1.6 x 22 cm) o f AH-Sepharose 48 coupled w i t h p e p s t a t i n , p r e v i o u s l y e q u i l i b r a t e d w i t h 0.1 M citrate-HC1 buffer, pH 3.0, c o n t a i n i n g 0.5 M NaCI. A f t e r extensive washing with the same b u f f e r (A), t h e enzyme was e l u t e d ( 6 ) w i t h 0.1 M NaHC03 b u f f e r , pH 7.0, c o n t a i n i n g 0.5 M NaC1. E l u t i o n r a t e 5 ml/h. F r a c t i o n volume, 5 m l . P r o t e i n () and cathepsin D enzyme a c t i v i t y ( - - - - ) were measured a t 280 nm (see t e x t ) .
TABLE I Cathepsin U y i e l d a f t e r p u r i f i c a t i o n w i t h AH-Sepharose 48 a f f i n i t y chromatography. The f i g u r e s are based on p u r i f i c a t i o n s t a r t i n g w i t h 2.5 kg r a t l i v e r . The t o t a l p r o t e i n ( 9 ) was assayed by the method of Lowry e t aZ. (10) using albumin as a standard. The t o t a l cathepsin D a c t i v i t y i s expreased as amount l i b e r a t e d p r o t e i n ( 9 ) d u r i n g 1 h i n c u b a t i o n a t 37 C w i t h haemoglobin as s u b s t r a t e and measured by the Lowry method (9). The s p e c i f i c a c t i v i t y describes the t o t a l enzyme a c t i v i t y p e r t o t a l p r o t e i n . The enzyme a c t i v i t y y i e l d ( % ) r e l a t e s the remaining enzyme a c t i v i t y a f t e r p u r i f i c a t i o n t o t h e t o t a l a c t i v i t y i n t h e hanogenate. Protein (9) Homogenat e AH-Sepharose 48
197 0.020
Activity (9) 44.0
5.4
Spec.act. 0.22 270
Y i e l d (%)
i00 12
LINDE AND PERSLIDEN
2 36
t o small columns (0.9 x 3.5 an). A f t e r 25 m l o f sample a p p l i c a t i o n , cathepsin D a c t i v i t y appeared i n t h e e l u a t e d u r i n g l o a d i n g of t h e columns. A f t e r e l u t i o n o f the enzyme w i t h 0.1 M NaHC03 and q u a n t i t a t i o n
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according t o Lowry e t
UZ. ( l o ) ,
the amount of enzyme bound t o t h e
pepstatin-Sepharose 48 m a t r i x was c a l c u l a t e d t o be i 6 0
-
20u pg pure
PO
lyacrylamide
enzyme/ml swollen g e l . P u r i t y Assay The enzyme p r e p a r a t i o n revealed o n l y one band on
d i s c e l e c t r o p h o r e s i s ( F i g u r e 2). The 7% g e l s w i t h an upper 3% g e l were r u n a t a pH o f 8.3 f o r 2 h and a t 3 mA per g e l . l o n exchange chromatography on UEAt-cellulose columns, developed w i t h a 0
-
200 mM
continuous NaCl g r a d i e n t ( 9 ) , showed the presence o t o n l y one peak appearing a t a 55 mM NaCl concentration. S i m i l a r l y , a CM-cellulose column
e uted w i t h 250 mM acetate b u f f e r ( 9 ) d i s c l o s e d a s i n g l e peak.
Molecular sieve chromatography on a Sephacryl S-200 Superfine ( 1 . 6 x 76 cm) column revealed one s i n g l e p r o t e i n c o n t a i n i n g peak
when e l u t e d w i t h PBS b u f f e r , pH 7.2, a t 5 ml/h. On the basis o t molecular sieve chromatography on Sephadex G-100 (91 t h e apparent molecular weight was c a l c u l a t e d as 43,000.
Albumin, ovalbumin,
a-chymotrypsi nogen and cytochrome C were used as reference substances. I n a l l column f r a c t i o n s t h e o p t i c a l d e n s i t y a t 280 nm and cathepsin D a c t i v i t y were determined. Rabbit antiserum a g a i n s t the cathepsin U p r e p a r a t i o n was produced and the IgG f r a c t i o n p u r i f i e d as described ( Y ) .
