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Bovine alpha-fetoprotein
BIOCHIMIE, 1978, 60, 663-672.
Bovine alpha-tbtoprotein. Isolation
and
characterization
Serge AL~AU <~, Jacques ~AlqTI and Jean MORETTI. (3-5-1978).
Laboratoire de B i o c h i m i e des Prot~ines, Universit~ des Sciences et T e c h n i q u e s du Languedoc, Montpellier, France.
R~sum6.
Summary.
L'AFP b o v i n e a v a i t 6t6 sicyaal6e d a n s le s 6 r u m foetal bovin. Ce travail d6crit son identification et s o n isolement p a r c h r o m a t o q r a p h i e sur c a r b o x y m e t h y l c e l l u l o s e DEAE S e p h a d e x A-50, gel de filtration et colonne d ' i m m u n o a d s o r b a n t . L'AFP se r6v~le h o m o g ~ n e , p a r blectrophorbse en gel d ' a c r y l a m i d e d a n s d e s conditions d 6 n a t u r a n t e s et non d 6 n a t u r a n t e s , p a r ultracentrifugation et en immuno61ectrophor~se. Les r6sultats s u i v a n t s ont 6t6 o b t e n u s : 1. La s 6 d i m e n t a t i o n & l'6quilibre indique u n poids m o l 6 c u l a i r e de 66 500 p o u r u n e c o n s t a n t e de S v e d b e r q s ° 20, w - - 4,71 s, u n v o l u m e partiel sp6cifique v = 0,737 m l g l e s t fir6 des m e s u r e s de densit6s. D ' a p r ~ s ces d o n n 6 e s un r a y o n de Stokes d e 3,26 nm, un coefficient de diffusion D20, ~ ----6,61 10 .7 crn ~ sec -~ et un coefficient d e friction f/to ---- 1,21 ont 6t6 calcul6s. 2. L'61ectrophor~se e n gel d e p o l y a c r y l a m i d e SDS p e r m e t d ' e s t i m e r le poids m o l 6 c u l a i r e & 67 000. 3. La microh6t6rog6n6it6 est d6montr6e p a r 61ectrofocalisation. Le point iso61ectrique du v a r i a n t p r i n c i p a l est de 4,6. 4. La composition chimique a 6t6 d6termin6e. L'AFP est u n e glycoprot6ine c o n t e n a n t 7 p. cent de s u c r e s : 1,67 p. cent d ' h e x o s e s , 2,38 p. cent de N-ac6tyl g l u c o s a m i n e , et 1,8 p. cent d ' a c i d e N-ac6tyl n e u r a m i n i q u e .
Bovine AFP w a s puriiied b y ion e x c h a n g e c h r o m a t o g r a p h y on C.M. cellulose a n d DEAE S e p h a d e x A-50, qel filtration a n d i m m u n o s o r b e n t technique. AFP w a s h o m o g e n e o u s w h e n studied b y qel electrophoresis u n d e r n o n d e n a turing a n d d e n a t u r i n g conditions, b y ultracentrifuqation a n d b y i m m u n o l o g i c a l m e t h o d s . The following m o l e c u l a r d a t a w e r e o b t a i n e d :
Abbreviations. AFP, alpha-fetoprolein ; SDS, Sodium dodecylsulphate ; BSA, bovine serum albumine ; PBS, phosphate buffer saline ; PAS, periodic aeid-Sehiff ; NBS, N-Bromosuccinimide ; DTNB, dithio-nitrobenzoi'c acid ; PDAB, para dimelhylamino benzaldehyde ; P.A.G., Polyacrylamide get. To whom all correspondence should be addressed.
1. S e d i m e n t a t i o n m o l e c u l a r weight of velocity g a v e s°20, w = v o l u m e v = 0.737 m l sity m e a s u r e m e n t s .
equilibrium indicated a 66,500 a n d s e d i m e n t a t i o n 4.71 S. A partial specific g-1 w a s d e r i v e d from den-
From these d a t a , a Stokes r a d i u s of 3.26 nm, a diffusion coefficient D20. ~ ~ 6.61 10 -7 cm 2 sec -1 a n d a frictional ratio f/to = 1.21 w e r e calculated. 2. S o d i u m d o d e c y l s u l p h a t e disc electrophorests s u g q e s t s a m o l e c u l a r w e i g h t of 67,000. 3. G e l filtration pointed to a m o l e c u l a r weight of 75,000. 4. M i c r o h e t e r o q e n e i t y of AFP w a s demonstrated b y isoelectric focusing. The isoelectric point of the m a j o r c o m p o n e n t is 4.6. 5. The c h e m i c a l composition w a s d e t e r m i n e d . AFP is a q l y c o p r o t e i n containing 7 p e r cent c a r b o h y d r a t e including 1.67 p e r cent hexoses, 2.38 p e r cent N-acetyl g l u c o s a m i n e a n d 1.8 p e r cent N-acetyl n e u r a m i n i c acid.
Key-words : fetoprotein, molecular, parameters, amino acids composition, carbohydrate content.
S. A Iiau, J. M a r t i a n d J. M o r e t t i .
664
For several years analogies between embryonic a n d c a n c e r tiss~ues, a l r e a d y r e c o g n i z e d o n m o r p h o l o g i c a l b a s e s , h a v e b e e n c o n f i r m e d at t h e m o l e c u l a r level, b y t h e d i s c o v e r y of s e v e r a l c a r c i n o e m b r y o n i c a n t i g e n s . T h e b e s t k n o ~ v n of t h e s e a n t i g e n s is A F P , a g l o b u l i n s p e c i f i c of f e t a l s e r u m , b u t reappearing under pathological conditions such as h e p a t o c e l l u l a r c a r c i n o m a [1]. I n a d d i t i o n to i t s potential diagnostic importance for the adult ( h e p a t o m a ) a n d t h e f e t u s ( n e u r a l t u b e d e f e c t [2]) A F P is a n i n t e r e s t i n g m a r k e r of c e l l u l a r d i f f e r e n t i a t i o n a n d of a g r e a t v a l u e i n t h e u n d e r s t a n d i n g of t u m o r b i o l o g y [3]. A F P w e r e d e s c r i b e d i n m a n y v e r t e b r a t e species [4]. B u t t h e f i r s t f e t a l p r o t e i n d e s c r i b e d i n t h e c a l f ~vas f e t u i n [5.] a n d f o r m a n y y e a r s a c o n fusion was made between fetuin and A~P. The immunological difference between these two prot e i n s w a s r e c e n t l y e s t a b l i s h e d [6]. L i t t l e is k n o ~ v n a b o u t t h e b i o l o g i c a l f u n c t i o n of A F P e x c e p t i n r a t and mouse. Nevertheless some indications have been obtained through the finding of primary structure homologies between albumin and AFP in h u m a n s p e c i e s [7]. These studies need further development, but biochemical studies are often limited by the lack of m a t e r i a l . It is t h e r e a s o n w h y , i n a d d i t i o n to t h e i n t r i n s i c i n t e r e s t of c o m p a r a t i v e b i o c h e m i s t r y , w e t h o u g h t u s e f u l to l o o k f o r a n a b u n d a n t s o u r c e of A F P . W e c h o o s e d t h e f e t a l b o v i n e s e r u m s i n c e it is p o s s i b l e to o b t a i n l a r g e v o l u m e of b l o o d e v e n at e a r l y s t a g e s of f e t u s d e v e l o p m e n t . B u t b o v i n e AFP was not characterized and the present paper d e a l s w i t h its p u r i f i c a t i o n a n d p h y s i c o - c h e m i c a l properties.
Materials
and
Methods.
Serum.
Pooled fetal calf s e r u m was used as the s t a r t i n g m a t e r i a l for the isolation of AFP. Blood was obtained f r o m 2-4 m o n t h s old fetuses b y p u n c t i o n of the j u g u l a r vein. After clotting, the serum was separated b y cent r i f u g a t i o n . Sera are pooled in 20~ ml lots a n d stored at --20°C. In the fetal serum the level of A F P decreases f r o m 4 m g / m l for fifty days old fetuses to a b o u t 0,2 m g / m l at b i r t h [8], (photo 1). In t h e samples used for purification, the level averaged 2 m g / m l .
Chemicals. Agarose (Indubiose A 37) was supplied b y I n d u s t r i e Biologique Franqaise, S.A. F r e u n d ' s complete a d j u v a n t was p u r c h a s e d f r o m Difco (Detroit). Glutaraldehyde, acrylamide, N,N b i s a c r y l a m i d e were E a s t m a n products. Ion exchange cellulose was supplied by W h a t m a n Biochemicals Ltd (England). Sephadex G 200, DEAEBIOCHIMIE, 1978, 60, n ° 6-7.
Sephadex A-50, Sepharose 4 B were supplied by P h a r m a r i a (S~veden). Phosphorylase B, a l p h a - a m y l a s e (B. subtilis), BS~A, o v a l b u m i n , c h y m o t r y p s i n o g e n A a n d cytochrome c were o b t a i n e d f r o m B o e h r i n g e r (Mannhelm). D i p h e n y l a m i n e , a n t h r o n e , PDAB a n d SDS were p u s c h a s e d f r o m Merck (Darmstadt, Germany). Bovine fetuin, s i d e r o p h i l i n a n d h u m a n g a m m a - g l o b u l i n s were p r e p a r e d in the l a b o r a t o r y . IMMUNSERA PREPARATION.
1. Antibodies a g a i n s t fetal calf serum were p r e p a r e d in New Zealand r a b b i t s b y weekly s u b c u t a n e o u s injections of increasing volumes of s e r u m (0,1 to 1 ml) mixed w i t h complete F r e u n d ' s a d j u v a n t (v/v). Blood was collected two weeks a f t e r the f o u r t h injection. A n t i s e r u m was recovered b y c e n t r i f u g a t i o n a n stored w i t h sodium azide (0:,1 per cent) at 4°C. 2. A n t i s e r u m was m a d e specific b y a d s o r p t i o n w i t h insolubilized a d u l t serie proteins. Ox serum was polymerized w i t h g l u t a r a l d e h y d e according to A v r a m e a s a n d T e r n y n c k [9]. After w a s h i n g to remove glutaraldehyde t h i s p o l y m e r is g e n t l y grinded in a P o t t e r h o m o g e n e i z e r a n d m i x e d w i t h a n equal volume of anti fetal calf serum: The m i x t u r e is left at 4~C overnight u n d e r stirring. The procedure m a y be repeated twice u n t i l the serum is quite feto-specific. The specificity of the anti serum h a s been discussed in a previous p a p e r [8]. 3. Antibodies a g a i n s t ox s e r u m p r o t e i n s were prepared in the same way. The Ig f r a c t i o n was precipitated w i t h 2.0M a m m o n i u m sulfate. After dialysis, the Ig were purified b y c h r o m a t o g r a p h y on DEAE-cellulose column using 0.05 M p h o s p h a t e buffer pH 7.0. The i m m u n o s o r b e n t was p r e p a r e d b y coupling the i m m u n o g l o b u l i n f r a c t i o n to Sepharose 4B as previously described [10]. PREPARATIVE PURIFICATION OF A F P .
Step I : CM Cellulose Chromatography. In a typical p r e p a r a t i o n , ~0 m l of fetal calf serum were dialyzed overnight a g a i n s t 2 1 of 0.01 M sodium acetate buffer pFt 4.6' A precipitate was discarded by c e n t r i f u g a t i o n a n d the s u p e r n a t a n t was applied to a Clff-cellulose c o l u m n (30 × 2 cm) e q u i l i b r a t e d w i t h the same (acetate) buffer. A f r a c t i o n C1 (20,0 ml) was eluted w i t h the s t a r t i n g buffer. It c o n t a i n s essentially f e t u i n [11]. The buffer was t h e n m a d e 0.1 M in NaC1, allowing the e l u t i o n of a C 2 f r a c t i o n (2,~0 ml), w h i c h contains t h e m a j o r p a r t of the a l b u m i n . The t h i r d f r a c t i o n C 3 (120, ml) eluted w i t h 0.1 M sodium acetate buffer pH 5.6. c o n t a i n i n g the AFP was dialyzed a g a i n s t several changes of 0.1 M Tris-HC1 buffer pH 8.6.
