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Reversible and irreversible immobilization of car☐ypeptidase y using biospecific adsorption
315
Journal of Chromatography, 376 (1986) 315--321 Biomedical Applications Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
CHROMBIO. 2976
REVERSIBLE AND IRREVERSIBLE IMMOBILIZATION OF CARBOXYPEPTIDASE Y USING BIOSPECIFIC ADSORPTION
J. TURKOV~* and M. FUSEK
Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6 (Czechoslovakia) and J.J. MAKSIMOV and Yu. B. ALAKHOV
Institute of Protein Research, U.S.S.R. Academy of Sciences, Pushchino, Moscow Reg. (U.S.S.R.)
SUMMARY To find optimal biospecific hydroxyalkyl methacrylate (Spheron) derivatives for reversible and irreversible immobilization of carboxypeptidase Y, we elaborated the procedure for the biospecific sorption of carboxypeptidase Y to Spheron--Gly-Gly-p-aminobenzylsuecinic acid, mercury--Spheron and concanavalin A--Spheron. The highest yield was achieved in the case of concanavalin A--Spheron, which was also used for the irreversible, oriented immobilization by cross-linking of carboxypeptidase Y and concanavalin A with glutaraldehyde.
INTRODUCTION
The combination of many complementary binding sites on the surface of enzymes makes possible the formation of a number of biospecific complexes that can be used not only for efficient isolation but also for oriented immobilization. Fig. 1 shows the surface of an enzyme covered with several complementary binding sites. For example, this could be carboxypeptidase Y (CPY) containing a complementary binding site for the specific inhibitor Gly-Gly-paminobenzylsuccinic acid and one free SH group, which is placed in the active site of the enzyme. CPY also contains a carbohydrate moiety, which is a complementary binding site for the active site of lectin concanavalin A (Con A). The antigenic sites of the protein can be identified by determination of the 0378-4347/86/$03.50
© 1986 Elsevier Science Publishers B.V.
316
SUBSTRATE
OR INHIBITOR
ANTi--
BODY
~NZ~I'4E
Fig. 1. Possibilities of the enzyme to form biospecific complexes.
antigenic structures of the peptide chain after its interaction with specific antibodies according to Attasi [1] who described, for example, five precise antigenic sites in the molecule of myoglobin. The following may serve as another example: about 30% of the approximately 2000 enzymes found in the cell contain nucleotide coenzymes participating in the reaction with nucleotides. The specific recognition of certain nucleotide sequences by proteins is one of the reasons for studying peptide--nucleotide interactions [2]. It is generally known that many enzymes in the form of biospecific complexes exhibit higher stability. Well oriented attachment of enzymes to solid supports can result in a higher relative activity of the immobilized enzyme. Hsiao and Royer [3] compared several ways of attaching CPY to agarose and glass. Immobilized CPY with the highest activity was prepared by adsorption of the enzyme to Con A--Sepharose and subsequent cross-linkage by glutaraldehyde. Hsiao and Royer [3] also proved that the carbohydrate moiety can be used for oriented immobilization of enzymes, and oriented immobilization guarantees high activity of immobilized enzymes. Practically the same kinetic constants of peptidase and esterase activities of native and immobilized carboxypeptidase A were determined by Solomon et al. [4] when the enzyme was attached to the immobilized monoclonal antibody. Monoclonal antibodies were also used for specific immobilization of guinea pig liver transglutaminase by Ikura et al. [5]. The aim of the work reported here was to find optimal biospecific hydroxy-
