BIOCHIMIE, 1976, 58, 505-512.

Pig prothrombin : Purification and properties. F r a n c i s c o A. LEONE, Glaico GHIERICATO, Jos6 C. SAY a n d Jos6 R. GIGLIO ~.

Department of Biochemistry, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, SP, Brazil. (20-6-1975). Summary. - - A procedure for the preparation of highly purified pig prothrombin is described. Compared to the initial clotting activity of the starting plasma, this protein was purified 776 times 'with a final yield of 8 per cent. The purified zymogen showed a specific activity of 1,460 NIH units/mg of protein, a molecular weight of 65,000 as determined by SDS-polyacrylamide disc gel electroplloresis, E~]0 mg/mI era, 280 ..... = 1.45 at pH 7.0 and the following amino acid composition : Asx~, Thrzs, Ser~, Glx~,_~,Pro~, GIy~o Aia=~,, Half-Cyst0, Val~, Met3, Ile~., Leu.~._,, Tyr~,~, Phe,~_~, Lys~s, Hiss, Arg~ and Trp~3, which accounts for a minimum molecular weight of 59,370 (carbohydrates not computed). Alanine 'was found as the only N-terminal residue. Carboxypeplidases A and B failed 1o release any C-terminal residue. By hydrazinolysis however 0.4 mole of serine 'was released per mole of prothrombin. The activation of crude and chromatographed pig prothrombin was investigated.

INTRODUCTION. In a p r e v i o u s p a p e r [1] ~ve h a v e d e s c r i b e d the p r e p a r a t i o n and a c t i v a t i o n of p a r t i a l l y p u r i f i e d p i g p r o t h r o m b i n in different c o n d i t i o n s , a,nd rep o r t e d the a m i n o acid c o m p o s i t i o n and N-terminal r e s i d u e s of the r e s u l t i n g active t h r o m b i n . An i n c r e a s i n g n u m b e r o'f p a p e r s r e p o r t i n g the p u r i f i c a t i o n of p r o t h r o m b i n f o r m several a n i m a l species h a v e a l r e a d y been p u b l i s h e d , such as : ox [2-7], h o r s e [8], dog [9-11~, r a t [12], m a n [13241 an.d c h i c k e n [25], thus s h o w i n g the great interest into the k n o w l e d g e of this key blood clotting e n z y m e ~n.d of its e v o l u t i o n in the a n i m a l kingdom. This p a p e r describes a p r o c e d u r e for the p r e p a r a t i o n of h i g h l y p u r i f i e d p i g p r o t h r o m b i n , the i d e n t i f i c a t i o n of its N and C-terminal residues a n d the d e t e r m i n a t i o n o.f its m o l e c u l a r w e i g h t a n d a m i n o a c i d c o m p o s i t i o n . T h e a c t i v a t i o n of the c r u d e and the c h r o m a t o g r a p h e d z y m o g e n is briefly r e p o r t e d . Abbreviations : E = extinction coefficient; Tris ---- tris (hydroxymethyl) aminomethane ; TAME =- p-toluenesulphonylL-arginine methyl e s t e r ; DNP = 2,4-dinitrophenyl radical; EDTA = ethylenediamine tetra-acetate, sodium s a l t ; NIH = National Institute of t t e a l t h ; DNS-CI = 5-dimethylaminonaphthalene-l-sulphonyl (dansyl) chloride ; SDS = sodium dodecyl sulphate. To 'whom all correspondence should be addressed.

MATERIALS AND METHODS. P i g p r o t h r o m b i n w a s p r e p a r e d a c c o r d i n g to a m o d i f i e d p r o c e d u r e d e v e l o p e d in our l a b o r a t o r y based on that p r e v i o u s l y d e s c r i b e d by Moore et al. [2] for b o v i n e p r o t h r o m b i n . It is r e p o r t e d h e r e as follows : 8,800 ml of b l o o d f r o m adult animals, s t a r v e d for 24 h, w e r e collected, t h r o u g h d i r e c t h e a r t p u n c t u r e , into 1,200 ml of a 3 p e r c e n t ( w / v ) solution of t r i s o d i u m citrate (NaaC6HsO 7. 2H20) in an ice bath. All s u b s e q u e n t steps w e r e p e r f o r m e d at 0-2°C. The plasma, isolated by cent r i f u g a t i o n at 10,100 × g (7,000 r.p.m.) in a Sorval RC2-B r e f r i g e r a t e d c e n t r i f u g e for 20 rain., was imm e d i a t e l y t r e a t e d d r o p w i s e w i t h a 1M solution of BaC12. 2H20 (80 ml/1 o.f plasma) u n d e r m a g n e t i c s t i r r i n g w h i c h w a s c o n t i n u e d for 1 h after the addition of the salt solution. The s u s p e n s i o n cont a i n i n g the insoluble b a r i u m citrate w i t h the ads o r b e d p r o t h r o m b i n was c e n t r i f u g e d at 2,520 × g (3,500 r.p.m.) for 10 minutes, the total p r e c i p i t a t e w a s h e d once w i t h 600 ml of d e i o n i z e d w a t e r a n d s e p a r a t e d by c e n t r i f u g a t i o n as before. The final r e s i d u e was m a c e r a t e d w i t h a small v o l u m e of 0.4 M EDTA, pH 8.0, in o r d e r to t r a n s f o r m it into a h o m o g e n i z e d paste. More 0.4 M E D T A was a d d e d up to 60 ml/1 of the original p l a s m a and the susp e n s i o n was s t i r r e d for 1 h w h i l e k e e p i n g the p H at 8.0 by the c o n t i n u o u s a d d i t i o n of s a t u r a t e d Tris solution. This t r e a t m e n t dissolves the b a r i u m citrate a n d releases p r o t h r o m b i n . 34