Step 2 : DEAE-Sephadex A-50 Chromatography. After dialysis, C 3 was applied to a D,l~AE-Sephadex c o l u m n (2~5 × 2 cm) e q u i l i b r a t e d w i t h the Tris-HC1 buffer. The p r o t e i n s were eluted by a l i n e a r g r a d i e n t of NaC1 f r o m 0 to 0.1 M. The fractions c o n t a i n i n g A,F P were pooled, dialyzed and freeze dried.
Step 3 : Gel Filtration. The lyophilizcd protein was dissolved in a small volume of 0.05 Mr Tris-HCl buffer pH 8.3, 0.1 M NaCl,
Bovine alpha-fetoprotein. a p p l i e d to a K 1 5 / 9 0 c o l u m n filled w i t h S e p h a d e x G 21)0 a n d e l u t e d w i t h t h e s a m e b u f f e r . A F P c o n t a i n i n g f r a c t i o n s w e r e pooled, d i a l y z e d a n d l y o p h i l i z e d .
Step ~ : Use of the lmmunosorbent. T h e p r o t e i n w a s d i s s o l v e d in PBS a n d a l l o w e d to d i f f u s e i n t h e i m m u n o s o r b e n t c o l u m n (25 X 0,5 em) during 4 hours. After this time, the elution was perf o r m e d w i t h P B S b u f f e r . T h e p r o t e i n p e a k (20 ml) c o n t a i n i n g p u r e AF,P w a s collected, d i a l y z e d a n d l y o philized. ANALYTICAL METHODS.
Immunoelectrophoretic Methods. Immunoelectrophoreses were performed on glass p l a t e s (9 X 12 cm) ; t h e a n t i b o d i e s - c o n t a i n i n g gel w a s p r e p a r e d i n b a r b i t a l b u f f e r 0.02 M p H 8.6 w i t h 1 p e r cent agarose. a) (< R o c k e t >> i m m u n o e l e c t r o p h o r e s i s w a s p e r f o r m e d a t 4 V / c m f o r 2 h [12]. b) C r o s s e d i m m u n o e l e c t r o p h o r e s i s [13] w a s c a r r i e d o u t w i t h 1-5 ~1 s a m p l e i n t h e first d i m e n s i o n at 4 V / c m f o r 3 h a n d i n t h e s e c o n d d i m e n s i o n a t 1,2: V / c m f o r 12 h. T h e p l a t e s w e r e t h e n w a s h e d i n s a l i n e (0.1 M NaC1)s d r i e d a n d s t a i n e d .
Polyucrylumide Gel Electrophoresis. S o d i u m d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s (5-7.5-10 a n d 15 p e r c e n t a c r y l a m i d e ) w a s p e r f o r m e d a s d e s c r i b e d b y W e b e r a n d O s b o r n [14]. T h e gels w e r e 0.8 X 12~ c m i n size. T h e p r o t e i n s a m p l e s c o n t a i n e d 5 to 2,0 itg/lO ~tl. Electrophoresis was carried out at room temperature at 8 m A p e r gel d u r i n g 6, to 8 h. B r o m o p h e n o l b l u e w a s u s e d as t h e t r a c k e r dye. Six p r o t e i n s of k n o w m o l e c u l a r w e i g h t w e r e s u b j e c t e d to e l e e t r o p h o r e s i s u n d e r t h e same conditions : phosphorylase B (rabbit), siderophilin, BSA,, o v a l b u m i n , e h y m o t r y p s i n o g e n e A a n d c y t o c h r o m e c. Gels w e r e s t a i n e d w i t h 0.25 p e r c e n t C o o m a s sie b l u e i n m e t h a n o l / a c e t i c a c i d / w a t e r ( 5 / 1 / 4 b y vol.) a n d d e s t a i n e d b y e l e e t r o p h o r e s i s i n acetic acid 7.5 p e r cent.
lsoelectric Focusing. T h e i s o e l e c t r i e p o i n t w a s e s t i m a t e d a c c o r d i n g to V e s t e r b e r g a n d S v e n s s o n [15], u s i n g A m p h o l i n e w i t h a p H r a n g e of 4-6 i n a c o n c e n t r a t i o n of 2 p e r c e n t a n d a s u c r o s e g r a d i e n t of 5-5~) p e r c e n t i n a 110 m l c o l u m n (LKB P r o d u e t e r s , S t o c k h o l m ) . A f t e r 72 h a t 600 V a n d 4°G, t h e c o l u m n w a s e m p t i e d . F r a c t i o n s of 2 m l w e r e collected. T h e i r p H w a s m e a s u r e d w i t h a p ~ m e t e r I s i s 1000 ( T a e u s s e l , L y o n , F r a n c e ) . T h e o p t i c a l d e n s i t y o f t h e e f f l u e n t w a s m o n i t o r e d a t 280 n m (Isco UA 5 Recorder). A c r y l a m i d e p l a t e s (LKB A m p h o l i n e P A G p l a t e ) c o n t a i n i n g 2~ p e r c e n t A m p h n l i n e w i t h a pH, r a n g e 3.5 to 9.5 w e r e a l s o e m p l o y e d a c c o r d i n g to t h e m a n u f a c t u r e r i n s t r u c t i o n s a n d stained, w i t h C o o m a s s i e b l u e . I n a d d i t i o n , a n a r r o w s t r i p of gel w a s c u t off a n d l a y e r e d o n t o a n a g a r o s e gel p l a t e c o n t a i n i n g a n t i b o d i e s a g a i n s t AFP. The protein pattern was developped by electroimmunodiffusion.
Partial Specific Volume. T h e p a r t i a l specific v o l u m e of t h e p r o t e i n w a s d e t e r m i n e d a t 2 0 ° C _ 0.~1! u s i n g a D ~ A 10 d e n s i m e t e r ( A n t o u P a r r K. G., Graz, A u s t r i a ) . T h e d e n s i t i e s o f t h e s o l u t i o n s u s e d in v i s c o s i t y e x p e r i m e n t s w e r e d e t e r m i -
BIOCHIMIE, 1 9 7 8 ,
60, n ° 6 - 7 .
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n e d a f t e r c a l i b r a t i o n of t h e q u a r t z t u b e w i t h air, w a t e r and 0.05M phosphate b u f f e r p H 6.5 c o n t a i n i n g 0.1 M NaCI.
Viscosity. T h e v i s c o s i t y m e a s u r e m e n t s w e r e m a d e a t 20°C ± 0.01 in a U b b e l o h d e v i s c o s i m e t e r (Flea, P a r i s ) e q u i p p e d with an auto loading system and a electronic timer activated by two photocells. A stock solution was prepared by dissolving AFP i n t h e b u f f e r a t a c o n c e n t r a t i o n of 4,0 m g / m l . T h i s sample was dialyzed overnight against the same buffer (1000 t i m e s b y v o l u m e ) . T h e s o l u t i o n w a s clarified u s i n g a M i l l i p o r e filter (0.45 it). All t h e p h y s i c a l m e a s u rements were done starting from this stock solution. T h e l o a d i n g v o l u m e w a s 5. m l . S u c c e s s i v e d i l u t i o n s of the protein solution were made within the instrument b y r e p l a c i n g a v o l u m e of s o l u t i o n b y a n e q u a l v o l u m e of buffer. T h e d e n s i t y o f t h e w i t h d r a w n s a m p l e w a s i m m e d i a t e l y m e a s u r e d a n d t h e c o n c e n t r a t i o n of p r o t e i n e s t i m a t e d f r o m t h e a b s o r b a n c e at 278 n m o n a gravimetrically made dilution.
Ultracentrifugation. U l t r a e e n t r i f u g a t i o n w a s p e r f o r m e d a t 20°C b y u s i n g a MSE c e n t r i f u g e w i t h a 6 cells r o t o r b y s c a n n i n g t h e a b s o r b a n e e a t 280 n m . T h e s a m p l e w a s d i a l y z e d 24 h a g a i n s t t h e p h o s p h a t e - - NaC1 b u f f e r . S e d i m e n t a t i o n v e l o c i t y e x p e r i m e n t s w e r e r u n a t 54 200 r e v / m i n a n d scanned at 8 minutes intervals. Sedimentation equilibrium experiment was run for 40 h a t 1,0 000 r e v / m i n i n t h e s a m e r o t o r . A b s o r b a n c e of t h e s o l u t i o n i n t h e r u n w a s 0.23.
Analytical Gel Filtration. O n e m g of p u r i f i e d A F P w a s s u b j e c t e d to gel filtrat i o n o n a S e p h a d e x G 200 c o l u m n (K 25/90, P h a r m a c i a ) i n 0.05 Tris-HC1 b u f f e r p H 7.0 c o n t a i n i n g 0.2 p e r c e n t s o d i u m azide. T h e c o l u m n w a s c a l i b r a t e d w i t h c o m m e r c i a l p r o t e i n s as s t a n d a r d m a r k e r s ( B o e h r i n g e r ) . M o l e c u l a r r a d i u s of A F P w a s d e t e r m i n e d i n t h e s a m e c o n d i t i o n s . T h e S t o k e s r a d i i a of t h e m a r k e r p r o t e i n s w e r e t a k e n f r o m l i t t e r a t u r e [16]. D i f f u s i o n coefficient D o f A,F P w a s c a l c u l a t e d u s i n g t h e E i n s t e i n f o r m u l a : D~0.w : R T / N 6 ~ ~1 a where N = Avogadro's number ~1 : V i s c o s i t y a : Molecular radius
Absorption Spectra and Absorbance Coefficient. The absorption spectra was established with a Cary 118 s p e c t r o p h o t o m e t e r u s i n g b o v i n e A F P s o l u t i o n i n distilled water. The protein concentration was measured by weighing ( a f t e r e v a p o r a t i o n to c o n s t a n t w e i g h t ) a p r e c i s e v o l u m e of t h e p r o t e i n s o l u t i o n .
Fluorescence of the Protein. This study was made in the conditions described b y P a j o t [17]. A F P w a s i n c u b a t e d f o r 39 r a i n i n 6 M g u a n i d i n e h y d r o c h l o r i d e c o n t a i n i n g 30 m M m e r c a p t o ethanol. The fluorescence was measured using a M P F 3L P e r k i n - E l m e r s p e e t r o f l u o r i m e t e r . T h e fluorescence intensity scale was calibrated using solutions of t r y p t o p h a n ( i n t e r n a l c a l i b r a t i o n ) .
Amino Acids Analysis. A n a l y s e s w e r e c a r r i e d o u t o n a B e c k m a n 124 M. a u t o matie amino acid analyser. Samples were hydrolysed
S. Aliau, J. Marti and J. Morelti.
666
p a r t of a l b u m i n (fraction C 2). In a s e c o n d step (fig. 2) the r e m a i n i n g p e a k C 3 is f r a c t i o n a t e d on DEA~E-Sephadex. The p e a k F 2 c o n t a i n s A F P and m a n y i m p u r i t i e s . Even after a t h i r d step of gel filtration s e v e r a l o t h e r p r o t e i n s still r e m a i n in small amounts. T h e y i e l d of p u r i f i c a t i o n is n e a r 25 p e r cent.
i n v a c u u m - s e a l e d t u b e s w i t h 100 ~1 of c o n s t a n t b o i l i n g HC1 a t 110°C. T h e y w e r e s u b j e c t e d to h y d r o l y s i s f o r p e r i o d s of 24, 48 o r 72 h. Serine and threonine values were estimated by e x t r a p o l a t i o n to z e r o t i m e a n d i s o l e u c i n e a n d v a l i n e f r o m t h e 72' h h y d r o l y s i s [18]. C y s t e i n e a n d m e t h i o n i n e ~ e r e a n a l y s e d as c y s t e i c a c i d a n d m e t h i o n i n e s u l p h o n e a f t e r o x y d a t i o n of t h e s a m p l e s b y t h e m e t h o d of t~irs [19].
