600
Specialia
Rdsumd. L ' i n j e c t i o n i n t r a v e n t r i c u l a i r e d'acetylcholine p r o v o q u e chez le rat m~le une s6cr6tion accrue de LH. Cet effet de l'acetylcholine est illhib6 par l ' a d m i n i s t r a t i o n
EXPERIENTIA 31[5
intraventriculMre d'atr0pine, e t e s t a u g m e n t 6 p a r l'injection i n t r a v e n t r i c u l a i r e de prostigmine. G. JIJSTO 2t, M. MOTTA and L. MARTINI
21 Ford Foundation Fellow. On leave of absence from the Instituto de Neurobiologia, Buenos Aires, Argentina.
Departments of Endocrinology and o/Pharmacology, University o[ Milan, Via A. del Sarto 21, 1-20129 Milano (Italy), 30 December 7974.
Purification of Rabbit Angiotensin II-Antibodies by Affinity Chromatography Antibodies against angiotensin I I h a v e been used r e p e a t e d l y for in v i v o experiments, in which the participation of the renin-angiotensin system in various forms of e x p e r i m e n t a l h y p e r t e n s i o n was studied (for review see CIIRISTLIEB and HICKLER 1). I n m o s t of these studies crude antisera h a v e been injected into the test animal w i t h the obvious d i s a d v a n t a g e t h a t n o t only the antibody, b u t also considerable a m o u n t s of heterologous protein, renin, renin substrate and angiotensin I I (up to 300 ng/ml) were injected. I n order to provide a more d e f i n e d basis for in v i v o studies, t h e conditions for purification of angiotensin-antibodies b y affinity c h r o m a t o g r a p h y were studied. Materials and methods. Anti-sera were raised in male white N e w Zealand rabbits, weighing 2000-2500 g, b y i m m u n i z a t i o n w i t h aspl-ileu~-angiotensin I I (Schwarz/ Mann, Orangeburg, USA) coupled to porcine y-globulin b y the carbodiimide method, as described b y GooDFRIEND et al. a. A f t e r 3 to 5 m o n t h s all animals developed a n t i b o d y titers ranging b e t w e e n 1:13000 and 1:180000. Affillity columns for m o s t of the separation experiments were prepared b y coupling angiotensin I I to the sepharose m a t r i x via a l b u m i n as a spacer, a principle first proposed b y CUATRECASAS et al. ~. I n a typical e x p e r i m e n t 3 g of c y a n o g e n b r o m i d e - a c t i v a t e d Sepharose 4]3 (Pharmacia, Uppsala, Sweden) were washed ill 1 m M tris-HCl buffer p H 6.0, and a d j u s t e d to p H 8.0 w i t h N a H C O 3 buffer. B o v i n e serum a l b u m i n (Behring Werke, Marburg), 20 m g in 0.1 M NaHCO~ buffer p H 8.0, containing 0.5 M NaC1, was added. After 2 h of incubation, a b o u t 65% of the a l b u m i n had been b o u n d to the sepharose as judged b y the decrease in absorbancy at 280 n m and protein c o n t e n t ill the s u p e r n a t a n t 5. Following t r e a t m e n t w i t h 1 M ethanolamine at p H 8 for 2 h, t h e p r o d u c t was washed 4 times a l t e r n a t e l y w i t h 0.1 M borate buffer p H 8.0, and 0.1 M acetate buffer p H 4.0, and finally a d j u s t e d to p H 6.0. subsequently 2 m g angiotensin I I were coupled to t h e sepharose b o u n d a l b u m i n b y the addition of 1.2 g of N - e t h y l - N - d i m e t h y l - a m i n o p r o p y l - c a r b o d i i m i d e at p H 6.0. A small a m o u n t of I*25-1abelled angiotensin I I was added to t h e reaction m i x t u r e , and the r a d i o a c t i v i t y of t h e sepharose-free s u p e r n a t a n t was d e t e r m i n e d at various t i m e intervals. F u r t h e r m o r e , t h e a m o u n t of angiotensin I I remaining in the s u p e r n a t a n t was e s t i m a t e d b y r a t blood pressure bioassay% The coupling p r o d u c t was again washed a l t e r n a t e l y w i t h acidic and alkaline buffer was described above, and packed in a 1.1 • 21 cm c o l u m n and equilibrated w i t h 0.1 M tris-HC1 p H 7.4. Angiotensin I I a n t i s e r u m (9 ml) was diluted 1 : 3 w i t h the same buffer and poured t h r o u g h the column at a flow rate of a b o u t 0.5 m l / mill. The c o l u m n was washed w i t h 50 ml 0.1 M tris-HC1 p H 7.4 and Muted in batches w i t h 50 ml each of 0.1 M Na-citrate-HC1 buffer of p H 5, 4, 3, 2, 1.2 and finally p H 1.2 containing 0.5 M NaC1. The eluate was collected in 10 ml fractions into tubes k e p t at 0 ~ and containing 2 ml