317 alkyl methacrylate derivatives for reversible and irreversible immobilization of CPY. The first step was purification of CPY by biospecific sorption via different complementary binding sites. The results of these purification experiments served for the determination of the optimal hydroxyalkyl methacrylate derivative for immobilization. Con A--Spheron was used for oriented immobilization of CPY. EXPERIMENTAL CPY (EC 3.4.16.1) was obtained from Biolar (Olain, U.S.S.R.). N-Carboxybenzoxy-L-phenylalanyl-L-alanine (Z-Phe-Ala) was a product of Sigma (St. Louis, MO, U.S.A.), ribonuclease (EC 3.1.26.1)and 2-(N-morpholino)ethanesulphonic acid (MES) were from Reanal (Budapest, Hungary) and 2-mercaptoethanol and glutaraldehyde were from Fluka (Buchs, Switzerland). Sodium dodecyl sulphate (SDS) was from Serva (Heidelberg, F.R.G.). Spheron 1000 (100--200/~m), DEAE--Spheron 1000 (25--40 pm) and the remaining chemicals of analytical purity were supplied by Lachema (Brno, Czechoslovakia). The mercury derivative of the hydroxyalkyl methacrylate gel (mercury--Spheron) was prepared according to Turkov~ et al. [6] ; Gly-Gly-p-aminobenzylsuccinic acid--Spheron was prepared according to Turkov~ et al. [7]. Con A--Spheron was kindly donated by Dr. K. Filka (Department of Biochemistry, Charles University, Prague, Czechoslovakia). Purification o f CPY from a yeast autolysate on Spheron-Gly-Gly-p-aminobe nz y lsuccinic acid The autolysate from yeast Saccharomyces cerevisiae (50 ml) prepared according to Johansen et al. [8] was applied to a column (9 × 0.8 cm I.D.) of Spheron--Gly-Gly-p-aminobenzylsuccinic acid (content of immobilized inhibitor: 2.6 tzmol/g of dry carrier) equilibrated with 0.01 M sodium acetate (pH 5.0). The column was washed with 1 1 of 1 M sodium chloride-0.01 M sodium acetate (pH 4.3). CPY was desorbed with 0.01 M phosphate buffer (pH 7.0). The flow-rate was 50 ml/h; fraction volume 4.0 ml (see Fig. 2A). Chromatography o f CPY on mercury- Spheron A solution (10 ml) of CPY (0.1 mg/ml) was applied to a column (8 X 0.8 cm I.D.) of mercury--Spheron (prepared with 15% mercury derivative of methacrylanilide, 93 t~mol Hg per ml gel) equilibrated with 0.1 M sodium acetate (pH 6.0). The column was washed with 15 ml of starting buffer and CPY was desorbed with 0.05 M 2-mercaptoethanol--0.1 M sodium acetate (pH 6.0). The flow-rate was 15 ml/h; fraction volume 1 ml (see Fig. 2B). Chromatography o f CPY on Con A--Spheron A solution (200 ml) of CPY (0.1 mg/ml) was applied to a column (18 X 0.8 cm I.D.) of Con A--Spheron (content of immobilized Con A: 0.4 pmol/g of dry carrier) equilibrated with 0.4 M sodium chloride--0.1 M sodium acetate (pH 7). The column was washed with 20 ml of starting buffer and CPY was eluted with 0.1 M borate buffer (pH 6.5). The flow-rate was 10 ml/h; fraction volume 1 ml (see Fig. 2C).
318
Chromatography of CPY on DEAE--Spheron A solution (4 ml) of CPY (3 mg/ml) was applied to a column (35 X 0.6 cm I.D.) of DEAE--Spheron (1.5 mequiv./g) equilibrated with 0.1 M sodium chloride--0.1 M sodium acetate (pH 7.0). A gradient of sodium chloride was used for elution. The pressure was 1 MPa, flow-rate was 130 ml/h and fraction volume was 3.5 ml (see Fig. 3).
Immobilization of CPY on Con A--Spheron by cross-linking with glutaraldehyde The immobilization of CPY was performed according to Hsaio and R o y e r [3], with some modifications. A fraction of CPY from DEAE--Spheron chromatography (10 ml) was applied to a column (6 X 0.8 cm I.D.) o f Con A--Spheron (content of immobilized Con A: 0.4 ~mol/g of dry carrier) equilibrated with 0.4 M sodium chloride--0.1 M sodium acetate (pH 7.0). The carrier with adsorbed CPY was then p u t into a 25-ml flask and after sucking o u t the buffer was suspended in 10 ml of the equilibrating buffer with 0.5% (w/w) glutaraldehyde. The suspension was shaken at laboratory temperature for 45 min, then the carrier was washed with 1 1 of 0.1 M borate buffer (pH 6.5).