T h e s e i m p u r i t i e s are r e m o v e d b y using an i m m u n o s o r p t i ~ n technique. At this stage the prep a r a t i o n a p p e a r s h o m o g e n o u s w i t h r e s p e c t to electrophoresis and immunoelectrophoretic criteria (photo 2). P r e p a r a t i o n in a l a r g e r scale w e r e also m a d e s t a r t i n g f r o m 200 ml of serum.
Sugars analyses. I d e n t i f i c a t i o n of s u g a r s w a s p e r f o r m e d u s i n g a P e r k i n - E l m e r F 11 Gas l i q u i d c h r o m a t o g r a p h , e q u i p p e d w i t h a O V 21@, c o l u m n o n c h r o m o s o r b HMDS. M e t h a n o l y s i s w a s m a d e a c c o r d i n g to C l a m p et al [20] a n d t r i f l u o r o a c e t y l a t i o n a c c o r d i n g to Z a n e t t a el al [21]. H e x o s e s w e r e a n a l y z e d b y t h e a n t h r o n e m e t h o d [22], s i a l i c a c i d s b y d i p h e n y l a m i n e m e t h o d a c c o r d i n g to N i a z i a n d S t a t e [23] a n d o s a m i n e s a c c o r d i n g to E l s o n a n d M o r g a n [24].
Polyacrylamide gel electrophoresis. This m e t h o d s h o w e d the h o m o g e n e i t y of the final p r o d u c t . Gel electrophoresis in r e d u c i n g cond i t i o n s i n d i c a t e d t h a t A F P is a single p o l y p e p t i d e chain. P~ovine A,FP gives p o s i t i v e r e a c t i o n w i t h P.A.S. staining.
Results.
E l e c t r o p h o r e s i s at different a c r y l a m i d e concent r a t i o n from 6 to 1'5 p e r cent a c c o r d i n g to Segrest and J a c k s o n [25] gave a m o l e c u l a r w e i g h t of 67 000 ; no significative differences w e r e o b s e r v e d b e t w e e n the different m o l e c u l a r w e i g h t estimations.
Purification procedure. I n v i e w to p r e p a r e AFP, we used the k n o w l e d g e o b t a i n e d d u r i n g the isolation of ovine fetuin. This g l y c o p r o t e i n is like A F P an a l p h a g l o b u l i n and its level in fetal s e r u m is h i g h e r than the level of AFP. The first step (fig. 1) a l r e a d y used d u r i n g the t m r i f i c a t i o n of fetuin offers the a d v a n t a g e to d i s c a r d this p r o t e i n (fraction C 1) and the m a j o r
1,5 '
Ultraviolet Spectrum. The a b s o r p t i o n s p e c t r u m of the p r o t e i n is given figure 3. The a b s o r b a n c e coefficient A (1 p e r cent,
C1
C3 C2
E c o
0,PH1M4.6
u c = o
J~
I
0.1M
i
0,5
50
Fraction
'
l
,
'
100
'
'
Number
Chromatography of fetal calf Serum on c a r b o x y m e l h y l c e l l n l o s e ( W h a l m a n CM 23) c o l u m n (22 × 3 cm) ; f l o w rate 70 m l / h , 10 m l fractions. FI6.
1. - -
P r o t e i n s a r e m o n i t o r e d a t 280 rim. A F P w a s d e t e c t e d b y e l e c t r o i m m u n o d i f f u s i o n (¢ r o c k e t s >>). AI~P c o n t a i n i n g f r a c t i o n s a r e m a r k e d .
BIOCHIMIE, 1978, 60, n ° 6-7.
Bovine alpha-fetoprotein.
667
Buffer+NaCI O,1M
TrisHCI
E
1 O"
PH 8,6
O , I M ~
~"
J
J
~
F3
0 oo (N
fo
¢3 u
c 0,5 .Q 0 J~
<
I
I
I
I
lO
I
50
Fraction
I
I
I
I
i
!
!
lOO
Number
F1a. 2. - - C h r o m a t o g r a p h y of (:~ on D E A E - S e p h a d e x A-50 c o l u m n (25 cm X 2 cm) ; f l o w rate 12 m l / h , 3 ml fractions. L i n e a r g r a d i e n t T r i s HC1 i = 0.1, p H 8.6, 200 ml. S a m e b u f f e r ÷ 0.1 M 200 ml.
PHOTO I. Crossed i m m u n o e l e c t r o p h o r e s i s of fetal bovine s e r u m against rabbit whole fetal bovine a n t i s e r u m . A. S e r u m f r o m a 80 days o l d fetuse. B. S e r u m f r o m a 250 d a y s old fetuse. V o l u m e of s a m p l e : 5 ~1 of fetal s e r u m . E l e c t r o p h o r e s i s w e r e carried o u t f o r 3 h a t 40 v o l t s in the first d i m e n s i o n a n d f o r 12 h at 15 v o l t s in t h e second dimension. Curved a r r o w m a r k s A F P .
BIOCHIMIE, 1978, 60, n ° 6-7.
S. Aliau, J. Marti a n d J. Moretti.
6:68
PHOTO 2.
Crossed immunoelectrophoresis of AFP fraction after immunosorption. Same conditions as in photo 1.
278 nm) is f o u n d to be 4.60 dl.g lcln-1. T h e m o l a r e x t i n c t i o n coef,f i c i e n t is e = 3.08 104 M-1 cm-1 at 280 nm. T h e s p e c t r u m s h o w s c h a r a c t e r i s t i c p h e TABLE I.
Values of density ~ of the solutions employed for partial specific volume determination, at 6 different protein concentrations. g. m1-1
p
0.03630 0.02527 0.01600 0.00956 0.00633 0.0000
1.01563 1.01306 1.0105 1.0080 1.0078 1.00359
T h e d e n s i t i e s of t h e s o l u t i o n s and of t h e s o l v e n t a l o n e w e r e m e a s u r e d (table I). T h e d e p e n d e n c e o f d e n s i t y ~ on p r o t e i n c o n c e n t r a t i o n w a s l i n e a r and, b y a least s q u a r e s p r o c e d u r e , f o u n d to be : ? = 0.~60 02~5 C + 1.006 317 w h e r e C [26] is t h e c o n c e n t r a t i o n of t h e p r o t e i n s o l u t i o n in g.ml-1. T h e p a r t i a l s p e c i f c v o l u m e was determined from
~= --
C
T , ?, ~ , C
w h e r e ~ is a l i n e a r f u n c t i o n of C : ~
--
c
n y l a l a n i n e spikes. W h e n p r o t e i n w a s d i s s o l v e d in 0.1 M 4NaOH, n o s h o u l d e r w a s o b s e r v e d t h a t w o u l d i n d i c a t e t h e p r e s e n c e of tryptoph.an.
Partial Specific Volume. Four dilutions were prepared from the stock p r o t e i n s o l u t i o n ( c o n c e n t r a t i o n : 3.6,3 mg.ml-1).
BIOCHIMIE, 1978, 60, n ° 6-7.
s i t y w i t h r e s p e c t to C at c o n s t a n t t e m p e r a t u r e T, p r e s s i o n P, a n d c h e m i c a l p o t e n t i a l v of all substances except the protein. The mean value of~ so o b t a i n e d is 0.737 --4--- 0.0001 ( r e g r e s s i o n coeffic i e n t = 0.99898).
Bovine alpha-fetoprotein. Viscosity. F r o m t h e f l o w t i m e of t h e five s o l u t i o n s , t h e intrinsic viscosity can be calculated using the f o r m u l a [27] : (~) = l i m i t of -
t --
% to
-
669
The axial ratio obtained from the viscosity increment from Simha's relation assuming that t h e m o l e c u l e is a n u n h y d r a t e d e l l i p s o i d of r e v o l u t i o n , a r e 5 . 6 f o r a p r o l a t e e l l i p s o i d a n d 8.5 f o r an oblate ellipsoid.
w h e n C --> O
% to C
w h e r e ~o i s t h e d e n s i t y of s o l v e n t a n d ~ t h e d e n s i t y of s o l u t i o n , t a n d to, r e s p e c t i v e f l o w t i m e s , a r e t h e m e a n of t h e l a s t t e n v a l u e s f r o m a s e r i e s of measurements repeated until the differences b e c a m e less t h a n 10 .2 sec.
Molecular Parameters derived from Gel Filtration. T h e a p p r o x i m a t e m o l e c u l a r w e i g h t v a l u e of A F P o b t a i n e d f r o m t h e gel f i l t r a t i o n d a t a [29] is 75,000. T h e S t o k e s r a d i u s a m e a s u r e d i n S e p h a d e x G 200 c h r o m a t o g r a p h y is 3.52 n m . T h e d i f f u s i o n coeffic i e n t D, c a l c u l a t e d f r o m t h e v a l u e s of a a n d n is D20,w :
6.05 10-7. c m 2 . s -1
Analytical ultracentrifugation. A s e d i m e n t a t i o n c o e f f i c i e n t s:0 ' w : 4,71 _ 0.1 was calculated from the sedimentation velocity r u n . A b s o r b a n c e s of t h e s o l u t i o n s w e r e f r o m 0.21 to 0.50 O.D.
0,7
0.6
Sedimentation equilibrium studies demonstrated t h a t t h e r e l a t i o n s h i p b e t w e e n t h e l o g a r i t h m of t h e r e c o r d e r d e f l e c t i o n (d) a n d r 2 w a s l i n e a r indicating monodispersity of t h e p r e p a r a t i o n . U s i n g t h e v a l u e 0,73'7 ml.g-1 f o r t h e p a r t i a l s p e c i fic v o l u m e a n d t h e v a l u e 0.16923 of t h e s l o p e , a m o l e c u l a r w e i g h t of 66,500 ± 1,500 is o b t a i n e d from the formula
0.3
2RT
M =
0.2
w
•
240
i
,
:eO
g
,
:eO
Wavelength
i :00
, .
,
I
:=0
18
nm
U H r a v i o l e t spectrum of the p u r i f i e d AFP.
1,7
The m e a s u r e m e n t was obtained on a double b e a m s p e c t r o p h o t o m e t e r Gary 18. 1. S p e c t r u m obtained w i t h 1 m g / m l A,F P in water. 2. Spectrum o b t a i n e d i n NaOH 0.I N w i t h o u t an), correction for the t u r b i d i t y .
1,6
Fro. 3. - -
. 2.303 tg ~ [30]
~'~) to2 r
(1 - -
I,S 1.4
1,2 ¢O
T h e p l o t of ( 0 ) v e r s u s C is l i n e a r (r = 0.99946) a n d , b y a l e a s t s q u a r e p r o c e d u r e , f o u n d to b e : (0) = 1 6 . 5 6 2 5 7 2 C 4- 5.704 638 T h e v a l u e of t h e i n t e r c e p t g i v e s (~1) w h e n C --+ O 01) = 5.70 ± 0.05 ml.g-1 T h e v a l u e of t h e v i s c o s i t y i n c r e m e n t v c a l c u l a t e d f r o m t h e i n t r i n s i c v i s c o s i t y (0) a n d t h e p a r t i a l s p e c i f i c v o l u m e ~ f r o m t h e r e l a t i o n [28] v = ( ~ ) / v w a s f o u n d to b e 7.73.
BIOCHIMIE, 1978, 60, n ° 6-7.
O) .OO
1,~ 1,0 0.9 0,8" 0.~-' 06
o
,
•
•
,,
•
,;
so racm
2
Fro. 4. - - Sedimentation equilibrium ullraventrifuffation of AFP. 10{)50 r e v / m i n at 20°C. (Value of slope 0.16923, regression : 0.99968).
S. Aliau, J. Marli a n d J. Moretli.
670
c o n t r o l l e d by crossed i m m u n o e l e c t r o p h o r e s i s , The m e d i a n v a r i a n t has a p H i of 4.6.