3 M tris-HC1 p H 7.4, to i m m e d i a t e l y neutralize the acidic eluate. I n a separate e x p e r i m e n t angiotensin I I was coupled directly to C N B r - a c t i v a t e d Sepharose w i t h o u t using a l b u m i n as a spacer. A n t i b o d y concentrations were e s t i m a t e d b y titer determination. The titer is arbitrarily defined as the dilution of an a n t i b o d y preparation, at which exactly half of 10 pg of I125-angiotensin I I is b o u n d to the a n t i b o d y in a s t a n d a r d r a d i o i m m u n o a s s a y system v (250 ~1 of 0.1 M tris-acetate buffer p H 7.4 w i t h 0.1% h u m a n serum albumin, 20 h equilibration at 0~ Assuming t h a t under these conditions of e x t r e m e dilution (e.g. a n t i s e r u m is diluted at least 15000-fold), there is no interference b y substances present in the original material, t h e a m o u n t of a n t i b o d y which binds 5 pg of angiotensin I I can be t a k e n as an a r b i t r a r y a n t i b o d y unit, p r o v i d e d t h a t no significant differences in affinity exist. Thus, t h e a n t i b o d y concentration of a given p r e p a r a t i o n can be expressed as the reciprocal of the titer. The r a d i o i m m u n o a s s a y system for angiotensin I I and the e s t i m a t i o n of cross r e a c t i v i t y w i t h analogous peptides has been described p r e v i o u s l y L The a p p a r e n t affinity constants of t h e a n t i b o d y preparations were e s t i m a t e d from double-reciprocal plots according to NISONOFF and PRESSMAN 8. Results and discussion. The coupling of allgiotensin I I to the sepharose-albumin c o m p l e x appeared to be complete after 2 h of incubation, since at t h a t t i m e there was no detectable biological a c t i v i t y in the supernatant, as j u d g e d b y rat blood pressure assay. H o w e v e r , at the same time, half of the r a d i o a c t i v i t y of P25-1abelled angiotensin II, which had been added at the beginning of the coupling reaction, was still in t h e soluble form. This discrepancy b e t w e e n biological a c t i v i t y and r a d i o a c t i v i t y m a y find its explanation in a partial p o l y m e r i z a t i o n of angiotensin, i.e. the f o r m a t i o n of dimers or trimers Of angiotensin I I b y the carbodiimid reaction, since the immobilized a l b u m i n is less readily available as a reaction partner. This i n t e r p r e t a t i o n is supported b y the following experiment. The p r o d u c t of a reaction m i x t u r e identical
1 A. R. CHRISTLIEBand R. B. HICKLER, Endocrinology 91, 1064 (1972). 2 p. eSTER, E. HACKENTHAL, P. VECSEI, K. H. GLESS, J. M6HRING
and F. GROSS, in Hypertension (Eds. A. DISTLER and H. P. WOLFF; Thieme, Stuttgart 1974), p. 62. s T. GOODFRIENDand L. LEVlNE, Science 74d, 1344 (1964). 4 p. CUATRECASAS, M. WILCHEK and C. B. ANFINSEN, Froc. natn. Acad. Sei., USA 6I, 636 (1968). O. EI. LOWRY,N. J. ROSEBROUGH,A. L. FARRand 1~. J. RANDALL, J. biol. Chem. 193, 265 (1951). 6 W. S. PEART, J. Bioehem. 59, 300 (1955). 7 p. OSTER, E. HACKENTHALand R. HEpP, Experientia 29, 353 (1973). s A. NISONOFFand D. PRESSMAN, J. Immun. 80, 417 (1958).