The p u r i t y o f t h e
cathepsin D was t e s t e d a g a i n s t t h i s IgG by means o f immunoelectrophoresi ( 2 ) . Only one p r e c i p i t a t i o n l i n e c o u l d be seen ( f i g u r e 2).
237
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PURIFICATION OF CATHEPSIN D
FIGURE Z ( a j AH-Sepharose-purified cathepsin D was r u n on 7% polyacrylamide gels a t a pH o f 8.3 f o r 2 h a t 3 mA p e r g e l . ( b ) AH-Sepharose-purified cathepsin 0 (10 pg)was r u n e l e c t r o p h o r e t i c a l l y f o r 2 h a t a v o l t a g e o f 8 V/cm on a 1% agarose p l a t e . The a n t i g e n was allowed t o p r e c i p i t a t e a g a i n s t r a b b i t anti-cathepsin D IgG f o r 24 h a t 2OC, The p i c t u r e was taken i n a P o l a r o i d MP-3 Land dark f i e l d equipment w i t h P o l a r o i d 200 speed 42 f i Im. A: cathepsin D, B: r a b b i t a n t i - c a t h e p s i n D lgG, C: r a b b i t a n t i y a t h e p s i n D IgG a i l u t e d 1 : l w i t h PBS buffer, pH 7.7.
DISCUSS ION The present comnunication describes a r a p i d and r e l i a b l e standard technique f o r the p u r i f i c a t i o n o f cathepsin D. The r e l a t i v e u t i l i t y f o r p r e p a r a t i o n o f o t h e r enzymes would depend on t h e i r a f f i n i t y for pepstatin. The cathepsin D p u r i f i e d by AH-Sepharose 4B coupled p e p s t a t i n a f f i n i t y chromatography was pure by g e l electrophoresis,
two types
LINDE AM) PERSLIDEN
238
o f i o n exchange chromatograpny, molecuiar s i e v e chromatography and
immunologically homogenous by immunoelectrophoresi s. The apparent molecular weight obtained f o r the cathepsin D, 43,000,
i s the same
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as t h a t e a r l i e r r e p o r t e d f o r t h e r a t l i v e r enzyme (Y), and i s s i m i l a r t o preparations o f cathepsin D from o t h e r t i s s u e s (1, 12, 15). A considerably h i g h e r enzyme y i e l d was obtained compared w i t h conventional methods (1, 9): Coupling w i t h higher. amounts o f p e p s t a t i n , h i g h e r concentrations o f ethanol, longer i n c u b a t i o n times f o r c o u p l i n g and f u r t h e r a d d i t i o n of EUC during the i n c u b a t i o n p e r i o d d i d n o t improve t h e f i n a l product. The use o t methanol i n s t e a d of ethanol t o d i s s o l v e the p e p s t a t i n was n o t successful. Epoxy-activated Sepharose 68, i n s t e a d o f AH-Sepharose
48, was found t o be more e l a b o r a t e t o handle, and t h e amount o f p e p s t a t i n bound t o t h e m a t r i x was found t o be lower than w i t h t h e procedure described. The use of a f f i n i t y chromatography f o r t h e p u r i f i c a t i o n o f cathepsin
D has been r e p o r t e d e a r l i e r
by Kazakova and Orekhovich ( 6 ) . S i m i l a r l y
r e n i n was p u r i f i e d by p e p s t a t i n a f f i n i t y chromatography ( 3 j . These authors, however, used a d i f f e r e n t and laborious- coupling system based on the SOiutiOn of p e p s t a t i n i n dimethylformamide. This system was n o t sucessful i n our hands. P e p s t a t i n has a l s o been coupled t o aminoethylated polyacrylamide g e l f o r r e n i n p u r i f i c a t i o n (11 j . By a d i f f e r e n t p r i n c i p i e , Smith and Turk (12) p u r i f i e d cathepsin
D on haemoglobin-agarose r e n i n
followed by molecular sieve chromatography. The present technique has proven t o be easy t o handle and very reproducible. This method i s considerably l e s s laborious than most
PURIFICATION OF CATHEPSIN D
239
p r e v i o u s techniques. I t i s a l s o o u r f i r m c o n v i c t i o n t h a t t h e p u r i t y o f c a t h e p s i n D prepared b y new methods must be c a r e f u l l y assessed by
t h e combined use o f d i f f e r e n t s e p a r a t i o n p r i n c i p l e s . Seemingly p u r e
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c a t h e p s i n D p r e p a r a t i o n s may e.g. be r e v e a l e d as i m m u n o l o g i c a i l y non-homogenous ( Y ) . ACKNOWLEDGEMENTS The t e c h n i c a l a s s i s t a n c e o f Mrs Agneta Hoglund and Mrs Ann-Katrin Rhodin i s h i g h l y a p p r e c i a t e d . The i n v e s t i g a t i o n was supported by t h e Swedish Medical Research C o u n c i l and t h e F a c u l t y o f Odontology, U n i v e r s i t y of Goteborg. REFERENCES 1.
