Isolation of chicken ovoinhibitor by affinity chromatography on chymotrypsin-sepharose

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BIOCHIMICAET BIOPHYSICAACT3.

BBA 35830 ISOLATION OF CHICKEN O V O I N H I B I T O R BY A F F I N I T Y CHROMATOGRAPHY ON C H Y M O T R Y P S I N - S E P H A R O S E

GAD FILINSTEIN

Department of Biochemistry, Tel-Aviv University, Tel-Aviv (lsrael) (Received December 7th, 197 o)

SUMMARY

Chicken ovoinhibitor was purified from ovomucoid by a new procedure using a single step of affinity chromatography through water-insoluble bovine chymotrypsin-Sepharose. It is capable of inhibiting bovine trypsin and chymotrypsin and has a molecular weight of 52 400.

INTRODUCTION

LINEWEAVER AND MURRAY1 isolated ovomucoid from chicken egg white and characterized it as an inhibitor of bovine trypsin. They used a mixture of trichloroacetic acid and acetone to precipitate most of the egg white proteins and then acetone to precipitate the ovomucoid. MATSUSHIMA2 found that chicken egg white contains two trypsin inhibitors, ovomucoid and an additional one that he named ovoinhibitor. He isolated it from egg white by salt fractionation. RHODES et al. 3 reported that ovoinhibitor was also capable of inhibiting bovine chymotrypsin. FEENEY st al. 4 found that ovomucoid prepared by the method of LINEWEAVERAND MURRAY1 also contains the ovoinhibitor. TOMIMATSU el al. 5 isolated ovoiinhibitor from ovomucoid by salt fractionation. DAVIS et al. ~ purified ovoinhibitor from egg white by salt fractionation followed by chromatography on DEAE-cellulose. They reported that chicken ovoinhibitor was heterogeneous due to variation in the content of charged sugars like sialic acid. Otherwise, the several fractions of ovoinhibitor had the sanle amino acid composition, molecular weight and inhibitory activities. We have recently reported 7,s the purification of trypsin and chymotrypsin by affinity chromatography on insoluble protein inhibitors of proteases. The protein inhibitors were cross-linked to Sepharose after it was activated by CNBr as described by Ax~N et al. s. We now report the use of chymotrypsin-Sepharose for the isolation of chicken ovoinhibitor from crude ovomucoid by a single step of affinity chromatography. Abbreviations: BAPA, N-benzoyl-DL-arginine-p-nitroanilide; ATEE, N-acetyl-L-tyrosine ethyl ester.

Biochim. Biophys. Mcta, 236 (1971) 73-77

ISOLATION OF CHICKEN OVOINHIBITOR I

I

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75 i

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Fig. i. Chromatography of crude chicken ovomucoid on chymotrypsin-Sepharose column. Solid line, absorbance at 28o m/~. Vertical arrow, elution buffer change. First protein peak contains ovmnucoid and the second one contains ovoinhibitor. See text. Crude chicken o v o m u c o i d was p r e p a r e d b y the m e t h o d of LINEWEAVER AND MURRAY1. Bovine a - c h y m o t r y p s i n (3 times crystallized L o t CDI 7JC) a n d bovine t r y p s i n (2 times crystallized L o t T R L 7LA) were p u r c h a s e d from W o r t h i n g t o n Biochemical Corp. N-Benzoyl-DL-arginine-p-nitroanilide (BAPA) a n d N-acetyl-Lt y r o s i n e e t h y l ester (ATEE) were from Cyclo Chemical Corp. Sepharose 2B was from P h a r m a c i a . A T E E was used as a c h y m o t r y p s i n s u b s t r a t e 1° to d e t e r m i n e the i n h i b i t o r y activities of o v o m u c o i d a n d ovoinhibitor. Likewise, B A P A was used as a t r y p s i n s u b s t r a t e n. Insoluble c h y m o t r y p s i n cross-linked to Sepharose was p r e p a r e d as described b y PORATH et al. 12. The c h y m o t r y p s i n - S e p h a r o s e was p a c k e d into a column, 26 cm × 1.6 cm, a n d w a s h e d t h o r o u g h l y in the cold w i t h 0.20 M t r i e t h a n o l a m i n e buffer, p H 8.0. 3 g of crude o v o m u c o i d were dissolved in 50 ml of the same buffer a n d a p p l i e d to the column (fractions of 3.0 ml were collected). The column was w a s h e d w i t h t r i e t h a n o l a m i n e buffer, p H 8.0, until no more o v o m u e o i d protein was d e t e c t e d in the effluent (Fig. I). T h e n the d u r i n g buffer was changed to 0.20 M KC1-HC1, p H 2.0. A second protein, ovoinhibitor, emerged from the column. The fractions c o n t a i n i n g t h e two proteins were pooled s e p a r a t e l y , d i a l y z e d a g a i n s t w a t e r a n d lyophilized. B o t h proteins were c a p a b l e of i n h i b i t i n g t r y p s i n . A b o u t 9 0 % of the t r y p s i n i n h i b i t o r y a c t i v i t y of the original crude o v o m u c o i d was recovered in the TABLE I PURIFICATION