Peptidase activity of CPY Peptidase activity was measured according to Hayashi [9] in 0.05 M MES buffer (pH 6.75) with 2 mM Z-Phe-Ala as substrate.
Rate of release of amino acids from ribonuclease by CPY-Con A--Spheron The reaction was carried out at 25°C with a substrate concentration of 0.3 pmol/ml and substrate to enzyme ratio (mol/mol) of 2000:1 in 0.1 M sodium acetate--0.5% SDS buffer (pH 6.0). RESULTS AND DISCUSSION
For several years we have used Spheron--Gly-Gly-p-aminobenzylsuccinic acid for the isolation of CPY from yeast autolysate. Fig. 2A shows the purification of the enzyme under the conditions described by Johansen et al. [8]. Chromatography of yeast autolysate on Spheron--Gly-Gly-p-aminobenzylsuccinic acid has already been used for large-scale preparation of CPY by the Latvian c o m p a n y Biolar. Since the specific activity and homogeneity of the isolated CPY are largely influenced by the quality and conditions of yeast fermentation [ 1 0 ] , we used only the enzyme prepared by Biolar for our study of enzyme stabilization by immobilization, rather than o u r own preparations. To compare the individual specific adsorbents, the yield of active CPY isolated was determined by its rechromatography under identical conditions; the yield of CPY isolated on Spheron--Gly-Gly-p-aminobenzylsuccinic acid was 68%. Fig. 2B shows the chromatography of CPY from Biolar on m e r c u r y Spheron. We [6] described the preparation of hydroxyalkyl methacrylate copolymers containing various amounts of mercury derivatives of p-acetaminophenoxyethyl methacrylates or of methacrylanilide. The derivative best suited for isolation of SH-proteinases was the one containing 15% mercury derivative of methacrylanilide. The yield of the CPY isolated was 25%.
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Fig. 2. (A) Gly-Gly-p-aminobenzylsuccinic acid-Spheron: the column (5 ml) was equilibrated with 0.01 M sodium acetate (pH 5.0). The sample of autolysate (50 ml) was added and then the column was washed with 1 M sodium chloride--0.01 M sodium acetate (1 1), pH 4.3. The elution (arrow) was performed with 0.01 M phosphate buffer (pH 7). (B) Mercury-Spheron: the column was equilibrated with 0.1 M sodium acetate (pH 6). After the application of sample, elution (arrow) was performed with 0.05 M mercaptoethanol--0.1 M sodium acetate (pH 6.0). (C) Con A--Spheron: the column (10 ml) was washed with 0.1 M sodium acetate and 1 mM manganese chloride-calcium chloride-magnesium chloride and equilibrated with 0.4 M sodium chloride- 0.1 M sodium acetate (pH (pH 7). After the application of sample, elution (arrow) was performed with 0.1 M borate buffer (pH 6.5). A n o t h e r b i o s p e c f f i c a d s o r b e n t f o r CPY is i m m o b i l i z e d C o n A. Fig. 2C s h o w s t h e c h r o m a t o g r a m of CPY on a c o l u m n o f Con A - S p h e r o n . This specific a d s o r b e n t was p r e p a r e d b y the m o d i f i c a t i o n o f S p h e r o n with glucose [ 1 1 ] , to w h i c h C o n A was a t t a c h e d a f t e r p e r i o d a t e a c t i v a t i o n [ 1 2 ] . CPY was d e s o r b e d b y b o r a t e b u f f e r ; t h e y i e l d o f CPY isolated was 89%. T h e results o f b i