15.5. 1975
Speeialia
to t h a t d e s c r i b e d a b o v e , e x c e p t for o m i s s i o n of s e p h a r o s e a l b u m i n , was c h r o m a t o g r a p h e d o n a s e p h a d e x G 15 c o l u m n . Most of t h e r a d i o a c t i v i t y was M u t e d w i t h t h e v o i d v o l u m e , i n d i c a t i n g a m o l e c u l a r w e i g h t of m o r e t h a n 1500, w h e r e a s r a d i o a c t i v i t y d u e t o u n r e a c t e d a n g i o t e n s i n I I M u t e d l a t e r in t h e f r a c t i o n a t i o n r a n g e - c l e a r l y s e p a r a t e f r o m t h e v o i d v o l u m e . I t is t h e r e f o r e c o n c e i v a b l e t h a t t h e final s e p h a r o s e c o u p l i n g - p r o d u c t c o n t a i n s n o t o n l y a n g i o t e n s i n m o n o m e r s c o u p l e d to a l b u m i n , b u t also d i m e r s and, t o a lesser e x t e n t , o t h e r p o l y m e r i z e d conj u g a t e s of a n g i o t e n s i n l i n k e d t o t h e m a t r i x . C h r o m a t o g r a p h y of a n g i o t e n s i n I I - a n t i s e r a b y a d i s c o n t i n u o u s p H - g r a d i e n t r e s u l t e d in t h e e l u t i o n of s e v e r a l d i s t i n c t fractions. A t y p i c a l e l u t i o n profile i s s h o w n in t h e Figure. M a i n p e a k s a p p e a r a t a n e l u t i o n p H of 5, a t p t I 4 a n d 1.2. T h e s e p e a k s were seen in all experim e n t s , t h e q u a n t i t a t i v e d i s t r i b u t i o n of a n t i b o d i e s a m o n g t h e s e peaks, h o w e v e r , v a r i e d a m o n g d i f f e r e n t e x p e r i m e n t s . T o t a l r e c o v e r y of a n t i b o d y f r o m t h e c o l u m n was a b o u t 60~o. T h e p u r i f i c a t i o n was m o r e t h a n 100-fold in t e r m s of a n t i b o d y c o n t e n t p e r m g p r o t e i n . T h e p u r i f i e d a n t i b o d y p r e p a r a t i o n w a s free of r e n i n s u b s t r a t e , b u t
antibody titer
1:300
1:200
1:100
I
lOO pH
7.5
"•L•
_j . ~ . . . . . . j . ~
BB
5
, 4
I
I
I
200
3
2
............ I
I
I
300 1.2
1.2 +NaCI
4 0 0 ml eluate
601
c o n t a i n e d still some a n g i o t e n s i n II, w h i c h was r e m o v e d b y e x t r a c t i o n w i t h a c a t i o n e x c h a n g e resin ( B i o r a d A G 50 W X 2 H + - f o r m ) L I n c o n t r a s t , t h e r e c o v e r y f r o m sepharose-angiotensin II-columns (without albumin) was o n l y 20~o, m o s t of w h i c h M u t e d a t p H 1.2. I t was n o t possible to increase t h e yield f r o m t h e s e c o l u m n s b y u s i n g m o r e d r a s t i c e l u t i o n c o n d i t i o n s s u c h as a l k a l i n e e l u t i o n m e d i a , stepwise increase of a c i d i t y t o 3 N I-IC1 - a p r o c e d u r e u s e d succesfully b y CUATRECASAS9 w i t h i n s u l i n a n t i b o d i e s - or w i t h l a u r y l - s u l f a t e . T h e e l u t i o n of a n g i o t e n s i n a n t i b o d i e s in s e v e r a l d i s t i n c t f r a c t i o n s m a y reflect a n t i b o d y h e t e r o g e n e i t y , i.e. s e p a r a t i o n of d i f f e r e n t a n t i b o d y p o p u l a t i o n s b y differences in a f f i n i t y for a n g i o t e n s i n II. H o w e v e r , besides a m o d e r a t e increase in a p p a r e n t a f f i n i t y