F r o m the values of M, S, and v_a diffusion c.oefficient can be c a l c u l a t e d s RT D : __ o : 6.61 10 -7 cm 2 sea -1 M ( 1 - - - v g ) D2°'w T h e f r i c t i o n a l ratio f/fo calculated a c c o r d i n g to Oncley [31] is 1.2~1. T h e axial r a t i o s o b t a i n e d f r o m the f r i c t i o n a l ratio by P e r r i n ' s r e l a t i o n assuming that the p r o t e i n is an u n h y d r a t e d ellipsoid of r e v o l u t i o n is 4.2 for a p r o l a p s e and 5 for an oblate ellipsoid.
-5 '4~5 .4
3.5 mO
0.S
e~
T h e r a d i u s of the e q u i v a l e n t s p h e r e calculated from M
5,5
O,75 "
O O
O,=S O
(1 - - ~ )
a -6~
O
is 32~.6 ~ (3~.26 nm).
Ns20. w ,o
Isoelectric Focusing. The isoelectric pattern obtained from column e x p e r i m e n t s shows one p e a k a r o u n d p H 4.6 ; a s h o u l d e r is detected at pH 4.8.6 (£~g. 5). T h e r e is an i m m u n o l o g i c a l i d e n t i t y b e t w e e n the peak and the shoulder. No others v a r i a n t s can be s e p a r a t e d in the p H 4.6 scale. T h e ampholine~PAG plate exper i m e n t reveals a m i c r o h e t e r o g e n e i t y of AFP betw e e n pH 4.4 and 5.3. T h e r e are at least nine v a r i a n t s s h o w i n g an i m m u n o l o g i c a l i d e n t i t y ~ as
;o Ffa orion
'
;o
Number
FI6. 5. Isoelectric focusing of bovine AFP, pH 4-6 gradient. The experiment was carried out as described -
-
in methods ; the anode was at the bottom of the column, pH is represented by the dotted line.
Carbohydrate composition. In table III, the c a r b o h y d r a t e c o m p o s i t i o n of A F P is c o m p a r e d w i t h those of the t w o v a r i a n t s of rat A F P [32]. The f r a c t i o n a r y n u m b e r for
TABLE II.
Amino acid composition of AFP. The e x p e r i m e n t a l results h a v e been n o r m a l i z e d . Best values are as defined by A m b l e r El81. The n u m b e r of residues m o l e c u l e h a v e been c a l c u l a t e d by the m e t h o d of Delaage [41]. Amount found after hydrolysis ior Amino
acid
Aspartate asparagine Threonine Serine Glutamate glutamine Proline
"
24 h
48 h
72 h
50.08 33.70 44.36 102.64 27.38
50.81 32.66 40.43 104.86 28.84 32.29 43.82 28.34 38.43 9.28 34.30 60.25 16.02 29.16 46.10 16.80 24.83
50.55 32.54 40.56 101.46 27.80 32.24 43.29 27.54 49.94 9.26 35.82 63.59 17.30 28.57 45.52 16.86 26.79
Glyctne
33.17
Alanine Half-cystine Valine Methionine Isoleueine Leucine Tyrosine Phenylalanine Lysine Histidlne Arginine Tryptophan Total residues
43.90 28.13 37.52 9.00 33.79 58.33 17.18 30 20 45.51 16.58 26.99
value
Best
Residues molecule protein
integral
50.51 34.70 47.00 102.99 28.00 32.57 43.67 21.1 (a) 49.94 9.18 35.82 63.59 16.83 27.80 (b) 45.71 16.74 26.20
41 ~.99 28.86 39.07 85.62 23.61 27.46 36.30 17.54 42.10 7.74 29.78 52.86 13.99 23.11 38.00 13.92 22.09
42 29 39 86 24 27 36 18 42 8 30 53 14 23 38 14 22
0
a Value founded for cysteic acid after performic oxydation according to Hirs [19]. b Corrected for contamination by glucosamine, c From fluormnetric results.
BIOCHIMIE, 1978, 60, n ° 6-7.
Nearest
0 545
Bovine alpha-f etoprotein.
67'1
TABLE III.
Carbohgdrate composition of bovine AFP compared to rat AFP. Values (per cent by w e i g h t and m o l / m o l ) are c a l c u l a t e d on the basis of M = 67 000. Results of m o l a r ratios t ak en f r o m t h e basis of 3: m a n n o s e residues. CalI AFP
Per cent
Mannose Galactose N. Ac. Glueosamine NANA Total
1.61 1.07 2.38 (a) i.80 6.81 (b)
mol/mol
6 4 7.06 (a) 4 21
Rat AFPA and B variants molar ratio
3.0 2.08 3.65 2.0
tool/tool
raotar ratio
A
B
A
B
6.1 6.1 7.6 6.2
6.1 5.7 7.8 5.8
3.0 3.1 3.8 3.1
3.0 2.9 3.9 2.9
a The glucosamine content of AFP is certainly higher. This may be due to the incomplete release of glucosamine during hydrolysis. The correct values seems be 8 mol/mol and 2.6 per cent. b This value agree with 6.8 per cent and 7.1 per cent found for A and B variants of rat AFP. N - a c e t y l - G l u c o s a m i n e m a y be due to an i n c o m plete release of the sugar f r o m the c a r b o h y d r a t e m o i e t y d u r i n g h y d r o l y s i s . T h e m o l a r ratios on the basis of 3 m a n n o s e i n d i c a t e a possible structure closely s i m i l a r to rat AFP. This s t r u c t u r e m ay consist of tw o glycans, e a c h one c o n t a i n i n g 2 NaNa - ) M'an - 2 Gal - 4 NAcGluc (Bayard an d Kerckaert, p e r s o n a l c o m m u n i c a t i o n ) .
Amino acid composition. The amin,o acid c o m p o s i t i o n of the p r o t e i n as d e t e r m i n e d by the a u t o m a t e d analysis of acid h y d r o l y s a t e s is given in table II. As a l r e a d y p o i n ted out, optical studies, even the f i u o r o m e t r i c m e t h o d of Pajot, failed to r e v e a l the p r e s e n c e of t r y p t o p h a n in the protein. O i h e r m e t h o d s including the NIBS-oxydation a c c o r d i n g to P a t c h o r n i k et al. [33~ and the c o l o r i m e t r i c m e t h o d of Spies and C h a m b e r s [34] gave values far b e l o w one r e s i d u e p e r mole of protein. It w a s c o n c l u d e d to the absence of t r y p t o p h a n , w h i c h accounts for the l o w value ~)f the the e x t i n c t i o n coefficient. Any cys.teine r e s i d u e was available for t i t r a t i o n w i t h 5-5' DTND in the p r e s e n c e or absence of 8 M u r e a [35]. T h e 18 cysteines in the p r o t e i n are thus assum e d to be i n v o l v e d in 9 disulfide bridges.
Discussion .
.
.
.
This p a p e r d e s c r i b e s a p r o c e d u r e for the p u r i fication of b o v i n e AFP, to a p p a r e n t c h e m i c a l an d
BIOCHIMIE, 1978, 60, n ° 6-7.
i m m u n o l o g i c a l h o m o g e n e i t y , and r e p o r t s the results of m e a s u r e m e n t s of its m o l e c u l a r parameters. The m a j o r difficulty was tile e l i m i n a t i o n of fetuin and al b u m i n ~ , h i c h are the more i m p o r t a n t constituants of fetal serum. F o r a part, it is o v e r c o m e by a CM-cellulose c h r o m a t o g r a p h y , a l l o w i n g to d i s c a r d fetuin. Th e m a j o r losses o c c u r at this step, w h i c h is h o w e v e r an easy w a y to separate the t w o fetoproteins. T h e last step of p u r i f i c a t i o n i n v o l v i n g an i m m u n o s o r p t i o n is n e c e s s a r y to r e m o v e al b u m i n traces,, i m p o s s i b l e to d i s c a r d by classical p r o c e d u r e s , because of its s i m i l a r i t y w i t h AFP. Th e m o l e c u l a r w e i g h t s estimated by SDS-disc e l e c t r a p h o r e s i s and by u l t r a c e n t r i f u g a t i o n are similar, about 67,000. Gel filtration gives a h i g h e r value but this reslalt m a y be e x p l a i n e d by the fact that A FP is a g l y c o p r o t e i n [361. Molecular w e i g h t s are of the same o r d e r of m a g n i t u d e for o t h er A FP : 70,000 (rat, mouse [373, m an [38], pig [39], or ~8,000 (rabbit [40]). The statistical m e t h o d of Delaage [41] relies the a m i n o a c i d c o m p o s i t i o n to the m o l e c u l a r w e i g h t . In t h e p r e s e n t case this m e t h o d gives 2 values of almost equal p r o b a b i l i t y : 6.2,000 and 70,000. A d d i n g the i n c r e m e n t of the c a r b o h y d r a t e m o i e t y ~5,600), the m o l e c u l a r w ei g h t s are 67,500 and 75,600. T h e first value is the most p r o b a b l e and is in good a g r e e m e n t ~vith the e l e c t r o p h o r e s i s and c e n t r i f u g a f i o n m e a s u r e m e n t s .
672
S . A l i a u , J. M a r t i a n d J . M o r e l t i .
B o v i n e AI~P is r i c h i n a c i d i c r e s i d u e s , f r e e , o r in the amide form (aspartic + glutamic acid 2'3:5 p e r c e n t ) . T h i s a c c o u n t s f o r t h e a c i d i c isoe l e c t r i c p o i n t of t h e p r o t e i n ( p H i = 4.6). T h e s e t y p e s of r e s i d u e s a r e k n o w n to f a v o r h y d r o g e n bonding, and their high percentage may be indic a t i v e of a r a t h e r c o m p a c t t e r t i a r y s t r u c t u r e . T h e v a l u e of 1.4: f o r t h e r a t i o of p o l a r to a p o l a r r e s i d u e s , c a l c u l a t e d a c c o r d i n g to H a t c h [42] f a v o r s t h i s h y p o t h e s i s . T h e h i g h c o n t e n t of g l u t a m i c r e s i d u e s ( ~ l u + Gln) a p p e a r s to b e a c h a r a c t e r i s t i c s h a r e d i n c o m m o n w i t h A F P of o t h e r s p e c i e s . T h e p h y s i c a l m e a s u r e m e n t s (f/fo = 1,21) i n d i c a t e t h a t b o v i n e &FI ) i s a g l o b u l a r p r o t e i n . T w o m o l e c u l a r f o r m s a r e o b s e r v e d w i t h r a t A F P i n n,on d e n a t u r i n g P.A.G. e l e c t r o p h o r c s i s . T h i s p h e n o m e n o n h a s not been found with bovine AFP. Additional exper i m e n t s w o u l d b e n e c e s s a r y to e x p l a i n t h e m i c r o heterogeneity observed in our case but this phen o m e n o n s e e m s to b e i~nherent to t h e g l y c o s y l a t i o n of t h e p r o t e i n . T h e s e x h o r m o n b i n d i n g p r o p e r t i e s of r a t a n d m o u s e A F P [43] h a v e n o t b e e n f o u n d w i t h o u r p r e p a r a t i o n of b o v i n e AF,P (Vers6e, V., E ~ r e l , A. O., p e r s o n n a l c o m m u n i c a t i o n ) . This r e s u l t is c o n f i r m e d b y t h e o b s e r v a t i o n s o f A t t a r d i a n d R u o s l a h t i [44]. in conclusion bovine AFP can be isolated in l a r g e q u a n t i t i e s f r o m f e t a l s e r u m (100 m g / 2 0 0 m l ) . T h i s p r o t e i n is a c o n v e n i e n t m a t e r i a l f o r f u r t h e r structural studies.
Acknowledgements. This work was supported by a Grant o[ I N S E R M (ATP 36.76.68, contrat 008). The A u t h o r s are indebted to Pr. Biserle and Drs Bayard and g e r c k a e r t for sugars analysis b!l gasliquid chromatography. The A u t h o r s are very grateful to Mile MichMe Silhol ~?ind to Mme Bdatriee R o m e s t a n d for their s k i l f u l l technical and secretarial assistance.