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ISSN: 0032-7484 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/lpbb19
Purification of Cathepsin D by Ah-Sepharose Affinity Chromatography A. Linde & B. Persliden To cite this article: A. Linde & B. Persliden (1978) Purification of Cathepsin D by AhSepharose Affinity Chromatography, Preparative Biochemistry, 8:4, 231-240, DOI: 10.1080/00327487808065523 To link to this article: http://dx.doi.org/10.1080/00327487808065523
Published online: 05 Dec 2006.
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PREPARATIVE BIOCHEMISTRY, 8(4), 231-240 (1978)
PURIFICATION OF CATHEPSIN D BY AH-SEPHAROSE AFFINITY CHROMATOGRAPHY A. L i n d e and B. P e r s l i d e n
L a b o r a t o r y o f O r a l B i o l o g y , Department o f H i s t o l o g y , U n i v e r s i t y o f Goteborg, Fack, S-400 33, GUTEBORG, Sweden ABSTRACT A r a p i d and r e l i a b l e method f o r c o u p l i n g t h e p r o t e a s e i n -
h i b i t o r p e p s t a t i n t o AH-Sepharose 48 was developed. The m a t r i x prepared was used t o p u r i f y c a t h e p s i n D from r a t l i v e r . The enzyme was e l u t e d i n one f r a c t i o n and proved t o be p u r e b y g e l e l e c t r o p h o r e s i s , two t y p e s of i o n exchange chromatography, m o l e c u l a r s i e v e chromatography, and i m m u n o l o g i c a l l y homogenous b y immunoelectrophoresis. T h i s method i s more r a p i d and g i v e s a h i g h e r y i e l d t b a n p r e v i o u s techniques. The p o s s i b i l i t y t o use t h i s t e c h n i q u e f o r t h e p u r i f i c a t i o n o f o t h e r enzymes i n h i b i t a b l e by p e p s t a t i n s h o u l d be considered. INTRODUCTION The lysosomal p r o t e a s e c a t h e p s i n 0 (EC 3.4.23.5)
has drawn
c o n s i d e r a b l e a t t e n t i o n due t o i t s p o s s i b l e r o l e i n t h e degrad a t i o n o f p r o t e o g l y c a n s i n v a r i o u s t i s s u e s . Thus, t h e enzyme has been c o n s i d e r e d t o be t h e f a c t o r r e s p o n s i b l e f o r t h i s p r o c e s s
231 Copyright 0 1978 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, microfilming, and recording, or by any information storage and retriwal system, without permission in writing from the publisher.
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232
i n c a r t i l a g e ( 4 ) and a t t h e m i n e r a l i z a t i o n f r o n t i n b i o l o g i c a l c a l c i f i c a t i o n (8). I n o u r s t u d i e s o f b i o l o g i c a l c a l c i f i c a t i o n c a t h e p s i n D was p u r i f i e d from r a t l i v e r b y a method m o d i f i e d a f t e r
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B a r r e t t ( 1 ) and used t o produce s p e c i e s - s p e c i f i c a n t i b o d i e s f o r immunohistochemical l o c a l i z a t i o n o f t h e enzyme ( 9 ) . T h i s p r e p a r a t i o n method f o r c a t h e p s i n D i s r a t h e r l a b o r i o u s and g i v e s low enzyme y i e l d s . O c c a s i o n a l l y , t h e c a t h e p s i n D prepared w i t h i s o e l e c t r i c f o c u s i n g as l a s t s t e p was impure, g i v i n g two p r e c i p i t a t i o n l i n e s when t e s t e d by immunoelectrophoresis a g a i n s t a n t i b o d i e s o b t a i n e d from t h e same preparation. T h i s communication p r e s e n t s a t e c h n i q u e by which t h e a c i d p r o t e a s e i n h i b i t o r p e p s t a t i n i s bound t o an agarose m a t r i x f o r a f f i n i t y chromatography. By t h i s method c a t h e p s i n D has been r a p i d l y p u r i f i e d w i t h a high y i e l d . The method w i l l be o f v a l u e f o r t h e p u r i f i c a t i o n of o t h e r enzymes i n h i b i t a b l e b y p e p s t a t i n , e.g.