b y t h e p u r i f i c a t i o n f r o m 2.5 • 10 l~ 1/mole to 5 • 101~ 1/mole, t h e r e was n o difference in a f f i n i t y b e t w e e n a n t i b o d y f r a c t i o n s M u t e d a t p H 4 a n d 5 ( c o m b i n e d to pool 1) a n d p i t 1.2 (pool 2), w h i c h is also reflected in c o m p a r a b l e s e n s i t i v i t y in t h e s t a n d a r d radioimmunoassay system. I n a d d i t i o n , o n l y m i n o r differences in specificity were f o u n d (Table) w h e n t h e cross-reactions w i t h s e v e r a l a n g i o t e n s i n a n a l o g u e s were e s t i m a t e d . T h e s e m i n o r differences in t h e p r o p e r t i e s of pool 1 a n d 2 a n t i b o d i e s do n o t s a t i s f a c t o r i l y e x p l a i n t h e d r a s t i c difference in t h e e l u t i o n b e h a v i o u r , i.e. e l u t i o n a t p H 5 v e r s u s p H 1.2. I t h a s to b e c o n s i d e r e d t h a t t h e f r a c t i o n a t i o n of a n t i b o d y , as s h o w n in t h e Figure, is p a r t l y d u e to h e t e r o g e n e i t y of t h e a f f i n i t y ligands on t h e column, as h a s b e e n p o i n t e d o u t a b o v e . Since t h e a i m of t h e p r e s e n t s t u d y was p r i m a r i l y to o b t a i n p u r i f i e d a n t i b o d y for in v i v o studies, t h i s p r o b l e m w a s n o t f u r t h e r i n v e s t i g a t e d lo.
Zusammen/assung. A n t i s e r a gegen A n g i o t e n s i n I I w u r d e n d u r c h I m m u n i s i e r u n g y o n K a n i n c h e n m i t Angiotensin II-Schweineglobulin-Konjugaten erhalten. Aus diesen A n t i s e r e n k o n n t e n A n g i o t e n s i n I I A n t i k 6 r p e r d u r c h Affinit~Ltschromatographie a u f S e p h a r o s e - A l b u m i n A n g i o t e n s i n I I - S ~ u l e n m e h r als 100-fach a n g e r e i c h e r t werden. D u r c h d i s k o n t i n u i e r l i c h e p H - G r a d i e n t e n - E l u t i o n w u r d e n v e r s c h i e d e n e A n t i k 6 r p e r - F r a k t i o n e n e r h a l t e n , die sich j e d o c h n u r geringfiigig in Affinit/it u n d Spezifit~t u n t e r s c h i e d e n . Die m 6 g l i c h e B e t e i l i g u n g u n t e r s c h i e d l i c h e r Liganden der Affinit~tsmatrix an der Fraktionierung wird diskutiert.
pH
H. BAUKNECHT,
Affinity chromatography of angiotensin II-antibody on sepharosealbumin-angiotensin II-columns. Elution by discontinuous pHgradient. Antibody concentration was estimated by titer determination.
P. OSTER
and E. ~IACKENTHAL
I0
Department o/Pharmacology, University o/ Heidelberg, Im Neuenheimer-Feld 366, D-69 Heidelberg (German Federal Republic, BRD), 27 October 797,1.
Crossreactivity of angiotensin II-antiserum and purified antibody fractions with some angiotensin analogues Angiotensin II analogue
lleuS-angiotensin If VaP-angiotensin If Ileu~-angiotensin I Argl-heptapeptide
Vall-hexapeptide
Crossreaetion of antibody of ileu"~angiotensin II (%) Untreated antiserum
Pool i
Pool 2
i00 67 0.6 40
i00 69 0.5 48
I00 41 0.2 28
35
44
35
9 p. CUATRECASAS,Biochem. biophys. Res. Commun. 35, 531 (1969). lo Acknowledgements: This study was supported by the Deutsche Forschungsgemeinschaft within the SFB No. 90 .The excellent technical assistance of H. DICK is gratefully acknowledged.