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Bovine alpha-tbtoprotein. Isolation
and
characterization
Serge AL~AU <~, Jacques ~AlqTI and Jean MORETTI. (3-5-1978).
Laboratoire de B i o c h i m i e des Prot~ines, Universit~ des Sciences et T e c h n i q u e s du Languedoc, Montpellier, France.
R~sum6.
Summary.
L'AFP b o v i n e a v a i t 6t6 sicyaal6e d a n s le s 6 r u m foetal bovin. Ce travail d6crit son identification et s o n isolement p a r c h r o m a t o q r a p h i e sur c a r b o x y m e t h y l c e l l u l o s e DEAE S e p h a d e x A-50, gel de filtration et colonne d ' i m m u n o a d s o r b a n t . L'AFP se r6v~le h o m o g ~ n e , p a r blectrophorbse en gel d ' a c r y l a m i d e d a n s d e s conditions d 6 n a t u r a n t e s et non d 6 n a t u r a n t e s , p a r ultracentrifugation et en immuno61ectrophor~se. Les r6sultats s u i v a n t s ont 6t6 o b t e n u s : 1. La s 6 d i m e n t a t i o n & l'6quilibre indique u n poids m o l 6 c u l a i r e de 66 500 p o u r u n e c o n s t a n t e de S v e d b e r q s ° 20, w - - 4,71 s, u n v o l u m e partiel sp6cifique v = 0,737 m l g l e s t fir6 des m e s u r e s de densit6s. D ' a p r ~ s ces d o n n 6 e s un r a y o n de Stokes d e 3,26 nm, un coefficient de diffusion D20, ~ ----6,61 10 .7 crn ~ sec -~ et un coefficient d e friction f/to ---- 1,21 ont 6t6 calcul6s. 2. L'61ectrophor~se e n gel d e p o l y a c r y l a m i d e SDS p e r m e t d ' e s t i m e r le poids m o l 6 c u l a i r e & 67 000. 3. La microh6t6rog6n6it6 est d6montr6e p a r 61ectrofocalisation. Le point iso61ectrique du v a r i a n t p r i n c i p a l est de 4,6. 4. La composition chimique a 6t6 d6termin6e. L'AFP est u n e glycoprot6ine c o n t e n a n t 7 p. cent de s u c r e s : 1,67 p. cent d ' h e x o s e s , 2,38 p. cent de N-ac6tyl g l u c o s a m i n e , et 1,8 p. cent d ' a c i d e N-ac6tyl n e u r a m i n i q u e .
Bovine AFP w a s puriiied b y ion e x c h a n g e c h r o m a t o g r a p h y on C.M. cellulose a n d DEAE S e p h a d e x A-50, qel filtration a n d i m m u n o s o r b e n t technique. AFP w a s h o m o g e n e o u s w h e n studied b y qel electrophoresis u n d e r n o n d e n a turing a n d d e n a t u r i n g conditions, b y ultracentrifuqation a n d b y i m m u n o l o g i c a l m e t h o d s . The following m o l e c u l a r d a t a w e r e o b t a i n e d :
Abbreviations. AFP, alpha-fetoprolein ; SDS, Sodium dodecylsulphate ; BSA, bovine serum albumine ; PBS, phosphate buffer saline ; PAS, periodic aeid-Sehiff ; NBS, N-Bromosuccinimide ; DTNB, dithio-nitrobenzoi'c acid ; PDAB, para dimelhylamino benzaldehyde ; P.A.G., Polyacrylamide get. To whom all correspondence should be addressed.
1. S e d i m e n t a t i o n m o l e c u l a r weight of velocity g a v e s°20, w = v o l u m e v = 0.737 m l sity m e a s u r e m e n t s .
equilibrium indicated a 66,500 a n d s e d i m e n t a t i o n 4.71 S. A partial specific g-1 w a s d e r i v e d from den-
From these d a t a , a Stokes r a d i u s of 3.26 nm, a diffusion coefficient D20. ~ ~ 6.61 10 -7 cm 2 sec -1 a n d a frictional ratio f/to = 1.21 w e r e calculated. 2. S o d i u m d o d e c y l s u l p h a t e disc electrophorests s u g q e s t s a m o l e c u l a r w e i g h t of 67,000. 3. G e l filtration pointed to a m o l e c u l a r weight of 75,000. 4. M i c r o h e t e r o q e n e i t y of AFP w a s demonstrated b y isoelectric focusing. The isoelectric point of the m a j o r c o m p o n e n t is 4.6. 5. The c h e m i c a l composition w a s d e t e r m i n e d . AFP is a q l y c o p r o t e i n containing 7 p e r cent c a r b o h y d r a t e including 1.67 p e r cent hexoses, 2.38 p e r cent N-acetyl g l u c o s a m i n e a n d 1.8 p e r cent N-acetyl n e u r a m i n i c acid.
Key-words : fetoprotein, molecular, parameters, amino acids composition, carbohydrate content.
S. A Iiau, J. M a r t i a n d J. M o r e t t i .
664
For several years analogies between embryonic a n d c a n c e r tiss~ues, a l r e a d y r e c o g n i z e d o n m o r p h o l o g i c a l b a s e s , h a v e b e e n c o n f i r m e d at t h e m o l e c u l a r level, b y t h e d i s c o v e r y of s e v e r a l c a r c i n o e m b r y o n i c a n t i g e n s . T h e b e s t k n o ~ v n of t h e s e a n t i g e n s is A F P , a g l o b u l i n s p e c i f i c of f e t a l s e r u m , b u t reappearing under pathological conditions such as h e p a t o c e l l u l a r c a r c i n o m a [1]. I n a d d i t i o n to i t s potential diagnostic importance for the adult ( h e p a t o m a ) a n d t h e f e t u s ( n e u r a l t u b e d e f e c t [2]) A F P is a n i n t e r e s t i n g m a r k e r of c e l l u l a r d i f f e r e n t i a t i o n a n d of a g r e a t v a l u e i n t h e u n d e r s t a n d i n g of t u m o r b i o l o g y [3]. A F P w e r e d e s c r i b e d i n m a n y v e r t e b r a t e species [4]. B u t t h e f i r s t f e t a l p r o t e i n d e s c r i b e d i n t h e c a l f ~vas f e t u i n [5.] a n d f o r m a n y y e a r s a c o n fusion was made between fetuin and A~P. The immunological difference between these two prot e i n s w a s r e c e n t l y e s t a b l i s h e d [6]. L i t t l e is k n o ~ v n a b o u t t h e b i o l o g i c a l f u n c t i o n of A F P e x c e p t i n r a t and mouse. Nevertheless some indications have been obtained through the finding of primary structure homologies between albumin and AFP in h u m a n s p e c i e s [7]. These studies need further development, but biochemical studies are often limited by the lack of m a t e r i a l . It is t h e r e a s o n w h y , i n a d d i t i o n to t h e i n t r i n s i c i n t e r e s t of c o m p a r a t i v e b i o c h e m i s t r y , w e t h o u g h t u s e f u l to l o o k f o r a n a b u n d a n t s o u r c e of A F P . W e c h o o s e d t h e f e t a l b o v i n e s e r u m s i n c e it is p o s s i b l e to o b t a i n l a r g e v o l u m e of b l o o d e v e n at e a r l y s t a g e s of f e t u s d e v e l o p m e n t . B u t b o v i n e AFP was not characterized and the present paper d e a l s w i t h its p u r i f i c a t i o n a n d p h y s i c o - c h e m i c a l properties.
Materials
and
Methods.
Serum.
Pooled fetal calf s e r u m was used as the s t a r t i n g m a t e r i a l for the isolation of AFP. Blood was obtained f r o m 2-4 m o n t h s old fetuses b y p u n c t i o n of the j u g u l a r vein. After clotting, the serum was separated b y cent r i f u g a t i o n . Sera are pooled in 20~ ml lots a n d stored at --20°C. In the fetal serum the level of A F P decreases f r o m 4 m g / m l for fifty days old fetuses to a b o u t 0,2 m g / m l at b i r t h [8], (photo 1). In t h e samples used for purification, the level averaged 2 m g / m l .
Chemicals. Agarose (Indubiose A 37) was supplied b y I n d u s t r i e Biologique Franqaise, S.A. F r e u n d ' s complete a d j u v a n t was p u r c h a s e d f r o m Difco (Detroit). Glutaraldehyde, acrylamide, N,N b i s a c r y l a m i d e were E a s t m a n products. Ion exchange cellulose was supplied by W h a t m a n Biochemicals Ltd (England). Sephadex G 200, DEAEBIOCHIMIE, 1978, 60, n ° 6-7.
Sephadex A-50, Sepharose 4 B were supplied by P h a r m a r i a (S~veden). Phosphorylase B, a l p h a - a m y l a s e (B. subtilis), BS~A, o v a l b u m i n , c h y m o t r y p s i n o g e n A a n d cytochrome c were o b t a i n e d f r o m B o e h r i n g e r (Mannhelm). D i p h e n y l a m i n e , a n t h r o n e , PDAB a n d SDS were p u s c h a s e d f r o m Merck (Darmstadt, Germany). Bovine fetuin, s i d e r o p h i l i n a n d h u m a n g a m m a - g l o b u l i n s were p r e p a r e d in the l a b o r a t o r y . IMMUNSERA PREPARATION.
1. Antibodies a g a i n s t fetal calf serum were p r e p a r e d in New Zealand r a b b i t s b y weekly s u b c u t a n e o u s injections of increasing volumes of s e r u m (0,1 to 1 ml) mixed w i t h complete F r e u n d ' s a d j u v a n t (v/v). Blood was collected two weeks a f t e r the f o u r t h injection. A n t i s e r u m was recovered b y c e n t r i f u g a t i o n a n stored w i t h sodium azide (0:,1 per cent) at 4°C. 2. A n t i s e r u m was m a d e specific b y a d s o r p t i o n w i t h insolubilized a d u l t serie proteins. Ox serum was polymerized w i t h g l u t a r a l d e h y d e according to A v r a m e a s a n d T e r n y n c k [9]. After w a s h i n g to remove glutaraldehyde t h i s p o l y m e r is g e n t l y grinded in a P o t t e r h o m o g e n e i z e r a n d m i x e d w i t h a n equal volume of anti fetal calf serum: The m i x t u r e is left at 4~C overnight u n d e r stirring. The procedure m a y be repeated twice u n t i l the serum is quite feto-specific. The specificity of the anti serum h a s been discussed in a previous p a p e r [8]. 3. Antibodies a g a i n s t ox s e r u m p r o t e i n s were prepared in the same way. The Ig f r a c t i o n was precipitated w i t h 2.0M a m m o n i u m sulfate. After dialysis, the Ig were purified b y c h r o m a t o g r a p h y on DEAE-cellulose column using 0.05 M p h o s p h a t e buffer pH 7.0. The i m m u n o s o r b e n t was p r e p a r e d b y coupling the i m m u n o g l o b u l i n f r a c t i o n to Sepharose 4B as previously described [10]. PREPARATIVE PURIFICATION OF A F P .
Step I : CM Cellulose Chromatography. In a typical p r e p a r a t i o n , ~0 m l of fetal calf serum were dialyzed overnight a g a i n s t 2 1 of 0.01 M sodium acetate buffer pFt 4.6' A precipitate was discarded by c e n t r i f u g a t i o n a n d the s u p e r n a t a n t was applied to a Clff-cellulose c o l u m n (30 × 2 cm) e q u i l i b r a t e d w i t h the same (acetate) buffer. A f r a c t i o n C1 (20,0 ml) was eluted w i t h the s t a r t i n g buffer. It c o n t a i n s essentially f e t u i n [11]. The buffer was t h e n m a d e 0.1 M in NaC1, allowing the e l u t i o n of a C 2 f r a c t i o n (2,~0 ml), w h i c h contains t h e m a j o r p a r t of the a l b u m i n . The t h i r d f r a c t i o n C 3 (120, ml) eluted w i t h 0.1 M sodium acetate buffer pH 5.6. c o n t a i n i n g the AFP was dialyzed a g a i n s t several changes of 0.1 M Tris-HC1 buffer pH 8.6.