506

F. A. Leone, G. Chiericato, J. C. Sag and J. R. Giglio.

A smal~ amou,nt o~f insoluble gelatinous m a t e r i a l w h i c h f o r m e d w a s r e m o v e d b y c e n t r i f u g a t i o n at 5,140 × g (5,000 r.p.m.) for 10 rain a n d the clear y e l l o w i s h solution d i a l y z e d a g a i n s l 10 1 of a 0.05 M NHa-HC1-0.3 M NaG1 buffer solution, p~H 8.0, in ¥ i s k i n g tlrbing w h i c h w a s F r e v i o u s l y b o i l e d f o r 15 m i n in 0.2 M EDTA, p H 8.0, a n d then w a s h e d a n d k e p t in d e i o n i z e d w a t e r at 2"C. D i a l y s i s p r o c e e d e d for about 40 h w i t h changes e v e r y 4 h, until no Ba ++ cou~,d be d e t e c t e d in the d i a l y s i s solution b y testing w i t h 10 p e r cent (v/v) H2~O 4. Some d e b r i s w h i c h f o r m e d in this step w a s not r e m o v e d . To the slightly t u r b i d dialysate, p o w d e r e d (NH4)zSO ~ was s l o w l y s p r i n k l e d , u n d e r m a g n e t i c s t i r r i n g , u p to 50 p e r c e n t s a t u r a t i o n . The p r e c i p i t a t e w a s collected b y c e n t r i f u g a t i o n at 8,00.0 × g for 10 rain and d i s c a r d e d . To t h e c l e a r s u p e r n a t a n t m o r e (N'I-I4)2SO4 was s l o w l y a d d e d up to 70 p e r cent saturation. After 1 h s t a n d i n g the r e s u l t i n g p r e c i p i t a t e w a s c o l l e c t e d as above a n d d i s s o l v e d in 5 to 10 ml of d e i o n i z e d w a t e r . Crude p i g p r o t h r o m b i n so p r e p a r e d was convert e d at p H 8.0 into t h r o m b i n in 25 p e r cent ( w / v ) triso,dium c i t r a t e at 25°C b y m i x i n g one v o l u m e of the above solution w i t h one vo.~ume of 50 p e r c e n t ( w / v ) Naa,CGH~O7.2:H20 a n d then s e e d i n g the susp e n s i o n w i t h 0.1 to 0.5 ml (ff a t h r o m b i n solution c o n t a i n i n g a b o u t I p e r cent of the total e n z y m a t i c a c t i v i t y e x p e c t e d after m a x i m a l activation. E s t e r a s e a n d c l o t t i n g activities of the r e s u l t i n g t h r o m b i n w e r e d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d [1] u s i n g TAM~E a n d fibrinogen as suhstrates, respectively. l~urther p u r i t l c a t i o n o'f p i g p r o t h r o m b i n was acc o m p l i s h e d b y c o l u m n c h r o m a t o g r a p h y on Amb e r l i t e IRC-50 f o l l o w e d b y r e c h r o m a t o g r a p h y on D,EAE-Sepha~dex A-50. The columns w e r e c o n n e c ted to one a n o t h e r b y p o l y e t h y l e n e c a p i l l a r y tub i n g so t h a t .the effluent f r o m t h e A m b c r l i t e col u m n w a s c o n d u c t e d to a U.V. d e t e c t o r a n d then to the DEA.E~Sepha'dex column. A m b e r l i t e IRC-50 w a s e q u i l i b r a t e d b e f o r e use w i t h 0.05 M p o t a s s i u m p h o s p h a t e buffer, p H 7.0, a f t e r b e i n g p r e c y c l e d as p r e v i o u s l y d e s c r i b e d [1]. DEA,E - S e p h a d e x A-50 w a s w a s h e d w i t h 0.5 M HC1, d e i o n i z e d w a t e r , 0.5 M KOH, ,deionized w a l e r , 0.1 M KH.~PO~ till p H 7.0 a n d t h e n e q n f l i b r a t e d o v e r n i g h t w i t h 0.05 M p o t a s s i u m p h o s p h a t e buffer, p H 7.0. The s a m p l e w a s d i a l y z e d against 0..05 ,M p o t a s s i u m p h o s p h a t e buffer, p H 7.0, a n d c e n t r i f u g e d b e f o r e a p p l i c a t i o n to the A m b e r l i t e column, f r o m w h i c h elution o,f th e p r o t h v o m b i n f r a c t i o n was p e r f o r m e d w i t h the same buffer [1]. I m m e d i a t e l y a f t e r f r a c t i o n I, w h i c h contains the p r o t h r o i n b i n Corn-

BIOCHIMIE, 1976, 58, n ° 5.