p e p s i n and r e n i n .
EXPERIMENTAL Chemi ca 1s AH-Sepharose 48, Sephacryl S-200 S u p e r f i n e and Sephadex 6-100 were purchased from Pharmacia F i n e Chemicals, Uppsala, Sweden. DEAE-cellulose (Whatman DE52) and CM-cellulose (Whatman CM52) were o b t a i n e d from W . & B a l s t o n e ( M o d i f i e d C e l l u l o s e ) Ltd.,
S p r i n g f i e l d M i l l , Maidstone, Kent,
England. C e l i t e was from Kebo AB, Stockholm, Sweden. The p r o t e a s e i n h i b i t o r p e p s t a t i n (5, 7, 13, 14), a p r o d u c t f r o m Streptomyces
urgenteoZus v a r . toyonakensis was k i n d l y s u p p l i e d b y D r . H. Umezawa, I n s t . o f M i c r o b i a l Chemistry, Tokyo, Japan. A l l o t h e r reagents were of ana l y t i c a l grade.
R
PURIFICATION OF CATHEPSIN D
233
Tissue Preparation
A crude enzyme p r e p a r a t i o n was prepared t r o m r a t l i v e r ( 9 ) . Pooled l i v e r , 2.5 kg, was homogenized i n 5.0 L 1% NaCl c o n t a i n i n g 2% ( b y v o l . )
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n-butanol i n a S o r v a l l m n i - m i x e r . A t t e r d i s s o l v i n g 110 g CaCI2 i n t h e t o t a l homogenate, 750 m l 1 M Na2HP04
. 12 H20 were added.
of pH t o n e u t r a l i t y w i t h 2 M t r i s - H C 1 buffer,
A t t e r adjustment
pH 9 . 0 , t h e suspension was
c l e a r e d by c e n t r i f u g a t i o n . The s u p e r n a t a n t was a d j u s t e d t o p n 3.6 b y a
5 M f o r m i c a c i d b u f f e r , pH 3.0,
and i n c u b a t e d o v e r n i g h t a t 37OC f o r auto-
l y s i s . A f t e r c o o l i n g t o 2OC, 0.9 volumes o f -12OC acetone were added. The p r e c i p i t a t e was removed by c e n t r i f u g a t i o n and t h e same amount of acetone was added t o t h e supernatant. A f t e r t h e a d d i t i o n o f C e l i t e ( 2 g / l ) t h e p r e c i p i t a t e was c o l l e c t e d by f i l t r a t i o n and d i a l y z e d f o r 24 h a g a i n s t
50 mM EUTA i n 50 mM t r i s - H C 1 b u f f e r , pH 8.0.
F u r t h e r d i a l y s i s was per-
formed a g a i n s t 2 mM t r i s - H C 1 b u f f e r ,
u n t i l t h e pH i n t h e d i a l y s i s
pH 9.0,
n
bag was above 7.5 and t h e c o n d u c t i v i t y below 1.0 mmho/cmL. The C e l i t e was f i n a l l y removed by f i l t r a t i o n . The p r e p a r a t i o n o b t a i n e d was termed c r u d e c a t h e p s i n D and was f u r t h e r p u r i f i e d on t h e a f f i n i t y columns. P r i o r t o use, t h e m a t e r i a l was e x t e n s i v e l y d i a l y z e d a g a i n s t 0.1 M c i t r a t e - H C 1 b u f f e r , pH 3.0,
c o n t a i n i n g 0.5 M NaCI.