Step 2 : DEAE-Sephadex A-50 Chromatography. After dialysis, C 3 was applied to a D,l~AE-Sephadex c o l u m n (2~5 × 2 cm) e q u i l i b r a t e d w i t h the Tris-HC1 buffer. The p r o t e i n s were eluted by a l i n e a r g r a d i e n t of NaC1 f r o m 0 to 0.1 M. The fractions c o n t a i n i n g A,F P were pooled, dialyzed and freeze dried.
Step 3 : Gel Filtration. The lyophilizcd protein was dissolved in a small volume of 0.05 Mr Tris-HCl buffer pH 8.3, 0.1 M NaCl,
Bovine alpha-fetoprotein. a p p l i e d to a K 1 5 / 9 0 c o l u m n filled w i t h S e p h a d e x G 21)0 a n d e l u t e d w i t h t h e s a m e b u f f e r . A F P c o n t a i n i n g f r a c t i o n s w e r e pooled, d i a l y z e d a n d l y o p h i l i z e d .
Step ~ : Use of the lmmunosorbent. T h e p r o t e i n w a s d i s s o l v e d in PBS a n d a l l o w e d to d i f f u s e i n t h e i m m u n o s o r b e n t c o l u m n (25 X 0,5 em) during 4 hours. After this time, the elution was perf o r m e d w i t h P B S b u f f e r . T h e p r o t e i n p e a k (20 ml) c o n t a i n i n g p u r e AF,P w a s collected, d i a l y z e d a n d l y o philized. ANALYTICAL METHODS.
Immunoelectrophoretic Methods. Immunoelectrophoreses were performed on glass p l a t e s (9 X 12 cm) ; t h e a n t i b o d i e s - c o n t a i n i n g gel w a s p r e p a r e d i n b a r b i t a l b u f f e r 0.02 M p H 8.6 w i t h 1 p e r cent agarose. a) (< R o c k e t >> i m m u n o e l e c t r o p h o r e s i s w a s p e r f o r m e d a t 4 V / c m f o r 2 h [12]. b) C r o s s e d i m m u n o e l e c t r o p h o r e s i s [13] w a s c a r r i e d o u t w i t h 1-5 ~1 s a m p l e i n t h e first d i m e n s i o n at 4 V / c m f o r 3 h a n d i n t h e s e c o n d d i m e n s i o n a t 1,2: V / c m f o r 12 h. T h e p l a t e s w e r e t h e n w a s h e d i n s a l i n e (0.1 M NaC1)s d r i e d a n d s t a i n e d .
Polyucrylumide Gel Electrophoresis. S o d i u m d o d e c y l s u l p h a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s (5-7.5-10 a n d 15 p e r c e n t a c r y l a m i d e ) w a s p e r f o r m e d a s d e s c r i b e d b y W e b e r a n d O s b o r n [14]. T h e gels w e r e 0.8 X 12~ c m i n size. T h e p r o t e i n s a m p l e s c o n t a i n e d 5 to 2,0 itg/lO ~tl. Electrophoresis was carried out at room temperature at 8 m A p e r gel d u r i n g 6, to 8 h. B r o m o p h e n o l b l u e w a s u s e d as t h e t r a c k e r dye. Six p r o t e i n s of k n o w m o l e c u l a r w e i g h t w e r e s u b j e c t e d to e l e e t r o p h o r e s i s u n d e r t h e same conditions : phosphorylase B (rabbit), siderophilin, BSA,, o v a l b u m i n , e h y m o t r y p s i n o g e n e A a n d c y t o c h r o m e c. Gels w e r e s t a i n e d w i t h 0.25 p e r c e n t C o o m a s sie b l u e i n m e t h a n o l / a c e t i c a c i d / w a t e r ( 5 / 1 / 4 b y vol.) a n d d e s t a i n e d b y e l e e t r o p h o r e s i s i n acetic acid 7.5 p e r cent.
lsoelectric Focusing. T h e i s o e l e c t r i e p o i n t w a s e s t i m a t e d a c c o r d i n g to V e s t e r b e r g a n d S v e n s s o n [15], u s i n g A m p h o l i n e w i t h a p H r a n g e of 4-6 i n a c o n c e n t r a t i o n of 2 p e r c e n t a n d a s u c r o s e g r a d i e n t of 5-5~) p e r c e n t i n a 110 m l c o l u m n (LKB P r o d u e t e r s , S t o c k h o l m ) . A f t e r 72 h a t 600 V a n d 4°G, t h e c o l u m n w a s e m p t i e d . F r a c t i o n s of 2 m l w e r e collected. T h e i r p H w a s m e a s u r e d w i t h a p ~ m e t e r I s i s 1000 ( T a e u s s e l , L y o n , F r a n c e ) . T h e o p t i c a l d e n s i t y o f t h e e f f l u e n t w a s m o n i t o r e d a t 280 n m (Isco UA 5 Recorder). A c r y l a m i d e p l a t e s (LKB A m p h o l i n e P A G p l a t e ) c o n t a i n i n g 2~ p e r c e n t A m p h n l i n e w i t h a pH, r a n g e 3.5 to 9.5 w e r e a l s o e m p l o y e d a c c o r d i n g to t h e m a n u f a c t u r e r i n s t r u c t i o n s a n d stained, w i t h C o o m a s s i e b l u e . I n a d d i t i o n , a n a r r o w s t r i p of gel w a s c u t off a n d l a y e r e d o n t o a n a g a r o s e gel p l a t e c o n t a i n i n g a n t i b o d i e s a g a i n s t AFP. The protein pattern was developped by electroimmunodiffusion.
Partial Specific Volume. T h e p a r t i a l specific v o l u m e of t h e p r o t e i n w a s d e t e r m i n e d a t 2 0 ° C _ 0.~1! u s i n g a D ~ A 10 d e n s i m e t e r ( A n t o u P a r r K. G., Graz, A u s t r i a ) . T h e d e n s i t i e s o f t h e s o l u t i o n s u s e d in v i s c o s i t y e x p e r i m e n t s w e r e d e t e r m i -
BIOCHIMIE, 1 9 7 8 ,
60, n ° 6 - 7 .
665
n e d a f t e r c a l i b r a t i o n of t h e q u a r t z t u b e w i t h air, w a t e r and 0.05M phosphate b u f f e r p H 6.5 c o n t a i n i n g 0.1 M NaCI.
Viscosity. T h e v i s c o s i t y m e a s u r e m e n t s w e r e m a d e a t 20°C ± 0.01 in a U b b e l o h d e v i s c o s i m e t e r (Flea, P a r i s ) e q u i p p e d with an auto loading system and a electronic timer activated by two photocells. A stock solution was prepared by dissolving AFP i n t h e b u f f e r a t a c o n c e n t r a t i o n of 4,0 m g / m l . T h i s sample was dialyzed overnight against the same buffer (1000 t i m e s b y v o l u m e ) . T h e s o l u t i o n w a s clarified u s i n g a M i l l i p o r e filter (0.45 it). All t h e p h y s i c a l m e a s u rements were done starting from this stock solution. T h e l o a d i n g v o l u m e w a s 5. m l . S u c c e s s i v e d i l u t i o n s of the protein solution were made within the instrument b y r e p l a c i n g a v o l u m e of s o l u t i o n b y a n e q u a l v o l u m e of buffer. T h e d e n s i t y o f t h e w i t h d r a w n s a m p l e w a s i m m e d i a t e l y m e a s u r e d a n d t h e c o n c e n t r a t i o n of p r o t e i n e s t i m a t e d f r o m t h e a b s o r b a n c e at 278 n m o n a gravimetrically made dilution.
Ultracentrifugation. U l t r a e e n t r i f u g a t i o n w a s p e r f o r m e d a t 20°C b y u s i n g a MSE c e n t r i f u g e w i t h a 6 cells r o t o r b y s c a n n i n g t h e a b s o r b a n e e a t 280 n m . T h e s a m p l e w a s d i a l y z e d 24 h a g a i n s t t h e p h o s p h a t e - - NaC1 b u f f e r . S e d i m e n t a t i o n v e l o c i t y e x p e r i m e n t s w e r e r u n a t 54 200 r e v / m i n a n d scanned at 8 minutes intervals. Sedimentation equilibrium experiment was run for 40 h a t 1,0 000 r e v / m i n i n t h e s a m e r o t o r . A b s o r b a n c e of t h e s o l u t i o n i n t h e r u n w a s 0.23.
Analytical Gel Filtration. O n e m g of p u r i f i e d A F P w a s s u b j e c t e d to gel filtrat i o n o n a S e p h a d e x G 200 c o l u m n (K 25/90, P h a r m a c i a ) i n 0.05 Tris-HC1 b u f f e r p H 7.0 c o n t a i n i n g 0.2 p e r c e n t s o d i u m azide. T h e c o l u m n w a s c a l i b r a t e d w i t h c o m m e r c i a l p r o t e i n s as s t a n d a r d m a r k e r s ( B o e h r i n g e r ) . M o l e c u l a r r a d i u s of A F P w a s d e t e r m i n e d i n t h e s a m e c o n d i t i o n s . T h e S t o k e s r a d i i a of t h e m a r k e r p r o t e i n s w e r e t a k e n f r o m l i t t e r a t u r e [16]. D i f f u s i o n coefficient D o f A,F P w a s c a l c u l a t e d u s i n g t h e E i n s t e i n f o r m u l a : D~0.w : R T / N 6 ~ ~1 a where N = Avogadro's number ~1 : V i s c o s i t y a : Molecular radius
Absorption Spectra and Absorbance Coefficient. The absorption spectra was established with a Cary 118 s p e c t r o p h o t o m e t e r u s i n g b o v i n e A F P s o l u t i o n i n distilled water. The protein concentration was measured by weighing ( a f t e r e v a p o r a t i o n to c o n s t a n t w e i g h t ) a p r e c i s e v o l u m e of t h e p r o t e i n s o l u t i o n .
Fluorescence of the Protein. This study was made in the conditions described b y P a j o t [17]. A F P w a s i n c u b a t e d f o r 39 r a i n i n 6 M g u a n i d i n e h y d r o c h l o r i d e c o n t a i n i n g 30 m M m e r c a p t o ethanol. The fluorescence was measured using a M P F 3L P e r k i n - E l m e r s p e e t r o f l u o r i m e t e r . T h e fluorescence intensity scale was calibrated using solutions of t r y p t o p h a n ( i n t e r n a l c a l i b r a t i o n ) .
Amino Acids Analysis. A n a l y s e s w e r e c a r r i e d o u t o n a B e c k m a n 124 M. a u t o matie amino acid analyser. Samples were hydrolysed
S. Aliau, J. Marti and J. Morelti.
666
p a r t of a l b u m i n (fraction C 2). In a s e c o n d step (fig. 2) the r e m a i n i n g p e a k C 3 is f r a c t i o n a t e d on DEA~E-Sephadex. The p e a k F 2 c o n t a i n s A F P and m a n y i m p u r i t i e s . Even after a t h i r d step of gel filtration s e v e r a l o t h e r p r o t e i n s still r e m a i n in small amounts. T h e y i e l d of p u r i f i c a t i o n is n e a r 25 p e r cent.
i n v a c u u m - s e a l e d t u b e s w i t h 100 ~1 of c o n s t a n t b o i l i n g HC1 a t 110°C. T h e y w e r e s u b j e c t e d to h y d r o l y s i s f o r p e r i o d s of 24, 48 o r 72 h. Serine and threonine values were estimated by e x t r a p o l a t i o n to z e r o t i m e a n d i s o l e u c i n e a n d v a l i n e f r o m t h e 72' h h y d r o l y s i s [18]. C y s t e i n e a n d m e t h i o n i n e ~ e r e a n a l y s e d as c y s t e i c a c i d a n d m e t h i o n i n e s u l p h o n e a f t e r o x y d a t i o n of t h e s a m p l e s b y t h e m e t h o d of t~irs [19].