p o n e n t , c o m p l e t e l y e m e r g e d from the A m b e r l i t e c o l u m n a n d e n t e r e d the I~EA~E A-50, the A m b e r l i t e column, still c o n t a i n i n g f r a c t i o n s I I a n d III, was d i s c o n n e c t e d from the system a n d elution p r o c e e ded d i r e c t l y on the D E A E . S e p h a d e x c o l u m n w i t h 0.2.0 M, 0.25 M a n d 0.30 M NaC1, all in 0.10~ M p o t a s sium p h o s p h a t e buffer, p H 7.0. A l t e r n a t i v e l y , fraction I f r o m tile Am~beNite c o l u m n was c a r e f u l l y p r e c i p i t a t e d w i t h p o w d e r e d (,NH4)2SO 4 up to 70 p e r cent s a t u r a t i o n a n d the p r e c i p i t a t e collected b y centri'fugalion at 8,000 X g for 10 minutes, then d i s s o l v e d in the smallest volume of 0.10 M p o t a s s i u m p h o s p h a t e - 0.10 M NaCI buffer, p H 7.0, d i a l y z e d a g a i n s t this buffer a n d r e c h r o m a t o g r a p h e d on D E A E - S e p h a d e x A-50 p r e v i o u s l y equilib r a t e d w i t h the same buffer. P r o t h r o m b i n p r e p a r a t i o n s w e r e tested for pur i t y b y d i s c gel e l e c t r o p h o r e s i s in 7 p e r cent ( w / v ) p o l y a c r y l a m i d e b y the m e l h o d o.f Davis for acidi:c p r o t e i n s [26] as p r e v i o u s l y d e s c r i b e d [1]. Ami,doschwarz ( n a p t h a l e n e b l a c k ) a n d Coomassie B r i l l i a n t Blue R (both f r o m Sigma Chem. Co.) w e r e used for staining. S c a n n i n g s of the d e s t a i n e d gels w e r e p e r f o r m e d u s i n g an Acta I I I B e c k m a n spect r o p h o t o m e t e r fitted w i t h a s c a n n i n g d e v i c e for gels. P r o t e i n c o n c e n t r a t i o n was estimated b y the mic r o b i u r e t m e t h o d of I t z h a k i a n d Gill [27] ; N-term i n a l a n a l y s i s f o l l o w e d the Sanger d i n i t r o p h e n y l alton as descri,be,d b y Bise,rte et al. [28] ; C-terminal analysis w a s per'formed b y the h y d r a z i n o l y s i s m e t h o d of A k a b o r i [29] a n d the c a r b o x y p e p t i d a s e m e t h o d [30]. C o n d i t i o n s for the N - t e r m i n a l analysis w e r e the same as those d e s c r i b e d for p i g t h r o m b i n V1]. H y d r a z i n e (Carlo E r b a , 85 p e r cent w / v ) was p u r i fied b y d i s t i l l a t i o n w i t h N a 0 H u n d e r N 2 b y the m e t h o d of B r a u n a n d S c h o r o e d e r [31] f o l l o w e d b y a r e d i s t i l l a t i o n w i t h C;aO [32]. The final p r o d u c t w a s t i t r a t e d w i t h a s t a n d a r d solution of KBrO 3 [33]. H y d r a z i n o l y s i s was p e r f o r m e d w i t h 6 m g of p r o t h r o m b i n p l u s 0.5 m l o~f p u r i f i e d by_ d r a z i n e ( a p p r o x i m a t e l y 99 p e r cent p u r i t y ) in evac u a t e d p y r e x a m p o u l e s at 110°C ¢or 2,6, a n d 10 h. After r e m o v i n g the excess h y d r a z i n e b y l y o p h i l i zation, the s o l i d r e s i d u e w a s d i s s o l v e d in 1 ml w a t e r a n d the solution v i g o r o u s l y s h a k e n w i t h 0.5 ml of b e n z a l d e h y d e for 2 h b y m e a n s o.f a TB-1 a u t o m a t i c m i x e r (Taiyo Bussan Co. Ltd. 5apan). After r e m o v i n g the hydrazi,des b y c e n t r i f u g a t i o n , a n a l y s i s of the C-terminal r e s i d u e in the s u p e r n a tant w a s p e r f o r m e d b y t h e d a n s y l a t i o n r e a c t i o n of G r a y a n d H a r t l e y [34] as d e s c r i b e d b y G r a y [35]. F o r that, 0.1 ml o,f the s u p e r n a t a n t w a s t r e a t e d w i t h 0.1 ml of 0.5 M N,aH:CO 3 p l u s 0.2 ml of 0.25 p e r

Pig prothrombin. cent (w/v) o'f DN,S-C1 i n acetone, a n d the r e a c t i o n p e r f o r m e d at room t e m p e r a t u r e i n the dark for 2 h. The DNS-amino acid was then identified by t h i n layer c h r o m a t o g r a p h y on 20 X 20 cm silica gel-G plates, u s i n g as solvent tert-butanol-cbloroform-acetic acid (3:6:1, v/v). Alternatively, after s h a k i n g w i t h benzal:dehyde for on,ly 30 rain, lhe s u p e r n a t a n t was q u i c k l y lyophilized a n d then subjected to a m i n o acid analysis i n the auto-analyser. Degradation with ca,r:boxypeptidases A a n d B was tried t h r o u g h i n c u b a t i o n of 6 mg of p r o t h r o m b i n ,

507

(8 mA p e r tube) for 5 h. B r o m o p h e n o l blue a n d Coomassie Brillant Blue R were used as m a r k e r a n d stain, respectively. A m i n o acid composition was d e t e r m i n e d w i t h the aid of the auto-analyser described by Alonzo a n d Hirs [37], operated by the m e t h o d of Spaekm a n et al. [38] with Aminex A-5 r e s i n i n the short column (0.9 cni × 16 cm) a n d Pa-28 r e s i n i n the long one (0.9 exn × 60 cm) at a flow rate of 30 m l / h at 52°C. Samples were h y d r o l y z e d w i t h constant boiling, glass distilled HC1 for 24, 48 a n d

TABLE 1.