A f f i n i t y Matrix Preparation Two l i t r e s o f 0.5 M NaCl were added i n a l i q u o t s o f 200 m l t o 10 g o f AH-Sepharose 48 f o r washing and r e s w e l l i n g . P e p s t a t i n , 50 mg, was d i s s o l v e d i n 250 m l 50% e t h a n o l and added t o t h e AH-Sepharose t o g e t h e r w i t h 0.4 g o f l-ethyl-3-(3-dimethyl-amino-propyl) c a r b o d i i m i d e h y d r o c h l o r i d e (EDC).
The pH was a d j u s t e d t o 5.5 w i t h 0.05 M NaOH. The
m i x t u r e was i n c u b a t e d on a shaking w a t e r b a t h f o r 48 h a t 37OC. A f t e r c o u p l i n g , b l o c k i n g of excess amino groups was pertormed b y i n c u b a t i n g
234
LINDE AND PERSLIDEN
the m a t r i x i n 1 M a c e t i c a c i d f o r 4 h. A f t e r blocking, the AH-Sepharose was washed w i t h 500 m l p o r t i o n s of 50%, 402, 30%, 20% and 10% ethanol and f i n a l l y washed w i t h t h e e q u i l i b r a t i o n b u f f e r , 0.1
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b u f f e r , pH 3.0,
containing 0.5
M citrate-HL1
M NaC1.
I n p r e l i m i n a r y experiments, various parameters were studied: the r e a c t i o n time f o r coupling p e p s t a t i n t o AH-Sepharose 46, temperature dependence of the r e a c t i o n and the e f f e c t s o f using d i f f e r e n t concentrations o f pepstatin, EDC and ethanol i n t h e i n c u b a t i o n m i x t u r e . The method described was, however, found t o be optimal. A f f in i t y Chromatography
The crude cathepsin D preparation was a p p l i e d t o a column of the AH-Sepharose coupled p e p s t a t i n . A f t e r extensive washing w i t h t h e e q u i l i b r a t i o n b u f f e r , the enzyme was e l u t e d i n one homogenous peak w i t h 0.1
M NaHC03, pH 7.0, c o n t a i n i n g 0.5 M NaCl (Figure 1 ) . F r a c t i o n s were c o l l e c t e d and assayed f o r p r o t e i n by absorhance a t
280 nm. Cathepsin D a c t i v i t y i n 25 111 samples from each f r a c t i o n was assayed ( 9 ) . The sample was d i l u t e d t o 200 111 w i t h 0.1 pH 3.0,
M Na-citrate buffer
and 300 111 2% a c i d denatured haemoglobin i n the same b u f f e r
were added as substrate. A f t e r i n c u b a t i o n f o r I h a t 37OC and t e r m i n a t i n d the r e a c t i o n by adding 1 m l 5.2% t r i c h l o r o a c e t i c acid, t h e p r e c i p i t a t e was discarded and the supernatant was assayed a t 280 nm. The y i e l d from a r e p r e s e n t a t i v e p u r i f i c a t i o n i s given i n Table I . There i s no simple method f g r r o u t i n e l y assaying p e p s t a t i n
,
q u a n t i t a t i v e l y , so i n order t o c a l c u l a t e the capacity o f t h e p e p s t a t i n Sepharose m a t r i x , 40 m l o f the crude enzyme p r e p a r a t i o n were a p p l i e d
PURIFICATION OF CATHEPSIN D
0
235
1.0.
1
a)
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cu
a
0.5.
A
'I
B
I
elution volume(m1) FIGURE 1 A f f i n i t y chromatography on AH-Sepharose 48. 135 n i l o f a crude enzyme p r e p a r a t i o n were a p p l i e d t o a column (1.6 x 22 cm) o f AH-Sepharose 48 coupled w i t h p e p s t a t i n , p r e v i o u s l y e q u i l i b r a t e d w i t h 0.1 M citrate-HC1 buffer, pH 3.0, c o n t a i n i n g 0.5 M NaCI. A f t e r extensive washing with the same b u f f e r (A), t h e enzyme was e l u t e d ( 6 ) w i t h 0.1 M NaHC03 b u f f e r , pH 7.0, c o n t a i n i n g 0.5 M NaC1. E l u t i o n r a t e 5 ml/h. F r a c t i o n volume, 5 m l . P r o t e i n () and cathepsin D enzyme a c t i v i t y ( - - - - ) were measured a t 280 nm (see t e x t ) .