T h e s e i m p u r i t i e s are r e m o v e d b y using an i m m u n o s o r p t i ~ n technique. At this stage the prep a r a t i o n a p p e a r s h o m o g e n o u s w i t h r e s p e c t to electrophoresis and immunoelectrophoretic criteria (photo 2). P r e p a r a t i o n in a l a r g e r scale w e r e also m a d e s t a r t i n g f r o m 200 ml of serum.
Sugars analyses. I d e n t i f i c a t i o n of s u g a r s w a s p e r f o r m e d u s i n g a P e r k i n - E l m e r F 11 Gas l i q u i d c h r o m a t o g r a p h , e q u i p p e d w i t h a O V 21@, c o l u m n o n c h r o m o s o r b HMDS. M e t h a n o l y s i s w a s m a d e a c c o r d i n g to C l a m p et al [20] a n d t r i f l u o r o a c e t y l a t i o n a c c o r d i n g to Z a n e t t a el al [21]. H e x o s e s w e r e a n a l y z e d b y t h e a n t h r o n e m e t h o d [22], s i a l i c a c i d s b y d i p h e n y l a m i n e m e t h o d a c c o r d i n g to N i a z i a n d S t a t e [23] a n d o s a m i n e s a c c o r d i n g to E l s o n a n d M o r g a n [24].
Polyacrylamide gel electrophoresis. This m e t h o d s h o w e d the h o m o g e n e i t y of the final p r o d u c t . Gel electrophoresis in r e d u c i n g cond i t i o n s i n d i c a t e d t h a t A F P is a single p o l y p e p t i d e chain. P~ovine A,FP gives p o s i t i v e r e a c t i o n w i t h P.A.S. staining.
Results.
E l e c t r o p h o r e s i s at different a c r y l a m i d e concent r a t i o n from 6 to 1'5 p e r cent a c c o r d i n g to Segrest and J a c k s o n [25] gave a m o l e c u l a r w e i g h t of 67 000 ; no significative differences w e r e o b s e r v e d b e t w e e n the different m o l e c u l a r w e i g h t estimations.
Purification procedure. I n v i e w to p r e p a r e AFP, we used the k n o w l e d g e o b t a i n e d d u r i n g the isolation of ovine fetuin. This g l y c o p r o t e i n is like A F P an a l p h a g l o b u l i n and its level in fetal s e r u m is h i g h e r than the level of AFP. The first step (fig. 1) a l r e a d y used d u r i n g the t m r i f i c a t i o n of fetuin offers the a d v a n t a g e to d i s c a r d this p r o t e i n (fraction C 1) and the m a j o r
1,5 '
Ultraviolet Spectrum. The a b s o r p t i o n s p e c t r u m of the p r o t e i n is given figure 3. The a b s o r b a n c e coefficient A (1 p e r cent,
C1
C3 C2
E c o
0,PH1M4.6
u c = o
J~
I
0.1M
i
0,5
50
Fraction
'
l
,
'
100
'
'
Number
Chromatography of fetal calf Serum on c a r b o x y m e l h y l c e l l n l o s e ( W h a l m a n CM 23) c o l u m n (22 × 3 cm) ; f l o w rate 70 m l / h , 10 m l fractions. FI6.
1. - -
P r o t e i n s a r e m o n i t o r e d a t 280 rim. A F P w a s d e t e c t e d b y e l e c t r o i m m u n o d i f f u s i o n (¢ r o c k e t s >>). AI~P c o n t a i n i n g f r a c t i o n s a r e m a r k e d .
BIOCHIMIE, 1978, 60, n ° 6-7.
Bovine alpha-fetoprotein.
667
Buffer+NaCI O,1M
TrisHCI
E
1 O"
PH 8,6
O , I M ~
~"
J
J
~
F3
0 oo (N
fo
¢3 u
c 0,5 .Q 0 J~
<
I
I
I
I
lO
I
50
Fraction
I
I
I
I
i
!
!
lOO
Number
F1a. 2. - - C h r o m a t o g r a p h y of (:~ on D E A E - S e p h a d e x A-50 c o l u m n (25 cm X 2 cm) ; f l o w rate 12 m l / h , 3 ml fractions. L i n e a r g r a d i e n t T r i s HC1 i = 0.1, p H 8.6, 200 ml. S a m e b u f f e r ÷ 0.1 M 200 ml.
PHOTO I. Crossed i m m u n o e l e c t r o p h o r e s i s of fetal bovine s e r u m against rabbit whole fetal bovine a n t i s e r u m . A. S e r u m f r o m a 80 days o l d fetuse. B. S e r u m f r o m a 250 d a y s old fetuse. V o l u m e of s a m p l e : 5 ~1 of fetal s e r u m . E l e c t r o p h o r e s i s w e r e carried o u t f o r 3 h a t 40 v o l t s in the first d i m e n s i o n a n d f o r 12 h at 15 v o l t s in t h e second dimension. Curved a r r o w m a r k s A F P .
BIOCHIMIE, 1978, 60, n ° 6-7.
S. Aliau, J. Marti a n d J. Moretti.
6:68
PHOTO 2.
Crossed immunoelectrophoresis of AFP fraction after immunosorption. Same conditions as in photo 1.
278 nm) is f o u n d to be 4.60 dl.g lcln-1. T h e m o l a r e x t i n c t i o n coef,f i c i e n t is e = 3.08 104 M-1 cm-1 at 280 nm. T h e s p e c t r u m s h o w s c h a r a c t e r i s t i c p h e TABLE I.
Values of density ~ of the solutions employed for partial specific volume determination, at 6 different protein concentrations. g. m1-1
p
0.03630 0.02527 0.01600 0.00956 0.00633 0.0000
1.01563 1.01306 1.0105 1.0080 1.0078 1.00359
T h e d e n s i t i e s of t h e s o l u t i o n s and of t h e s o l v e n t a l o n e w e r e m e a s u r e d (table I). T h e d e p e n d e n c e o f d e n s i t y ~ on p r o t e i n c o n c e n t r a t i o n w a s l i n e a r and, b y a least s q u a r e s p r o c e d u r e , f o u n d to be : ? = 0.~60 02~5 C + 1.006 317 w h e r e C [26] is t h e c o n c e n t r a t i o n of t h e p r o t e i n s o l u t i o n in g.ml-1. T h e p a r t i a l s p e c i f c v o l u m e was determined from
~= --
C
T , ?, ~ , C
w h e r e ~ is a l i n e a r f u n c t i o n of C : ~
--
c
n y l a l a n i n e spikes. W h e n p r o t e i n w a s d i s s o l v e d in 0.1 M 4NaOH, n o s h o u l d e r w a s o b s e r v e d t h a t w o u l d i n d i c a t e t h e p r e s e n c e of tryptoph.an.
Partial Specific Volume. Four dilutions were prepared from the stock p r o t e i n s o l u t i o n ( c o n c e n t r a t i o n : 3.6,3 mg.ml-1).
BIOCHIMIE, 1978, 60, n ° 6-7.
s i t y w i t h r e s p e c t to C at c o n s t a n t t e m p e r a t u r e T, p r e s s i o n P, a n d c h e m i c a l p o t e n t i a l v of all substances except the protein. The mean value of~ so o b t a i n e d is 0.737 --4--- 0.0001 ( r e g r e s s i o n coeffic i e n t = 0.99898).
Bovine alpha-fetoprotein. Viscosity. F r o m t h e f l o w t i m e of t h e five s o l u t i o n s , t h e intrinsic viscosity can be calculated using the f o r m u l a [27] : (~) = l i m i t of -
t --
% to
-
669
The axial ratio obtained from the viscosity increment from Simha's relation assuming that t h e m o l e c u l e is a n u n h y d r a t e d e l l i p s o i d of r e v o l u t i o n , a r e 5 . 6 f o r a p r o l a t e e l l i p s o i d a n d 8.5 f o r an oblate ellipsoid.
w h e n C --> O
% to C
w h e r e ~o i s t h e d e n s i t y of s o l v e n t a n d ~ t h e d e n s i t y of s o l u t i o n , t a n d to, r e s p e c t i v e f l o w t i m e s , a r e t h e m e a n of t h e l a s t t e n v a l u e s f r o m a s e r i e s of measurements repeated until the differences b e c a m e less t h a n 10 .2 sec.
Molecular Parameters derived from Gel Filtration. T h e a p p r o x i m a t e m o l e c u l a r w e i g h t v a l u e of A F P o b t a i n e d f r o m t h e gel f i l t r a t i o n d a t a [29] is 75,000. T h e S t o k e s r a d i u s a m e a s u r e d i n S e p h a d e x G 200 c h r o m a t o g r a p h y is 3.52 n m . T h e d i f f u s i o n coeffic i e n t D, c a l c u l a t e d f r o m t h e v a l u e s of a a n d n is D20,w :
6.05 10-7. c m 2 . s -1
Analytical ultracentrifugation. A s e d i m e n t a t i o n c o e f f i c i e n t s:0 ' w : 4,71 _ 0.1 was calculated from the sedimentation velocity r u n . A b s o r b a n c e s of t h e s o l u t i o n s w e r e f r o m 0.21 to 0.50 O.D.
0,7
0.6
Sedimentation equilibrium studies demonstrated t h a t t h e r e l a t i o n s h i p b e t w e e n t h e l o g a r i t h m of t h e r e c o r d e r d e f l e c t i o n (d) a n d r 2 w a s l i n e a r indicating monodispersity of t h e p r e p a r a t i o n . U s i n g t h e v a l u e 0,73'7 ml.g-1 f o r t h e p a r t i a l s p e c i fic v o l u m e a n d t h e v a l u e 0.16923 of t h e s l o p e , a m o l e c u l a r w e i g h t of 66,500 ± 1,500 is o b t a i n e d from the formula
0.3
2RT
M =
0.2
w
•
240
i
,
:eO
g
,
:eO
Wavelength
i :00
, .
,
I
:=0
18
nm
U H r a v i o l e t spectrum of the p u r i f i e d AFP.
1,7
The m e a s u r e m e n t was obtained on a double b e a m s p e c t r o p h o t o m e t e r Gary 18. 1. S p e c t r u m obtained w i t h 1 m g / m l A,F P in water. 2. Spectrum o b t a i n e d i n NaOH 0.I N w i t h o u t an), correction for the t u r b i d i t y .
1,6
Fro. 3. - -
. 2.303 tg ~ [30]
~'~) to2 r
(1 - -
I,S 1.4
1,2 ¢O
T h e p l o t of ( 0 ) v e r s u s C is l i n e a r (r = 0.99946) a n d , b y a l e a s t s q u a r e p r o c e d u r e , f o u n d to b e : (0) = 1 6 . 5 6 2 5 7 2 C 4- 5.704 638 T h e v a l u e of t h e i n t e r c e p t g i v e s (~1) w h e n C --+ O 01) = 5.70 ± 0.05 ml.g-1 T h e v a l u e of t h e v i s c o s i t y i n c r e m e n t v c a l c u l a t e d f r o m t h e i n t r i n s i c v i s c o s i t y (0) a n d t h e p a r t i a l s p e c i f i c v o l u m e ~ f r o m t h e r e l a t i o n [28] v = ( ~ ) / v w a s f o u n d to b e 7.73.
BIOCHIMIE, 1978, 60, n ° 6-7.
O) .OO
1,~ 1,0 0.9 0,8" 0.~-' 06
o
,
•
•
,,
•
,;
so racm
2
Fro. 4. - - Sedimentation equilibrium ullraventrifuffation of AFP. 10{)50 r e v / m i n at 20°C. (Value of slope 0.16923, regression : 0.99968).
S. Aliau, J. Marli a n d J. Moretli.
670
c o n t r o l l e d by crossed i m m u n o e l e c t r o p h o r e s i s , The m e d i a n v a r i a n t has a p H i of 4.6.