Yield of pig prothrombin and speci[ic actioity [ollowing the puri[ication procedure. Volume (ml) Original plasma ( I ) . . .

Nlll units/ml

Total NIH units

mg tolal INItl units/mg protein/ml of protein

5,000

137

685,000 [ (I00 percent)i

73.0

After dialysis against NH~CI-NaCI buffer (H) . . . . . . . . . . . . . . .

720

920

662,400 (96.7 percent)

7.3

After preeipitation with 50-70 per cent saturated ammonium su~ fate (IfI) . . . . . . . . . . '

44

10,000

440,000 (64.2 percent)

25.8

388

After chromatography (*) on amberlite IRC-50 (Iv) . . . . . . . .

40

5,480

219,200 (32.0 per cent)

10.7

512

8,600

55,900 (8.2 per cent) i

After chromatography (') on DEAE-sephadex A-50 (V) . . .

6.5

1.88

i i

5.9

126

1,458

(*) Followed by precipitation with 70 per cent saturated (NH~)zSOI and dialysis.

i n 1 ml 0.2 M N - e t h y l m o r p h o l i n e acetate buffer, pH 8.5, at 37°C, c o n t a i n i n g 0.05:6 M SDS, w i t h 0.05 mg o,f DFP-treated c a r b o x y p e p t i d a s e A (Sigma Chemical Co.) or 0.03 mg of c a r b o x y p e p t i dase B ( W o r t h i n g t o n Biochemical Corp.) exactly as d e s c r i b e d by Ambler [39], except that analysis for free a m i n o acids was p e r f o r m e d i n the autoanalyser. Molecular weights w e r e d e t e r m i n e d b y SDSp o l y a c r y l a m i d e gel electrophoresis as described b y W e b e r a n d Osborn [36] using, as s t a n d a r d s , b o v i n e s e r u m a l b u m i n (Carlbiochemical), egg alb u m i n , c a r b o x y p e p t i d a s e A, a - c h y m o t r y p s i n o g e n a n d c y t o c h r o m e c (.all from Sigma Chemical Co.). R u n n i n g gels, 10 p e r cent (w/v) i n a c r y l a m i d e a n d measuring 6 mm× 10.0 ram, w e r e used at room temperature and constant current conditions

BIOCHIMIE, 1976, 58, n ° 5.

72 h at l l 0 ° C in evacuated P y r e x ampoules. Tyrosine, t h r e o n i n e a n d serine were d e t e r m i n e d b y e x t r a p o l a t i o n of the h y d r o l y s i s curve to zero time intercept. Half-eystine was analysed in the form of cysteie acid after o x i d a t i o n w i t h p e r f o r m i c acid [39] and 18 h of h y d r o l y s i s at l l 0 ° C . T r y p t o p h a n was estimated by the s p e c t r o p h o t o m e t r i c method of G o o d w i n a n d Morton [40J i n the whole protein. Due to the c o n s t a n t yield of p r o l i n e values at different times of hydrolysis, this r e s i d u e was used as re.terence for the calculation of the m o l a r ratios. RESULTS. Yields of pig p r o t h r o m b i n a n d specific activities following the described p u r i f i c a t i o n p r o c e d u r e are s h o w n i n table I.

508

F. A. Leone, G. Chiericato, J. C. Sag and J. R. Giglio.

The n o n - c h r o m a t o g r a p h e d zymogen so p r e p a r e d couLld be c o n v e r t e d into t h r o m b i n i n 25 per cent triso'dium citrate, p H 8.0, at 25°C, w i t h i n a relatively short time, p r o v i d e d the m ~ d i u m was seeded w i t h a small a m o u n t (rf t h r o m b i n i n or,der to decrease the ,lag phrase. The sigmoid a c t i v a t i o n

0.05 M potassium p h o s p h a t e buffer, pH 7.0. Although two other fractions m a y be eluted by the 0.20 a n d 0.30 M buffers, only f r a c t i o n I shows clotting activity on fibrinogen w h e n processed b y the two-~stage metho.d of W a r e a n d Seegers [41]. This fraction, w h e n analysed b y disc gel eleetrophoresis i n 7 per cent polyacrylam~de was h o w n to be heterogenous (fig. 1A). As described in the section, fraction I was d i r e c t l y r e e h r o m a t o g r a p h e d on a D~SA,E-Sephadex A-50 c o l u m n c o n n e c ted to the Amberlite c o l u m n (fig. 2). Only fraction VI f r o m this second c o l u m n showed p r o t h r o m b i n activity on fibrinogen. If elution of the p r o t h r o m b i n f r a c t i o n i n the D't~AE-Sepha.dex c o l m n n is p e r f o r m e d ~,ith 0.20 M i n s t e a d of 0.2!5 M NaCi buffer, the elution volume a n d time are significantly i n c r e a s e d a n d a second fraction, p a r t i a l l y resolved from the p r o t h r o m b i n one, is eluted later. The electrophoretic parterres of both fractions are s h o w n i n figure 1B a n d figure 1C, respectively.