OF O V O I N H I B I T O R

Crude ovomucoid Ovomucoid Ovoinhibitor

ON C H Y M O T R Y P S I N - - S E P H A R O S E

Chymotrypsin (inhibition units*)

Yield ( %)

Specific activity (inhibition units~rag)

84. 3 o 39.8

IOO

0.024

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36.6

47.2

Purification

* An inhibition unit is an amount that inhibits I m g of chymotrypsin. Biochim. Biophys. Mcta, 236 (1971) 73-77

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Fig. 2. S e d i m e n t a t i o n e q u i l i b r i u m of o v o i n h i b i t o r in t h e a n a l y t i c a l u l t r a c e n t r i f u g e , a t 17 ooo re v./ min, o. Io M t r i e t h a n o l a m i n e buffer, p H 8.o.

ovomucoid and about 2 ~o in the ovoinhibitor. Table I gives the results in recovering the chymotrypsin inhibitory activity. About 50% of the chymotrypsin inhibitory activity was associated with tile ovoinhibitor fraction while none was associated with the ovomucoid. These yields of inhibitory activity in different experiments were 40 700/0 • The specific activity of ovoinhibitor was about i. In some preparations it was I. 4. Aerylamide disc gel electrophoresis had shown that the ovoinhibitor was free of ovomucoid or any other protein contamination. A single broad band was obtained containing several sub-bands in agreement with the results of DAvis et al. ~. The amino acid composition of ovoinhibitor after complete acidic hydrolysis was determined on Beckman amino acid analyzer (moles amino acid per IO ooo g). It was found to be in very good agreement with the results reported by other workers 5,°. Equilibrium sedimentation studies in Beckman Model E analytical ultracentrifuge (17 ooo rev./rnin) had shown that ovoinhibitor was homogeneous (Fig. 2). The molecular weight of ovoinhibitor at infinite concentration was found to be 52 400. The molecular weights of ovoinhibitor reported by TOMIMATSU et al. ~ and by DAvis et al. 6 were 46 500 and 49 ooo, respectively. Sepharose, being a polydextran free of charge (no ion exchange), was found to be a good insoluble carrier of chymotrypsin. The difference in stabilities of ovoinhibitor chymotrypsin complex at various p H values made it possible to isolate a highly pure ovoinhibitor from crude chicken ovomucoid by a single step of affinity chromatography on chymotrypsin-Sepharose. At the same time, we obtained ow)mucoid free of ovoinhibitor. R I ' ; F I i RIgNCI';S I H. LINEWEAVER AND C. W. ~{URRAY, J. Biol. Chem., 171 (1947) 565 . 2 K. ),'[ATSUSHIMA, Science, 127 (1958) 1178. 3 M. B. RHODES, N. BENNETT AND R. E. FEENEY, J. Biol. Chem., 235 (196o) 1686.

Biochim. Biophys. Acta, 236 (1971) 73-77

ISOLATION OF CHICKEN OVOINHIBITOR 4 5 6 7 8 9 IO II 12

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R. E. FEENEY, F. C. STEVENES AND D. T. OSUGA,J. Biol. Chem., 238 (1963) 1415. Y. TOMIMATSU, J. J. CLARY AND J. J. BARTULOVlCH, Arch. Biochem. Biophys., 115 (1966) 536. J. G. DAVIS, J. C. ZAHNLEY AND j. w . DONOVAN, Biochemistry, 8 (1969) 2044. G. FEINSTEIN, F E B S Letters, 7 (197 °) 353. G. FEINSTEIN, Biochim. Biophys. Acta, 214 (197 o) 224. R. AXI~N, J. PORATH AND S. ERNBACK, Nature, 214 (1967) 13o2. G. V~. SCH\VERT AND ~'. TAKENAKA, Biochim. Biophys. Acta, 16 (I955) 57 °. B. F. ERLANGER, N. KOKOWSKY AND W. COHEN', Arch. Biochem. Biophys., 95 (1961) 271. J. PORATH, R. AXt~N AND S. ERNBACK, Nature, 215 (1967) 1491.

Biochim. Biophys. Acta, 236 (1971) 73-77