F r o m the values of M, S, and v_a diffusion c.oefficient can be c a l c u l a t e d s RT D : __ o : 6.61 10 -7 cm 2 sea -1 M ( 1 - - - v g ) D2°'w T h e f r i c t i o n a l ratio f/fo calculated a c c o r d i n g to Oncley [31] is 1.2~1. T h e axial r a t i o s o b t a i n e d f r o m the f r i c t i o n a l ratio by P e r r i n ' s r e l a t i o n assuming that the p r o t e i n is an u n h y d r a t e d ellipsoid of r e v o l u t i o n is 4.2 for a p r o l a p s e and 5 for an oblate ellipsoid.
-5 '4~5 .4
3.5 mO
0.S
e~
T h e r a d i u s of the e q u i v a l e n t s p h e r e calculated from M
5,5
O,75 "
O O
O,=S O
(1 - - ~ )
a -6~
O
is 32~.6 ~ (3~.26 nm).
Ns20. w ,o
Isoelectric Focusing. The isoelectric pattern obtained from column e x p e r i m e n t s shows one p e a k a r o u n d p H 4.6 ; a s h o u l d e r is detected at pH 4.8.6 (£~g. 5). T h e r e is an i m m u n o l o g i c a l i d e n t i t y b e t w e e n the peak and the shoulder. No others v a r i a n t s can be s e p a r a t e d in the p H 4.6 scale. T h e ampholine~PAG plate exper i m e n t reveals a m i c r o h e t e r o g e n e i t y of AFP betw e e n pH 4.4 and 5.3. T h e r e are at least nine v a r i a n t s s h o w i n g an i m m u n o l o g i c a l i d e n t i t y ~ as
;o Ffa orion
'
;o
Number
FI6. 5. Isoelectric focusing of bovine AFP, pH 4-6 gradient. The experiment was carried out as described -
-
in methods ; the anode was at the bottom of the column, pH is represented by the dotted line.
Carbohydrate composition. In table III, the c a r b o h y d r a t e c o m p o s i t i o n of A F P is c o m p a r e d w i t h those of the t w o v a r i a n t s of rat A F P [32]. The f r a c t i o n a r y n u m b e r for
TABLE II.
Amino acid composition of AFP. The e x p e r i m e n t a l results h a v e been n o r m a l i z e d . Best values are as defined by A m b l e r El81. The n u m b e r of residues m o l e c u l e h a v e been c a l c u l a t e d by the m e t h o d of Delaage [41]. Amount found after hydrolysis ior Amino
acid
Aspartate asparagine Threonine Serine Glutamate glutamine Proline
"
24 h
48 h
72 h
50.08 33.70 44.36 102.64 27.38
50.81 32.66 40.43 104.86 28.84 32.29 43.82 28.34 38.43 9.28 34.30 60.25 16.02 29.16 46.10 16.80 24.83
50.55 32.54 40.56 101.46 27.80 32.24 43.29 27.54 49.94 9.26 35.82 63.59 17.30 28.57 45.52 16.86 26.79
Glyctne
33.17
Alanine Half-cystine Valine Methionine Isoleueine Leucine Tyrosine Phenylalanine Lysine Histidlne Arginine Tryptophan Total residues
43.90 28.13 37.52 9.00 33.79 58.33 17.18 30 20 45.51 16.58 26.99
value
Best
Residues molecule protein
integral
50.51 34.70 47.00 102.99 28.00 32.57 43.67 21.1 (a) 49.94 9.18 35.82 63.59 16.83 27.80 (b) 45.71 16.74 26.20
41 ~.99 28.86 39.07 85.62 23.61 27.46 36.30 17.54 42.10 7.74 29.78 52.86 13.99 23.11 38.00 13.92 22.09
42 29 39 86 24 27 36 18 42 8 30 53 14 23 38 14 22
0
a Value founded for cysteic acid after performic oxydation according to Hirs [19]. b Corrected for contamination by glucosamine, c From fluormnetric results.
BIOCHIMIE, 1978, 60, n ° 6-7.
Nearest
0 545
Bovine alpha-f etoprotein.
67'1
TABLE III.
Carbohgdrate composition of bovine AFP compared to rat AFP. Values (per cent by w e i g h t and m o l / m o l ) are c a l c u l a t e d on the basis of M = 67 000. Results of m o l a r ratios t ak en f r o m t h e basis of 3: m a n n o s e residues. CalI AFP
Per cent
Mannose Galactose N. Ac. Glueosamine NANA Total
1.61 1.07 2.38 (a) i.80 6.81 (b)
mol/mol
6 4 7.06 (a) 4 21
Rat AFPA and B variants molar ratio
3.0 2.08 3.65 2.0
tool/tool
raotar ratio
A
B
A
B
6.1 6.1 7.6 6.2
6.1 5.7 7.8 5.8
3.0 3.1 3.8 3.1
3.0 2.9 3.9 2.9
a The glucosamine content of AFP is certainly higher. This may be due to the incomplete release of glucosamine during hydrolysis. The correct values seems be 8 mol/mol and 2.6 per cent. b This value agree with 6.8 per cent and 7.1 per cent found for A and B variants of rat AFP. N - a c e t y l - G l u c o s a m i n e m a y be due to an i n c o m plete release of the sugar f r o m the c a r b o h y d r a t e m o i e t y d u r i n g h y d r o l y s i s . T h e m o l a r ratios on the basis of 3 m a n n o s e i n d i c a t e a possible structure closely s i m i l a r to rat AFP. This s t r u c t u r e m ay consist of tw o glycans, e a c h one c o n t a i n i n g 2 NaNa - ) M'an - 2 Gal - 4 NAcGluc (Bayard an d Kerckaert, p e r s o n a l c o m m u n i c a t i o n ) .
Amino acid composition. The amin,o acid c o m p o s i t i o n of the p r o t e i n as d e t e r m i n e d by the a u t o m a t e d analysis of acid h y d r o l y s a t e s is given in table II. As a l r e a d y p o i n ted out, optical studies, even the f i u o r o m e t r i c m e t h o d of Pajot, failed to r e v e a l the p r e s e n c e of t r y p t o p h a n in the protein. O i h e r m e t h o d s including the NIBS-oxydation a c c o r d i n g to P a t c h o r n i k et al. [33~ and the c o l o r i m e t r i c m e t h o d of Spies and C h a m b e r s [34] gave values far b e l o w one r e s i d u e p e r mole of protein. It w a s c o n c l u d e d to the absence of t r y p t o p h a n , w h i c h accounts for the l o w value ~)f the the e x t i n c t i o n coefficient. Any cys.teine r e s i d u e was available for t i t r a t i o n w i t h 5-5' DTND in the p r e s e n c e or absence of 8 M u r e a [35]. T h e 18 cysteines in the p r o t e i n are thus assum e d to be i n v o l v e d in 9 disulfide bridges.
Discussion .
.
.
.
This p a p e r d e s c r i b e s a p r o c e d u r e for the p u r i fication of b o v i n e AFP, to a p p a r e n t c h e m i c a l an d
BIOCHIMIE, 1978, 60, n ° 6-7.
i m m u n o l o g i c a l h o m o g e n e i t y , and r e p o r t s the results of m e a s u r e m e n t s of its m o l e c u l a r parameters. The m a j o r difficulty was tile e l i m i n a t i o n of fetuin and al b u m i n ~ , h i c h are the more i m p o r t a n t constituants of fetal serum. F o r a part, it is o v e r c o m e by a CM-cellulose c h r o m a t o g r a p h y , a l l o w i n g to d i s c a r d fetuin. Th e m a j o r losses o c c u r at this step, w h i c h is h o w e v e r an easy w a y to separate the t w o fetoproteins. T h e last step of p u r i f i c a t i o n i n v o l v i n g an i m m u n o s o r p t i o n is n e c e s s a r y to r e m o v e al b u m i n traces,, i m p o s s i b l e to d i s c a r d by classical p r o c e d u r e s , because of its s i m i l a r i t y w i t h AFP. Th e m o l e c u l a r w e i g h t s estimated by SDS-disc e l e c t r a p h o r e s i s and by u l t r a c e n t r i f u g a t i o n are similar, about 67,000. Gel filtration gives a h i g h e r value but this reslalt m a y be e x p l a i n e d by the fact that A FP is a g l y c o p r o t e i n [361. Molecular w e i g h t s are of the same o r d e r of m a g n i t u d e for o t h er A FP : 70,000 (rat, mouse [373, m an [38], pig [39], or ~8,000 (rabbit [40]). The statistical m e t h o d of Delaage [41] relies the a m i n o a c i d c o m p o s i t i o n to the m o l e c u l a r w e i g h t . In t h e p r e s e n t case this m e t h o d gives 2 values of almost equal p r o b a b i l i t y : 6.2,000 and 70,000. A d d i n g the i n c r e m e n t of the c a r b o h y d r a t e m o i e t y ~5,600), the m o l e c u l a r w ei g h t s are 67,500 and 75,600. T h e first value is the most p r o b a b l e and is in good a g r e e m e n t ~vith the e l e c t r o p h o r e s i s and c e n t r i f u g a f i o n m e a s u r e m e n t s .
672
S . A l i a u , J. M a r t i a n d J . M o r e l t i .
B o v i n e AI~P is r i c h i n a c i d i c r e s i d u e s , f r e e , o r in the amide form (aspartic + glutamic acid 2'3:5 p e r c e n t ) . T h i s a c c o u n t s f o r t h e a c i d i c isoe l e c t r i c p o i n t of t h e p r o t e i n ( p H i = 4.6). T h e s e t y p e s of r e s i d u e s a r e k n o w n to f a v o r h y d r o g e n bonding, and their high percentage may be indic a t i v e of a r a t h e r c o m p a c t t e r t i a r y s t r u c t u r e . T h e v a l u e of 1.4: f o r t h e r a t i o of p o l a r to a p o l a r r e s i d u e s , c a l c u l a t e d a c c o r d i n g to H a t c h [42] f a v o r s t h i s h y p o t h e s i s . T h e h i g h c o n t e n t of g l u t a m i c r e s i d u e s ( ~ l u + Gln) a p p e a r s to b e a c h a r a c t e r i s t i c s h a r e d i n c o m m o n w i t h A F P of o t h e r s p e c i e s . T h e p h y s i c a l m e a s u r e m e n t s (f/fo = 1,21) i n d i c a t e t h a t b o v i n e &FI ) i s a g l o b u l a r p r o t e i n . T w o m o l e c u l a r f o r m s a r e o b s e r v e d w i t h r a t A F P i n n,on d e n a t u r i n g P.A.G. e l e c t r o p h o r c s i s . T h i s p h e n o m e n o n h a s not been found with bovine AFP. Additional exper i m e n t s w o u l d b e n e c e s s a r y to e x p l a i n t h e m i c r o heterogeneity observed in our case but this phen o m e n o n s e e m s to b e i~nherent to t h e g l y c o s y l a t i o n of t h e p r o t e i n . T h e s e x h o r m o n b i n d i n g p r o p e r t i e s of r a t a n d m o u s e A F P [43] h a v e n o t b e e n f o u n d w i t h o u r p r e p a r a t i o n of b o v i n e AF,P (Vers6e, V., E ~ r e l , A. O., p e r s o n n a l c o m m u n i c a t i o n ) . This r e s u l t is c o n f i r m e d b y t h e o b s e r v a t i o n s o f A t t a r d i a n d R u o s l a h t i [44]. in conclusion bovine AFP can be isolated in l a r g e q u a n t i t i e s f r o m f e t a l s e r u m (100 m g / 2 0 0 m l ) . T h i s p r o t e i n is a c o n v e n i e n t m a t e r i a l f o r f u r t h e r structural studies.
Acknowledgements. This work was supported by a Grant o[ I N S E R M (ATP 36.76.68, contrat 008). The A u t h o r s are indebted to Pr. Biserle and Drs Bayard and g e r c k a e r t for sugars analysis b!l gasliquid chromatography. The A u t h o r s are very grateful to Mile MichMe Silhol ~?ind to Mme Bdatriee R o m e s t a n d for their s k i l f u l l technical and secretarial assistance.
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