FI6. 1. - - Polgacrglamide disc gel electrophoresis of pig prothrombin. A - - Fraction I eluted from the Amberlite IRC-50 column, followed by precipitation by 70 per cent saturated (NH,)~SO~ and dialysis. B - - Prothrombin fraction eluted from the DEAESephadex A-50 column by 0.20 M NaC1-0.10 M potassium phosphate, pH 7.0. C - - Contaminant component which follows immediately after prothrombin when elution is continued with the 0.20 M NaC1 buffer. D - - Prothrombin fraction eluted by the 0.25 M NaC1- 0.10 M potassium phosphate buffer, pH 7.0. E - - Same as D, except that the sample was aged one month at --20°C before the chromatography on Amberlite. B to E - - Amberlite and DEA,E-Sephadex columns connected to one another. Electrophoretic running was dowr~ward, to'ward the anode. The thin line near the bottom of the gels are of the marker.

curve [1] showed a m a x i m u m activity c o r r e s p o n d i n g to 8 h of i n c u b a t i o n a n d a yield of about 22,000 TAME units/1 of plasma. Chromatography, w h e n p e r f o r m e d only on Amberlite IRC-50 c o l u m n gives a n elution profile essentially s i m i l a r to that p r e v i o u s l y r e p o r t e d [1], p r o t h r o m b i n b e i n g eluted i n fraction I b y the

BIOCHIMIE, 1976, 58, n ° 5.

F i n a l l y , figure 1 shows the electrophoretie patt e r n s o,f two p r e p a r a t i o n s w h i c h were chromatog r a p h e d i m m e d i a t e l y after step III of table I (fig. 1D). a n d after b e i n g aged for one m o n t h at - - 2 0 ° C (fig. 1E). I n both cases, the c o l u m n s were c o n n e c t e d to one a n o t h e r a n d elution of p r o t h r o m b i n was p e r f o r m e d w i t h the 0.2'5 M NaC] buffer. A very faint fast movin.g b a n d , w h i c h a m o u n t s to less t h a n 0.5 per cent of the p r o t h r o m b i n component, could still be detected both i n figure 113 a n d figm-e 1D. Tile e l e c t r o p h o r e t i c m o b i l i t y of this b a n d was the same as that o,f the c o n t a m i n a n t of figure 1C. This c o n t a m i n a n t in.creases to about 3 p e r c e n t if the Amberlite a n d D EAE-Sephadex c o l u m n s are operated separately, w i t h i n t e r m e diale steps of p r e c i p i t a t i o n b y ¢NH4)2SO4 a n d dialysis as described. This highly purified pig p r o t h r o m b i n showed rag/nil a n E~:0 °e~ = 1.45 at pH 7.0 a n d 280 nm, a n d only a l a n i n e as N-terminal. C - t e r m i n a l analysis revealed serine b y d a n s y l a t i o n of the released a m i n o aci.d a,fter h y d r a z i n o l y s i s , although traces of glycine were also visible. Direct d e t e r m i n a t i o n of a m i n o acids in the auto-ana,lyser, following the alternative d e s c r i b e d p r o c e d u r e , confirmed serine as the only resi~due released i n significant a m o u n t (about 0.4 m o l e / m o l e ) . Car~boxypeptidases A a n d B failed to split a n y C-terminal r e s i d u e from pig p r o t h r o m b i n even after 3,6 h of i n c u b a t i o n . A final specific activity of 1,460 NIH u n i t s / m g p r o t e i n and 8 p e r cent yield c o m p a r e d to the initial p r o t h r o m b i n activity of the s t a r t i n g plasma

Pig prolhrombin. was obtained, which corresponds to a 776 fold purification, /

I

~

]

509

The molecular weight of pig prothrombin (fraetion VI) determined by SD,S-polyaerylamide disc

l

o i

E w

2.5-

i E

b

"E 2 . 0 - c O

~

!

m O c~ o9

FIG. 2. - - Column rechromatography ! on DEAE-Sephadex A - 50 of fraction I f r o m the A m b e r l i t e I R C - 5 0 c o l u m n i El]. The gel w a s first e q u i l i b r a t e d w i t h 0.05 M p o t a s s i u m p h o s p h a t e b u f f e r pH 7.0, a n d e l u t i o n p r o c e e d e d w i t h t h e ~ i n d i c a t e d buffers. This c o l u m n w a s I first c o n n e c t e d to the A m b e r l i t e coI l u m n to collect f r a c t i o n I a n d t h e n l ~ developed separately. F l o w rate, 20 m l / h ; f r a c t i o n vol u m e s , 10 m l ; gel bed, 2.5 × 60 c m ; t e m p e r a t u r e , 2 ° C ; load, 200 m g t o t a l t protein.

~ ~ ~

1.5

z 0

g,-

1.0

HI ~

0

70

A

v

I l l I ~

~ d ~

I

I

~

9O

PPO

~30

FRACTION

o_

~

~ o ~ ~ \ ~ ~ ~ / o / d ~ ~ i ~50

.

~

~ J70

J90

NUMBER

TABLE II.