o a f f i n i t y c h r o m a t o g r a p h y e x p e r i m e n t s s h o w t h a t t h e best yields o f active CPY w e r e o b t a i n e d with C o n A - - S p h e r o n . The h o m o g e n e i t y o f all t h e e n z y m e s isolated was assayed b y S D S - p o l y a c r y l a m i d e gel e l e c t r o phoresis ( P A G E ) (10% a c r y l a m i d e gel, p H 7.0). N o n e o f the e n z y m e s were completely pure. Fig. 3 s h o w s t h e c h r o m a t o g r a p h y p r o c e d u r e t h a t y i e l d e d t h e p u r e s t e n z y m e , n a m e l y i o n e x c h a n g e c h r o m a t o g r a p h y o f CPY o n D E A E - S p h e r o n . A g r a d i e n t o f s o d i u m c h l o r i d e was used f o r the e l u t i o n a n d t h e yield o f t h e e n z y m e i s o l a t e d was 72%. T h e e n z y m e was h o m o g e n e o u s w h e n a s s a y e d b y S D S - P A G E ; t h e specific a c t i v i t y was 100 U / m g . T h e f r a c t i o n o f C P Y isolated b y c h r o m a t o g r a p h y o n D E A E - - S p h e r o n was u s e d f o r irreversible i m m o b i l i z a t i o n o n Con A - - S p h e r o n a c c o r d i n g t o H s i a o and R o y e r [ 3 ] . CPY a f t e r a d s o r p t i o n o n Con A - - S p h e r o n was cross-linked w i t h C o n A b y g l u t a r a l d e h y d e . T h e a m o u n t o f i m m o b i l i z e d CPY d e t e r m i n e d by a m i n o acid analysis was 1.6 m g / g o f d r y s u p p o r t ; t h e p e p t i d a s e a c t i v i t y was 130 U / g of d r y derivative.
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Fig. 3. P u r i f i c a t i o n o f CPY o n D E A E - - S p h e r o n . T h e c o l u m n ( 1 0 m l ) was e q u i l i b r a t e d w i t h 0.1 M s o d i u m a c e t a t e ( p H 7.0). T h e flow-rate was 1 3 0 m l / h ; f r a c t i o n v o l u m e 3.5 ml; pressure 1 MPa. A g r a d i e n t o f s o d i u m c h l o r i d e was used f o r t h e e l u t i o n . I00-
>-- 5O
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Fig. 4. T h e p H rate p r o f i l e s o f p e p t i d a s e activities o f i m m o b i l i z e d ( e ) a n d n a t i v e (o) CPY. T h e a c t i v i t y was m a s u r e d in 50 m M MES buffer.
321 T h e pH profile o f peptidase activity of native CPY and o f CPY a t t a c h e d to Con A - - S p h e r o n is s h o w n in Fig. 4. T h e pH o p t i m u m o f the b o u n d e n z y m e is shifted t o the acid side w i t h respect t o t h e o p t i m u m o f t h e native e n z y m e in the peptidase assay. F o r d e t e r m i n a t i o n o f the C-terminal s e q u e n c e of ribonuclease b y CPY a t t a c h e d to Con A - - S e p h a r o s e , an u l t r a f i l t r a t i o n cell was f o u n d very useful. Practically the same results as described b y Hsiao and R o y e r [3] were achieved. T h e rates at which a m i n o acids were released are in a g r e e m e n t with the k n o w n a m i n o acid s e q u e n c e o f t h e C-terminal p a r t o f t h e ribonuclease chain [ 1 3 ] . CONCLUSIONS