Specialia
Rdsumd. L ' i n j e c t i o n i n t r a v e n t r i c u l a i r e d'acetylcholine p r o v o q u e chez le rat m~le une s6cr6tion accrue de LH. Cet effet de l'acetylcholine est illhib6 par l ' a d m i n i s t r a t i o n
EXPERIENTIA 31[5
intraventriculMre d'atr0pine, e t e s t a u g m e n t 6 p a r l'injection i n t r a v e n t r i c u l a i r e de prostigmine. G. JIJSTO 2t, M. MOTTA and L. MARTINI
21 Ford Foundation Fellow. On leave of absence from the Instituto de Neurobiologia, Buenos Aires, Argentina.
Departments of Endocrinology and o/Pharmacology, University o[ Milan, Via A. del Sarto 21, 1-20129 Milano (Italy), 30 December 7974.
Purification of Rabbit Angiotensin II-Antibodies by Affinity Chromatography Antibodies against angiotensin I I h a v e been used r e p e a t e d l y for in v i v o experiments, in which the participation of the renin-angiotensin system in various forms of e x p e r i m e n t a l h y p e r t e n s i o n was studied (for review see CIIRISTLIEB and HICKLER 1). I n m o s t of these studies crude antisera h a v e been injected into the test animal w i t h the obvious d i s a d v a n t a g e t h a t n o t only the antibody, b u t also considerable a m o u n t s of heterologous protein, renin, renin substrate and angiotensin I I (up to 300 ng/ml) were injected. I n order to provide a more d e f i n e d basis for in v i v o studies, t h e conditions for purification of angiotensin-antibodies b y affinity c h r o m a t o g r a p h y were studied. Materials and methods. Anti-sera were raised in male white N e w Zealand rabbits, weighing 2000-2500 g, b y i m m u n i z a t i o n w i t h aspl-ileu~-angiotensin I I (Schwarz/ Mann, Orangeburg, USA) coupled to porcine y-globulin b y the carbodiimide method, as described b y GooDFRIEND et al. a. A f t e r 3 to 5 m o n t h s all animals developed a n t i b o d y titers ranging b e t w e e n 1:13000 and 1:180000. Affillity columns for m o s t of the separation experiments were prepared b y coupling angiotensin I I to the sepharose m a t r i x via a l b u m i n as a spacer, a principle first proposed b y CUATRECASAS et al. ~. I n a typical e x p e r i m e n t 3 g of c y a n o g e n b r o m i d e - a c t i v a t e d Sepharose 4]3 (Pharmacia, Uppsala, Sweden) were washed ill 1 m M tris-HCl buffer p H 6.0, and a d j u s t e d to p H 8.0 w i t h N a H C O 3 buffer. B o v i n e serum a l b u m i n (Behring Werke, Marburg), 20 m g in 0.1 M NaHCO~ buffer p H 8.0, containing 0.5 M NaC1, was added. After 2 h of incubation, a b o u t 65% of the a l b u m i n had been b o u n d to the sepharose as judged b y the decrease in absorbancy at 280 n m and protein c o n t e n t ill the s u p e r n a t a n t 5. Following t r e a t m e n t w i t h 1 M ethanolamine at p H 8 for 2 h, t h e p r o d u c t was washed 4 times a l t e r n a t e l y w i t h 0.1 M borate buffer p H 8.0, and 0.1 M acetate buffer p H 4.0, and finally a d j u s t e d to p H 6.0. subsequently 2 m g angiotensin I I were coupled to t h e sepharose b o u n d a l b u m i n b y the addition of 1.2 g of N - e t h y l - N - d i m e t h y l - a m i n o p r o p y l - c a r b o d i i m i d e at p H 6.0. A small a m o u n t of I*25-1abelled angiotensin I I was added to t h e reaction m i x t u r e , and the r a d i o a c t i v i t y of t h e sepharose-free s u p e r n a t a n t was d e t e r m i n e d at various t i m e intervals. F u r t h e r m o r e , t h e a m o u n t of angiotensin I I remaining in the s u p e r n a t a n t was e s t i m a t e d b y r a t blood pressure bioassay% The coupling p r o d u c t was again washed a l t e r n a t e l y w i t h acidic and alkaline buffer was described above, and packed in a 1.1 • 21 cm c o l u m n and equilibrated w i t h 0.1 M tris-HC1 p H 7.4. Angiotensin I I a n t i s e r u m (9 ml) was diluted 1 : 3 w i t h the same buffer and poured t h r o u g h the column at a flow rate of a b o u t 0.5 m l / mill. The c o l u m n was washed w i t h 50 ml 0.1 M tris-HC1 p H 7.4 and Muted in batches w i t h 50 ml each of 0.1 M Na-citrate-HC1 buffer of p H 5, 4, 3, 2, 1.2 and finally p H 1.2 containing 0.5 M NaC1. The eluate was collected in 10 ml fractions into tubes k e p t at 0 ~ and containing 2 ml