TABLE I Cathepsin U y i e l d a f t e r p u r i f i c a t i o n w i t h AH-Sepharose 48 a f f i n i t y chromatography. The f i g u r e s are based on p u r i f i c a t i o n s t a r t i n g w i t h 2.5 kg r a t l i v e r . The t o t a l p r o t e i n ( 9 ) was assayed by the method of Lowry e t aZ. (10) using albumin as a standard. The t o t a l cathepsin D a c t i v i t y i s expreased as amount l i b e r a t e d p r o t e i n ( 9 ) d u r i n g 1 h i n c u b a t i o n a t 37 C w i t h haemoglobin as s u b s t r a t e and measured by the Lowry method (9). The s p e c i f i c a c t i v i t y describes the t o t a l enzyme a c t i v i t y p e r t o t a l p r o t e i n . The enzyme a c t i v i t y y i e l d ( % ) r e l a t e s the remaining enzyme a c t i v i t y a f t e r p u r i f i c a t i o n t o t h e t o t a l a c t i v i t y i n t h e hanogenate. Protein (9) Homogenat e AH-Sepharose 48
197 0.020
Activity (9) 44.0
5.4
Spec.act. 0.22 270
Y i e l d (%)
i00 12
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2 36
t o small columns (0.9 x 3.5 an). A f t e r 25 m l o f sample a p p l i c a t i o n , cathepsin D a c t i v i t y appeared i n t h e e l u a t e d u r i n g l o a d i n g of t h e columns. A f t e r e l u t i o n o f the enzyme w i t h 0.1 M NaHC03 and q u a n t i t a t i o n
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according t o Lowry e t
UZ. ( l o ) ,
the amount of enzyme bound t o t h e
pepstatin-Sepharose 48 m a t r i x was c a l c u l a t e d t o be i 6 0
-
20u pg pure
PO
lyacrylamide
enzyme/ml swollen g e l . P u r i t y Assay The enzyme p r e p a r a t i o n revealed o n l y one band on
d i s c e l e c t r o p h o r e s i s ( F i g u r e 2). The 7% g e l s w i t h an upper 3% g e l were r u n a t a pH o f 8.3 f o r 2 h and a t 3 mA per g e l . l o n exchange chromatography on UEAt-cellulose columns, developed w i t h a 0
-
200 mM
continuous NaCl g r a d i e n t ( 9 ) , showed the presence o t o n l y one peak appearing a t a 55 mM NaCl concentration. S i m i l a r l y , a CM-cellulose column
e uted w i t h 250 mM acetate b u f f e r ( 9 ) d i s c l o s e d a s i n g l e peak.
Molecular sieve chromatography on a Sephacryl S-200 Superfine ( 1 . 6 x 76 cm) column revealed one s i n g l e p r o t e i n c o n t a i n i n g peak
when e l u t e d w i t h PBS b u f f e r , pH 7.2, a t 5 ml/h. On the basis o t molecular sieve chromatography on Sephadex G-100 (91 t h e apparent molecular weight was c a l c u l a t e d as 43,000.
Albumin, ovalbumin,
a-chymotrypsi nogen and cytochrome C were used as reference substances. I n a l l column f r a c t i o n s t h e o p t i c a l d e n s i t y a t 280 nm and cathepsin D a c t i v i t y were determined. Rabbit antiserum a g a i n s t the cathepsin U p r e p a r a t i o n was produced and the IgG f r a c t i o n p u r i f i e d as described ( Y ) .
The p u r i t y o f t h e
cathepsin D was t e s t e d a g a i n s t t h i s IgG by means o f immunoelectrophoresi ( 2 ) . Only one p r e c i p i t a t i o n l i n e c o u l d be seen ( f i g u r e 2).