B i o a f f i n i t y c h r o m a t o g r a p h y can be used for e l a b o r a t i o n o f the c o n d i t i o n s f o r biospecific s o r p t i o n o f e n z y m e s t h a t can be used for their o r i e n t e d i m m o b i l i z a t i o n . T h e f o r m a t i o n o f biospecific c o m p l e x e s with d i f f e r e n t parts of the e n z y m e surface c o u l d be v e r y useful f o r the p r e p a r a t i o n o f i m m o b i l i z e d e n z y m e s f o r use as active catalysts. REFERENCES 1 M.Z. Atassi, Eur. J. Biochem., 145 (1984) 1. 2 H. Schott, Affinity Chromatography, Template Chromatography of Nucleic Acids and Proteins, Marcel Dekker, New York, 1984, pp. 1- 234. 3 H. Hsiao and G.P. Royer, Arch. Biochem. Biophys., 198 (1979) 379. 4 B. Solomon, R. Koppel and E. Katchalski-Katzir, Biotechnology, 2 (1984) 709. 5 K. Ikura, K. Okumura, M. Yoshikawa, R. Sasaki and H. Chiba, J. Appl. Biochem., 6 (1984) 222. 6 J. Turkov~, S. Vavreinov~,M. Kriv{kov~and J. Coupek, Biochim. Biophys. Acta, 386 (1975) 503. 7 J. Turkov~, K. Bl~ha, J. Hor~Sek, J. Vaj~ner, A. FrydryShov~ and J. Coupek, J. Chromatogr., 215 (1981) 165. 8 J.T. Johansen, K. Breddam and M. Ottesen, Carlsberg Res. Commun., 41 (1976) 1. 9 R. Hayashi, Methods Enzymol., 47 (1977) 86. 10 J. Turkov~i, Third European Congress on Biotechnology, Vol. 1, Verlag Chemic, Weinheim, 1984,~. 761. 11 K. Filka, J. Coupek and J. Kocourek, Biochim. Biophys. Acta, 539 (1978) 518. 12 D. VanSurov~i, J. Turkov~, J. ~oupek and A. Frydrychov~, Collect. Czech. Chem. Commun., 44 (1979) 3405. 13 D.G. Smyth, W.H. Stein and S. Moore, J. Biol. Chem., 238 (1963) 227.
Journal of Chromatography, 376 (1986) 315--321 Biomedical Applications Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
CHROMBIO. 2976
REVERSIBLE AND IRREVERSIBLE IMMOBILIZATION OF CARBOXYPEPTIDASE Y USING BIOSPECIFIC ADSORPTION
J. TURKOV~* and M. FUSEK
Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6 (Czechoslovakia) and J.J. MAKSIMOV and Yu. B. ALAKHOV
Institute of Protein Research, U.S.S.R. Academy of Sciences, Pushchino, Moscow Reg. (U.S.S.R.)
SUMMARY To find optimal biospecific hydroxyalkyl methacrylate (Spheron) derivatives for reversible and irreversible immobilization of carboxypeptidase Y, we elaborated the procedure for the biospecific sorption of carboxypeptidase Y to Spheron--Gly-Gly-p-aminobenzylsuecinic acid, mercury--Spheron and concanavalin A--Spheron. The highest yield was achieved in the case of concanavalin A--Spheron, which was also used for the irreversible, oriented immobilization by cross-linking of carboxypeptidase Y and concanavalin A with glutaraldehyde.
INTRODUCTION
The combination of many complementary binding sites on the surface of enzymes makes possible the formation of a number of biospecific complexes that can be used not only for efficient isolation but also for oriented immobilization. Fig. 1 shows the surface of an enzyme covered with several complementary binding sites. For example, this could be carboxypeptidase Y (CPY) containing a complementary binding site for the specific inhibitor Gly-Gly-paminobenzylsuccinic acid and one free SH group, which is placed in the active site of the enzyme. CPY also contains a carbohydrate moiety, which is a complementary binding site for the active site of lectin concanavalin A (Con A). The antigenic sites of the protein can be identified by determination of the 0378-4347/86/$03.50
© 1986 Elsevier Science Publishers B.V.