3 M tris-HC1 p H 7.4, to i m m e d i a t e l y neutralize the acidic eluate. I n a separate e x p e r i m e n t angiotensin I I was coupled directly to C N B r - a c t i v a t e d Sepharose w i t h o u t using a l b u m i n as a spacer. A n t i b o d y concentrations were e s t i m a t e d b y titer determination. The titer is arbitrarily defined as the dilution of an a n t i b o d y preparation, at which exactly half of 10 pg of I125-angiotensin I I is b o u n d to the a n t i b o d y in a s t a n d a r d r a d i o i m m u n o a s s a y system v (250 ~1 of 0.1 M tris-acetate buffer p H 7.4 w i t h 0.1% h u m a n serum albumin, 20 h equilibration at 0~ Assuming t h a t under these conditions of e x t r e m e dilution (e.g. a n t i s e r u m is diluted at least 15000-fold), there is no interference b y substances present in the original material, t h e a m o u n t of a n t i b o d y which binds 5 pg of angiotensin I I can be t a k e n as an a r b i t r a r y a n t i b o d y unit, p r o v i d e d t h a t no significant differences in affinity exist. Thus, t h e a n t i b o d y concentration of a given p r e p a r a t i o n can be expressed as the reciprocal of the titer. The r a d i o i m m u n o a s s a y system for angiotensin I I and the e s t i m a t i o n of cross r e a c t i v i t y w i t h analogous peptides has been described p r e v i o u s l y L The a p p a r e n t affinity constants of t h e a n t i b o d y preparations were e s t i m a t e d from double-reciprocal plots according to NISONOFF and PRESSMAN 8. Results and discussion. The coupling of allgiotensin I I to the sepharose-albumin c o m p l e x appeared to be complete after 2 h of incubation, since at t h a t t i m e there was no detectable biological a c t i v i t y in the supernatant, as j u d g e d b y rat blood pressure assay. H o w e v e r , at the same time, half of the r a d i o a c t i v i t y of P25-1abelled angiotensin II, which had been added at the beginning of the coupling reaction, was still in t h e soluble form. This discrepancy b e t w e e n biological a c t i v i t y and r a d i o a c t i v i t y m a y find its explanation in a partial p o l y m e r i z a t i o n of angiotensin, i.e. the f o r m a t i o n of dimers or trimers Of angiotensin I I b y the carbodiimid reaction, since the immobilized a l b u m i n is less readily available as a reaction partner. This i n t e r p r e t a t i o n is supported b y the following experiment. The p r o d u c t of a reaction m i x t u r e identical
1 A. R. CHRISTLIEBand R. B. HICKLER, Endocrinology 91, 1064 (1972). 2 p. eSTER, E. HACKENTHAL, P. VECSEI, K. H. GLESS, J. M6HRING
and F. GROSS, in Hypertension (Eds. A. DISTLER and H. P. WOLFF; Thieme, Stuttgart 1974), p. 62. s T. GOODFRIENDand L. LEVlNE, Science 74d, 1344 (1964). 4 p. CUATRECASAS, M. WILCHEK and C. B. ANFINSEN, Froc. natn. Acad. Sei., USA 6I, 636 (1968). O. EI. LOWRY,N. J. ROSEBROUGH,A. L. FARRand 1~. J. RANDALL, J. biol. Chem. 193, 265 (1951). 6 W. S. PEART, J. Bioehem. 59, 300 (1955). 7 p. OSTER, E. HACKENTHALand R. HEpP, Experientia 29, 353 (1973). s A. NISONOFFand D. PRESSMAN, J. Immun. 80, 417 (1958).