237
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PURIFICATION OF CATHEPSIN D
FIGURE Z ( a j AH-Sepharose-purified cathepsin D was r u n on 7% polyacrylamide gels a t a pH o f 8.3 f o r 2 h a t 3 mA p e r g e l . ( b ) AH-Sepharose-purified cathepsin 0 (10 pg)was r u n e l e c t r o p h o r e t i c a l l y f o r 2 h a t a v o l t a g e o f 8 V/cm on a 1% agarose p l a t e . The a n t i g e n was allowed t o p r e c i p i t a t e a g a i n s t r a b b i t anti-cathepsin D IgG f o r 24 h a t 2OC, The p i c t u r e was taken i n a P o l a r o i d MP-3 Land dark f i e l d equipment w i t h P o l a r o i d 200 speed 42 f i Im. A: cathepsin D, B: r a b b i t a n t i - c a t h e p s i n D lgG, C: r a b b i t a n t i y a t h e p s i n D IgG a i l u t e d 1 : l w i t h PBS buffer, pH 7.7.
DISCUSS ION The present comnunication describes a r a p i d and r e l i a b l e standard technique f o r the p u r i f i c a t i o n o f cathepsin D. The r e l a t i v e u t i l i t y f o r p r e p a r a t i o n o f o t h e r enzymes would depend on t h e i r a f f i n i t y for pepstatin. The cathepsin D p u r i f i e d by AH-Sepharose 4B coupled p e p s t a t i n a f f i n i t y chromatography was pure by g e l electrophoresis,
two types
LINDE AM) PERSLIDEN
238
o f i o n exchange chromatograpny, molecuiar s i e v e chromatography and
immunologically homogenous by immunoelectrophoresi s. The apparent molecular weight obtained f o r the cathepsin D, 43,000,
i s the same
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as t h a t e a r l i e r r e p o r t e d f o r t h e r a t l i v e r enzyme (Y), and i s s i m i l a r t o preparations o f cathepsin D from o t h e r t i s s u e s (1, 12, 15). A considerably h i g h e r enzyme y i e l d was obtained compared w i t h conventional methods (1, 9): Coupling w i t h higher. amounts o f p e p s t a t i n , h i g h e r concentrations o f ethanol, longer i n c u b a t i o n times f o r c o u p l i n g and f u r t h e r a d d i t i o n of EUC during the i n c u b a t i o n p e r i o d d i d n o t improve t h e f i n a l product. The use o t methanol i n s t e a d of ethanol t o d i s s o l v e the p e p s t a t i n was n o t successful. Epoxy-activated Sepharose 68, i n s t e a d o f AH-Sepharose
48, was found t o be more e l a b o r a t e t o handle, and t h e amount o f p e p s t a t i n bound t o t h e m a t r i x was found t o be lower than w i t h t h e procedure described. The use of a f f i n i t y chromatography f o r t h e p u r i f i c a t i o n o f cathepsin
D has been r e p o r t e d e a r l i e r
by Kazakova and Orekhovich ( 6 ) . S i m i l a r l y
r e n i n was p u r i f i e d by p e p s t a t i n a f f i n i t y chromatography ( 3 j . These authors, however, used a d i f f e r e n t and laborious- coupling system based on the SOiutiOn of p e p s t a t i n i n dimethylformamide. This system was n o t sucessful i n our hands. P e p s t a t i n has a l s o been coupled t o aminoethylated polyacrylamide g e l f o r r e n i n p u r i f i c a t i o n (11 j . By a d i f f e r e n t p r i n c i p i e , Smith and Turk (12) p u r i f i e d cathepsin
D on haemoglobin-agarose r e n i n
followed by molecular sieve chromatography. The present technique has proven t o be easy t o handle and very reproducible. This method i s considerably l e s s laborious than most
PURIFICATION OF CATHEPSIN D
239
p r e v i o u s techniques. I t i s a l s o o u r f i r m c o n v i c t i o n t h a t t h e p u r i t y o f c a t h e p s i n D prepared b y new methods must be c a r e f u l l y assessed by
t h e combined use o f d i f f e r e n t s e p a r a t i o n p r i n c i p l e s . Seemingly p u r e
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c a t h e p s i n D p r e p a r a t i o n s may e.g. be r e v e a l e d as i m m u n o l o g i c a i l y non-homogenous ( Y ) . ACKNOWLEDGEMENTS The t e c h n i c a l a s s i s t a n c e o f Mrs Agneta Hoglund and Mrs Ann-Katrin Rhodin i s h i g h l y a p p r e c i a t e d . The i n v e s t i g a t i o n was supported by t h e Swedish Medical Research C o u n c i l and t h e F a c u l t y o f Odontology, U n i v e r s i t y of Goteborg. REFERENCES 1.
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