Amino acid composition of pig prothrombin. Ox [&8]

Pig Amino acid

g residue/ [experiment t00 g protein Imolar ratio

9.36 50.£0 Thr 6.15 c 37.95 Set 4.99, 35.65 Glx 12.91 62.10 Pro 3.58 23.00 Gly 4.03 43.93 Ala 2.91 25.30 Half-Gys 4.96 a 29.90 Val 5.57 34.96 Met 0.57 2.76 5.35 29.44 Ile 5.80 31.74 Leu Tyr 4.97,~ 18.86 5.27 22.31 Phe 7.39 35.88 Lys His 1.86 8.28 5.76 23.00 Arg 3.42 t, Trp Total 94.85 M i n i m u m molecular weightd

Asx

Rat [12]

[

Man [14]

g residue/t00 g protein

integer

51 38 36 62 23 44 25 30 35 3 30 32 19 22 36 8 23 13 53O 59,375

.

8.80

10.22

4.20 5.01 11.98 5.24 3.58 3.36 2.57 4.95

6.32 4.47 11.98 3.83 3.12 2.38 1.36 4.56

1.36

1.55

2.91 6.69 3.88 4.39 4.77 2.05 7.25

3.05 5.88 3.76 5.52 5.31 1.99 5.34 2.82 83.46

--

82.99

9.85 4.90 4.36 13.17 4.28 3.83 3.75 2.51 4.53 1.29 3.30 6.52 4.31 5.07 5.19 1 91 8.34 3.28

90.39

(a) D e t e r m i n e d as c y s t e i c acid a f t e r p e r f o r m i c acid o x i d a t i o n a n d 18 h h y d r o l y s i s at ll0°C. (b) D e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y . (c) V a l u e s o b t a i n e d b y e x t r a p o l a t i o n o f t h e h y d r o l y s i s c u r v e to zero t i m e i n t e r c e p t . (d) C a r b o h y d r a t e s n o t i n c l u d e d . Asx, Glx- a s p a r t i c acid o r a s p a r a g i n e , g l u t a m i c acid o r g l u t a m i n e , r e s p e c t i v e l y . (*) P r o l i n e w a s u s e d as r e f e r e n c e f o r t h e c a l c u l a t i o n o f t h e m o l a r r a t i o .

BIOCHIMIE, 1976, 58, n ° 5.

510

F. A. Leone, G. Chiericato, J. C. Sag and J. R. Giglio.

gel e l e c t r o p h o r e s i s w a s s h o w n to be 65,090 (50,000 for the 2nd, fast m o v i n g c o m p o n e n t m e n t i o n e d above). The a m i n o a c i d c o m p o s i t i o n of p i g p r o t h r o m bin, as well as those of ox, rat a n d m a n are compareld on table II. C h r o m a t o g r a p h e d p i g p r o t h r o m b i n , e i t h e r the h e t e r o g e n e o u s f r a c t i o n I from the A m b e r l i t e col u m n or the h i g h l y p u r i f i e d f r a c t i o n VI form the D,EAE-Sephadex c o l u m n , di,d not activate in 25 p e r cent t r i s o d i u m c i t r a t e s e e d e d w i t h t h r o m b i n even after p e r i o d s of incu.balion as l o n g as 24 h. DISCUSSION. The p r o c e d u r e d e s c r i b e d h e r e for the p r e p a r a tion c~f c r u d e ( n o n - c h r o m a t o g r a p h e d ) p i g p r o t h r o m b i n is r e l a t i v e l y s i m p l e r a n d less l a b o r i o u s t h a n that p r e v i o u s l y d e s c r i b e d [1]. Table I s h o w s that except f o r the last step o'f the p u r i f i c a t i o n p r o c e d u r e , c h r o m a t o g r a p h y on D~EAE-Sephadex, the specific activities at the steps w h i c h are comm o n for both p r o c e d u r e s are l o w e r in the p r e s e n t method. This m a y be e x p l a i n e d b y the fact t h a t the p r e v i o u s m e t h o d involves p r e l i m i n a r y f r a c t i o n a t i o n s u n d e r specific c o n d i t i o n s , i n c l u d i n g iso, e l e c t r i c p r e c i p i t a t i o n at p H 5.0. I n spite of that, the p r e s e n t p r o c e d u r e affords, after c h r o m a t o g r a p h y on D E A E - S e p h a d e x , a final p,roduct w i t h 1,4,60 clottin,g u n i t s / r a g an~d a h i g h level of p u r i t y as .determined b y d i s c gel p o l y a c r y l a m i d e electrop h o r e s i s (fig. 1B a n d 1D) anld t h e r e f o r e a v e r y suitable m a t e r i a l to be s u b m i t t e d to s t r u c t u r a l analysis. F o u r c o n d i t i o n s w e r e s h o w n to be f u n d a m e n t a l for a goo,d p e r f o r m a n c e of this n e w p r o c e d u r e : h i g h c o n c e n t r a t i o n (if the EDTA solution, complete dissolution of the b a r i u m c i t r a t e c o m p l e x p r e l i m i n a r y to the d i a l y s i s step, m a i n t e n a n c e of the p H at 8.0 d u r i n g d i a l y s i s a n d a sufficiently h i g h i o n i c s t r e n g t h to keep the large a m o u n t of globuline in solution. The p r e l i m i n a r y i n c r e a s e a n d m a i n t e n a n c e of the p,H at 8.0 in the s t a r t i n g b a r i u m citrate suspension, b y m e a n s of a s a t u r a t e d T r i s solution, a n d d i a l y s i s of the r e s u l t i n g solution against the 0.05 .M NHa-I~Cil-0.3 M NaC1 buffer, p H 8.0, a f f o r d e d the r e q u i r e d c o n d i t i o n s . T a b l e I shows a h i g h y i e l d (9,6.7 p e r cent) of the clotting a c t i v i t y after step II, so i n d i c a t i n g that d e n a t u r a t i o n d i d not o c c u r a u d the c o n d i t i o n s for almost c o m p l e t e r e c o v e r y of p:rothrombin f r o m the b a r i u m c i t r a t e w e r e m e t in the p r e s e n t p r o c e dure. It is clear h o w e v e r t r o m table I that signific a n t losses of t h e e n z y m a t i c units o c c u r d u r i n g