15.5. 1975
Speeialia
to t h a t d e s c r i b e d a b o v e , e x c e p t for o m i s s i o n of s e p h a r o s e a l b u m i n , was c h r o m a t o g r a p h e d o n a s e p h a d e x G 15 c o l u m n . Most of t h e r a d i o a c t i v i t y was M u t e d w i t h t h e v o i d v o l u m e , i n d i c a t i n g a m o l e c u l a r w e i g h t of m o r e t h a n 1500, w h e r e a s r a d i o a c t i v i t y d u e t o u n r e a c t e d a n g i o t e n s i n I I M u t e d l a t e r in t h e f r a c t i o n a t i o n r a n g e - c l e a r l y s e p a r a t e f r o m t h e v o i d v o l u m e . I t is t h e r e f o r e c o n c e i v a b l e t h a t t h e final s e p h a r o s e c o u p l i n g - p r o d u c t c o n t a i n s n o t o n l y a n g i o t e n s i n m o n o m e r s c o u p l e d to a l b u m i n , b u t also d i m e r s and, t o a lesser e x t e n t , o t h e r p o l y m e r i z e d conj u g a t e s of a n g i o t e n s i n l i n k e d t o t h e m a t r i x . C h r o m a t o g r a p h y of a n g i o t e n s i n I I - a n t i s e r a b y a d i s c o n t i n u o u s p H - g r a d i e n t r e s u l t e d in t h e e l u t i o n of s e v e r a l d i s t i n c t fractions. A t y p i c a l e l u t i o n profile i s s h o w n in t h e Figure. M a i n p e a k s a p p e a r a t a n e l u t i o n p H of 5, a t p t I 4 a n d 1.2. T h e s e p e a k s were seen in all experim e n t s , t h e q u a n t i t a t i v e d i s t r i b u t i o n of a n t i b o d i e s a m o n g t h e s e peaks, h o w e v e r , v a r i e d a m o n g d i f f e r e n t e x p e r i m e n t s . T o t a l r e c o v e r y of a n t i b o d y f r o m t h e c o l u m n was a b o u t 60~o. T h e p u r i f i c a t i o n was m o r e t h a n 100-fold in t e r m s of a n t i b o d y c o n t e n t p e r m g p r o t e i n . T h e p u r i f i e d a n t i b o d y p r e p a r a t i o n w a s free of r e n i n s u b s t r a t e , b u t
antibody titer
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c o n t a i n e d still some a n g i o t e n s i n II, w h i c h was r e m o v e d b y e x t r a c t i o n w i t h a c a t i o n e x c h a n g e resin ( B i o r a d A G 50 W X 2 H + - f o r m ) L I n c o n t r a s t , t h e r e c o v e r y f r o m sepharose-angiotensin II-columns (without albumin) was o n l y 20~o, m o s t of w h i c h M u t e d a t p H 1.2. I t was n o t possible to increase t h e yield f r o m t h e s e c o l u m n s b y u s i n g m o r e d r a s t i c e l u t i o n c o n d i t i o n s s u c h as a l k a l i n e e l u t i o n m e d i a , stepwise increase of a c i d i t y t o 3 N I-IC1 - a p r o c e d u r e u s e d succesfully b y CUATRECASAS9 w i t h i n s u l i n a n t i b o d i e s - or w i t h l a u r y l - s u l f a t e . T h e e l u t i o n of a n g i o t e n s i n a n t i b o d i e s in s e v e r a l d i s t i n c t f r a c t i o n s m a y reflect a n t i b o d y h e t e r o g e n e i t y , i.e. s e p a r a t i o n of d i f f e r e n t a n t i b o d y p o p u l a t i o n s b y differences in a f f i n i t y for a n g i o t e n s i n II. H o w e v e r , besides a m o d e r a t e increase in a p p a r e n t a f f i n i t y b y t h e p u r i f i c a t i o n f r o m 