316
SUBSTRATE
OR INHIBITOR
ANTi--
BODY
~NZ~I'4E
Fig. 1. Possibilities of the enzyme to form biospecific complexes.
antigenic structures of the peptide chain after its interaction with specific antibodies according to Attasi [1] who described, for example, five precise antigenic sites in the molecule of myoglobin. The following may serve as another example: about 30% of the approximately 2000 enzymes found in the cell contain nucleotide coenzymes participating in the reaction with nucleotides. The specific recognition of certain nucleotide sequences by proteins is one of the reasons for studying peptide--nucleotide interactions [2]. It is generally known that many enzymes in the form of biospecific complexes exhibit higher stability. Well oriented attachment of enzymes to solid supports can result in a higher relative activity of the immobilized enzyme. Hsiao and Royer [3] compared several ways of attaching CPY to agarose and glass. Immobilized CPY with the highest activity was prepared by adsorption of the enzyme to Con A--Sepharose and subsequent cross-linkage by glutaraldehyde. Hsiao and Royer [3] also proved that the carbohydrate moiety can be used for oriented immobilization of enzymes, and oriented immobilization guarantees high activity of immobilized enzymes. Practically the same kinetic constants of peptidase and esterase activities of native and immobilized carboxypeptidase A were determined by Solomon et al. [4] when the enzyme was attached to the immobilized monoclonal antibody. Monoclonal antibodies were also used for specific immobilization of guinea pig liver transglutaminase by Ikura et al. [5]. The aim of the work reported here was to find optimal biospecific hydroxy-
317 alkyl methacrylate derivatives for reversible and irreversible immobilization of CPY. The first step was purification of CPY by biospecific sorption via different complementary binding sites. The results of these purification experiments served for the determination of the optimal hydroxyalkyl methacrylate derivative for immobilization. Con A--Spheron was used for oriented immobilization of CPY. EXPERIMENTAL CPY (EC 3.4.16.1) was obtained from Biolar (Olain, U.S.S.R.). N-Carboxybenzoxy-L-phenylalanyl-L-alanine (Z-Phe-Ala) was a product of Sigma (St. Louis, MO, U.S.A.), ribonuclease (EC 3.1.26.1)and 2-(N-morpholino)ethanesulphonic acid (MES) were from Reanal (Budapest, Hungary) and 2-mercaptoethanol and glutaraldehyde were from Fluka (Buchs, Switzerland). Sodium dodecyl sulphate (SDS) was from Serva (Heidelberg, F.R.G.). Spheron 1000 (100--200/~m), DEAE--Spheron 1000 (25--40 pm) and the remaining chemicals of analytical purity were supplied by Lachema (Brno, Czechoslovakia). The mercury derivative of the hydroxyalkyl methacrylate gel (mercury--Spheron) was prepared according to Turkov~ et al. [6] ; Gly-Gly-p-aminobenzylsuccinic acid--Spheron was prepared according to Turkov~ et al. [7]. Con A--Spheron was kindly donated by Dr. K. Filka (Department of Biochemistry, Charles University, Prague, Czechoslovakia). Purification o f CPY from a yeast autolysate on Spheron-Gly-Gly-p-aminobe nz y lsuccinic acid The autolysate from yeast Saccharomyces cerevisiae (50 ml) prepared according to Johansen et al. [8] was applied to a column (9 × 0.8 cm I.D.) of Spheron--Gly-Gly-p-aminobenzylsuccinic acid (content of immobilized inhibitor: 2.6 tzmol/g of dry carrier) equilibrated with 0.01 M sodium acetate (pH 5.0). The column was washed with 1 1 of 1 M sodium chloride-0.01 M sodium acetate (pH 4.3). CPY was desorbed with 0.01 M phosphate buffer (pH 7.0). The flow-rate was 50 ml/h; fraction volume 4.0 ml (see Fig. 2A). Chromatography o f CPY on mercury- Spheron A solution (10 ml) of CPY (0.1 mg/ml) was applied to a column (8 X 0.8 cm I.D.) of mercury--Spheron (prepared with 15% mercury derivative of methacrylanilide, 93 t~mol Hg per ml gel) equilibrated with 0.1 M sodium acetate (pH 6.0). The column was washed with 15 ml of starting buffer and CPY was desorbed with 0.05 M 2-mercaptoethanol--0.1 M sodium acetate (pH 6.0). The flow-rate was 15 ml/h; fraction volume 1 ml (see Fig. 2B). Chromatography o f CPY on Con A--Spheron A solution (200 ml) of CPY (0.1 mg/ml) was applied to a column (18 X 0.8 cm I.D.) of Con A--Spheron (content of immobilized Con A: 0.4 pmol/g of dry carrier) equilibrated with 0.4 M sodium chloride--0.1 M sodium acetate (pH 7). The column was washed with 20 ml of starting buffer and CPY was eluted with 0.1 M borate buffer (pH 6.5). The flow-rate was 10 ml/h; fraction volume 1 ml (see Fig. 2C).