BIOCHIM1E, 1976, 58, n ° 5.

e a c h step unti4 a final y i e l d o~f 8.2 p e r cent of the i n i t i a l clotting a c t i v i t y is r e a c h e d . F r a c t i o n a t i o n w i t h a m m o n i u m s u l p h a t e was p e r f o r m e d in l h e 50-70 p e r cent r a n g e in o r d e r to p r o v i d e a h i g h e r level of p u r i t y at this step [1]. Due to the b u f f e r i n g of the solution at p H 8.0, solid a m m o n i u m s u l p h a t e c o u l d be used i n s t e a d of a s a t u r e d solution of this salt, w h i c h w o u l d increase three times the i n i t i a l volume. After the p r e c i p i t a t i o n at 70 p e r cent s a t u r a t i o n a n d c e n t r i fugation, l h e p H of the d i s c a r d e d s u p e r n a t a n t was a b o u t 6.6. P r e l i m i n a r y c h r o m a t o g r a p h y of c r u d e p r o t h r o m b i n on A m b e r l i t e I~C-50 was s h o w n to be essential to p r o v i d e a h i g h l y p u r i f i e d z y m o g e n in the next step. Attempts to get the same p r o d u c t b y d i r e c t c h r o m a t o g r a p h y on D'EA.E-Sephadex w e r e unsuccessful. Tile m o l e c u l a r w e i g h t of the fast m o v i n g comp o n e n t (fig. 1C) is close to that d e t e r m i n e d b y Bajaj [42~ a n d E s n o u f [43] for F a c t o r X, but the fact t h a i F a c t o r X is eluted at a h i g h e r ionic s t r e n g t h [43] suggests o t h e r p o s s i b i l i t i e s too, such as a d e g r a d e d p r o t h r o m b i n . This h y p o t h e s i s is s i r o n g l y s u p p o r t e d b y the fact that l h e p r o p o r t i o n of this c o n t a m i n a n t in the s a m p l e m a y be i n c r e a sed w h e n b o t h c h r o m a t o g r a p h s a r e p e r f o r m e d s e p a r a t e l y as d e s c r i b e d , o r w h e n the s a m p l e is aged, even at - - 2 0 ° C , p r i o r to the A m b e r l i t e column. In this l a t t e r case a 3rd c o n t a m i n a n t a p p e a r s (fig. 1E). The same c o n c l u s i o n is d r a w n f r o m the o b s e r v a t i o n .that p r o l o n g e d time a n d large volume of elution w i t h the 0.20 M NaC1 buffers affords a 2n:d p e a k folio:wing the p r o t h r o m b i n one, f r o m lhe D E A E - S e p h a d e x column, w i t h the same m o b i l i t y as t h a t of the m e n t i o n e d fast m o v i n g c o m p o n e n t (fig. 1C). The above consid e r a t i o n s i n d i c a t e t h e r e f o r e that our p i g p r o t h r o m b i n p r e p a r a t i o n m a y be c o n s i d e r e d h i g h l y p u r e b y the e l e c t r o p h o r e t i c c r i t e r i u m a n d that the almost i n v i s i b l e c o n t a m i n a n t b a n d (fig. I~B a n d 1D) still d e t e c t a b l e b y p o l y a e r y l a m i d e gel e l e c t r o p h o rests (less t h a n 0.5 p e r cent) is a d e g r a d e d fragm e n t of m o l e c u l a r w e i g h t 50,000. The final y i e l d of 8 p e r cent is s i m i l a r to t h a t r e p o r t e d b y Esnou~f [43] a n d Cox [44] for b o v i n e p r o i h r o m b i n , but l o w e r t h a n thai of Stenn a n d Blout [45]. The fact that the c h r o m a t o g r a p h e d z y m o g e n does not activate significantly in 25 p e r cent tris o d i u m citrate, b u t activMes d u r i n g the c l o t t i n g a c t i v i t y test, is i n d i c a t i v e of l a c k (K c o n t a m i n a n t a c t i v e clotting factors, although the A m b e r l i l e f r a c t i o n I is c l e a r l y h e t e r o g e n e o u s (fig. 1A), The