2.5 • 10 l~ 1/mole to 5 • 101~ 1/mole, t h e r e was n o difference in a f f i n i t y b e t w e e n a n t i b o d y f r a c t i o n s M u t e d a t p H 4 a n d 5 ( c o m b i n e d to pool 1) a n d p i t 1.2 (pool 2), w h i c h is also reflected in c o m p a r a b l e s e n s i t i v i t y in t h e s t a n d a r d radioimmunoassay system. I n a d d i t i o n , o n l y m i n o r differences in specificity were f o u n d (Table) w h e n t h e cross-reactions w i t h s e v e r a l a n g i o t e n s i n a n a l o g u e s were e s t i m a t e d . T h e s e m i n o r differences in t h e p r o p e r t i e s of pool 1 a n d 2 a n t i b o d i e s do n o t s a t i s f a c t o r i l y e x p l a i n t h e d r a s t i c difference in t h e e l u t i o n b e h a v i o u r , i.e. e l u t i o n a t p H 5 v e r s u s p H 1.2. I t h a s to b e c o n s i d e r e d t h a t t h e f r a c t i o n a t i o n of a n t i b o d y , as s h o w n in t h e Figure, is p a r t l y d u e to h e t e r o g e n e i t y of t h e a f f i n i t y ligands on t h e column, as h a s b e e n p o i n t e d o u t a b o v e . Since t h e a i m of t h e p r e s e n t s t u d y was p r i m a r i l y to o b t a i n p u r i f i e d a n t i b o d y for in v i v o studies, t h i s p r o b l e m w a s n o t f u r t h e r i n v e s t i g a t e d lo.
Zusammen/assung. A n t i s e r a gegen A n g i o t e n s i n I I w u r d e n d u r c h I m m u n i s i e r u n g y o n K a n i n c h e n m i t Angiotensin II-Schweineglobulin-Konjugaten erhalten. Aus diesen A n t i s e r e n k o n n t e n A n g i o t e n s i n I I A n t i k 6 r p e r d u r c h Affinit~Ltschromatographie a u f S e p h a r o s e - A l b u m i n A n g i o t e n s i n I I - S ~ u l e n m e h r als 100-fach a n g e r e i c h e r t werden. D u r c h d i s k o n t i n u i e r l i c h e p H - G r a d i e n t e n - E l u t i o n w u r d e n v e r s c h i e d e n e A n t i k 6 r p e r - F r a k t i o n e n e r h a l t e n , die sich j e d o c h n u r geringfiigig in Affinit/it u n d Spezifit~t u n t e r s c h i e d e n . Die m 6 g l i c h e B e t e i l i g u n g u n t e r s c h i e d l i c h e r Liganden der Affinit~tsmatrix an der Fraktionierung wird diskutiert.
pH
H. BAUKNECHT,
Affinity chromatography of angiotensin II-antibody on sepharosealbumin-angiotensin II-columns. Elution by discontinuous pHgradient. Antibody concentration was estimated by titer determination.
P. OSTER
and E. ~IACKENTHAL
I0
Department o/Pharmacology, University o/ Heidelberg, Im Neuenheimer-Feld 366, D-69 Heidelberg (German Federal Republic, BRD), 27 October 797,1.
Crossreactivity of angiotensin II-antiserum and purified antibody fractions with some angiotensin analogues Angiotensin II analogue
lleuS-angiotensin If VaP-angiotensin If Ileu~-angiotensin I Argl-heptapeptide
Vall-hexapeptide
Crossreaetion of antibody of ileu"~angiotensin II (%) Untreated antiserum
Pool i
Pool 2
i00 67 0.6 40
i00 69 0.5 48
I00 41 0.2 28
35
44
35
9 p. CUATRECASAS,Biochem. biophys. Res. Commun. 35, 531 (1969). lo Acknowledgements: This study was supported by the Deutsche Forschungsgemeinschaft within the SFB No. 90 .The excellent technical assistance of H. DICK is gratefully acknowledged.