Pig prothrombin. h i g h l y p u r i f i e d p r o t h r o m b i n s h o w e d a n E a.0 1.0 mg/m'. cm = 1.45 a t p H 7.0 an,d 280 n m , s l i g h t l y d i f f e r e n t f r o m t h a t p r e v i o u s l y r e p o r t e d , n a m e l y , 1.50 [1!. A l a n i n e , f o u n d as t h e N - t e r m i n a l r e s i d u e of p i g p r o t h r o m b i n , s e e m s to b e t h e N - t e r m i n a l c o m m o n to t h e p r o t h r o m b i n of all a n i m a l s p e c i e s stu'died, s u c h as o x [3], h o r s e [8], d o g [10], r a t [12], m a n [17] a n d s h e e p ( C h i e r i c a t o , G., u n p u b l i s h e d da~a). S e r i n e , f o u n d as C - t e r m i n a l , is s i m i l a r l y t h e C - t e r m i n a l of b o v i n e p r o t h r o m b i n [46]. A l t h o u g h s e r i n e w a s r e l e a s e d i n a ,low y i e l d b y h y d r a z i n e a n d n o t at all b y c a r b o x y p e p t i d a s e A, i t w a s t h e o n l y r e s i d u e d e t e c t e d w h e n t h e t i m e of e x p o s u r e o f t h e h y d r a z i d e s to w a t e r w a s r e d u c e d to 30 niln u t e s a n d t h e 2 h i n c u b a t i o n at r o o m t e m p e r a t u r e and high pH required for dansylation was avoid e d . T h i s p r o c e d u r e a f f o r d e d 0.4 mo~e o f s e r i e / m o l e of p r o t e i n , b u t p r a c t i c a l l y n o g l y c i n e o r alan i n e , w h i c h a r e k n o w n to give, as s e r i n e , l a b i l e h y d r a z i d e s i n a q u e o u s so lu.tions [47]. T h e d i s a p p e a r a n c e of t r a c e s of ~ l y e i n e b u t p e r s i s t a n c e of s i g n i f i c a n t a m o u n t s of s e r i n e w h e n t h i s a l t e r n a t i v e p r o c e d u r e w a s p e r f o r m e d s u g g e s t s t h a t s e r i n e is t h e C - t e r m i n a l r e s i d u e of p i g p r o l h r o m b i n . T h e r e c o v e r y of 40 p e r c e n t i s c o m p a r a b l e to t h a t obtained for other proteins in similar conditions [48]. The finding that only alanine and serine are in t h e N anal C - t e r m i n a l p o s i t i o n s , r e s p e c t i v e l y , suggests that pig prothrombin has a single peptide c h a i n . T h e v a l u e o~ 65,000 f o r t h e m o l e c u l a r w e i g h t is c o m p a r a b l e to t h o s e f o u n d f o r o t h e r a n i m a l s p e c i e s ~4, 10, 12]. T h e d i f f e r e n c e 65,000 59,375 : 5,6,25 : 8.6 p e r c e n t , b e t w e e n t h e mo,lecular weight determined by SDS-polyacrylamide d i s c gel e l e c t r o p h o r e s i s an,d t h e m i n i m u m v a l u e g i v e n b y t h e a m i n o a c i d corn, p o s i t i o n , m a y b e a t t r i b u t e d t o t h e c a r b o h y d r a t e c o n t e n t , w h i c h is a b o u t 11 p e r c e n t i n b o v i n e p r o t h r o m b i n [31 a n d is o b v i o u s l y i n c i u d e d i n t h e e l e c t r o p h o r e t i c d e t e r ruination. Further studies on this subject are in progress in our laboratory. Ac,knamled!lments. We w i s h to t h a n k the for the c o n t i n u o u s financial support to o u r l a b o r a t o r y . R~SUMP, Nous d6crivons i c i u n e m~thode de p r 6 p a r a t i o n de p r o t h r o m h i n e de pore tr6s purifi~e. P a r c o m p a r a i s o n aver l'activit6 coagulante du p l a s m a initial, cette prot6ine a ~t6 purifi6e 776 fois, aver u n r e n d e m e n t final de 8 per cent. Le zynmg6ne purifi6 a uue activit6 spt~cifique de 1.460 unit~s NIH p a r nag de prot6ine, un poids moI~BIOCHIMIE, 1976, 58, n ° 5.

511

culaire de 65.000 apr6s d 6 t e r m i n a t i o n p a r 61ectrophor6se ca gel de polvacrylamide. S.D.S., F l'0mg/mll,0 era, 280 nm : 1,45 h pH 7.0 et la c o m p o s i t i o n en acides aminds suiv a n t e : Asx~, Thr,,, Ser~, Glx~,._., Pro~, Gly~,, Ala~, L'.'~ Cyst, Val~, Met3, Ile~o, Leua:, Tyro.,, Phe._,~, Lys~,;, His,, Arg~ et Trp~.~, ee qui correspond h u n poids mol6culaire, m i n i m u m de 59.370 (hydrates de carbone non compris). L ' a l a n i n e est le seul r(isidu N-terminal. Les earl)oxypeptidases A e t B n ' o n t lib~r~ a u c u n r6sidu C-leriuinal. Cependant, l ' h y d r a z i n o l y s e lib~re 0..4 mole de s6rine p a r mole de p r o t h r o m b i n e . Nous avons (~tudi6 l'aetiwltion de la p r o t h r o m h i n e de pore, b r u t e et chromatographi6e.

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Pig prothrombin: purification and properties.

BIOCHIMIE, 1976, 58, 505-512. Pig prothrombin : Purification and properties. F r a n c i s c o A. LEONE, Glaico GHIERICATO, Jos6 C. SAY a n d Jos6 R...
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