318
Chromatography of CPY on DEAE--Spheron A solution (4 ml) of CPY (3 mg/ml) was applied to a column (35 X 0.6 cm I.D.) of DEAE--Spheron (1.5 mequiv./g) equilibrated with 0.1 M sodium chloride--0.1 M sodium acetate (pH 7.0). A gradient of sodium chloride was used for elution. The pressure was 1 MPa, flow-rate was 130 ml/h and fraction volume was 3.5 ml (see Fig. 3).
Immobilization of CPY on Con A--Spheron by cross-linking with glutaraldehyde The immobilization of CPY was performed according to Hsaio and R o y e r [3], with some modifications. A fraction of CPY from DEAE--Spheron chromatography (10 ml) was applied to a column (6 X 0.8 cm I.D.) o f Con A--Spheron (content of immobilized Con A: 0.4 ~mol/g of dry carrier) equilibrated with 0.4 M sodium chloride--0.1 M sodium acetate (pH 7.0). The carrier with adsorbed CPY was then p u t into a 25-ml flask and after sucking o u t the buffer was suspended in 10 ml of the equilibrating buffer with 0.5% (w/w) glutaraldehyde. The suspension was shaken at laboratory temperature for 45 min, then the carrier was washed with 1 1 of 0.1 M borate buffer (pH 6.5).
Peptidase activity of CPY Peptidase activity was measured according to Hayashi [9] in 0.05 M MES buffer (pH 6.75) with 2 mM Z-Phe-Ala as substrate.
Rate of release of amino acids from ribonuclease by CPY-Con A--Spheron The reaction was carried out at 25°C with a substrate concentration of 0.3 pmol/ml and substrate to enzyme ratio (mol/mol) of 2000:1 in 0.1 M sodium acetate--0.5% SDS buffer (pH 6.0). RESULTS AND DISCUSSION
For several years we have used Spheron--Gly-Gly-p-aminobenzylsuccinic acid for the isolation of CPY from yeast autolysate. Fig. 2A shows the purification of the enzyme under the conditions described by Johansen et al. [8]. Chromatography of yeast autolysate on Spheron--Gly-Gly-p-aminobenzylsuccinic acid has already been used for large-scale preparation of CPY by the Latvian c o m p a n y Biolar. Since the specific activity and homogeneity of the isolated CPY are largely influenced by the quality and conditions of yeast fermentation [ 1 0 ] , we used only the enzyme prepared by Biolar for our study of enzyme stabilization by immobilization, rather than o u r own preparations. To compare the individual specific adsorbents, the yield of active CPY isolated was determined by its rechromatography under identical conditions; the yield of CPY isolated on Spheron--Gly-Gly-p-aminobenzylsuccinic acid was 68%. Fig. 2B shows the chromatography of CPY from Biolar on m e r c u r y Spheron. We [6] described the preparation of hydroxyalkyl methacrylate copolymers containing various amounts of mercury derivatives of p-acetaminophenoxyethyl methacrylates or of methacrylanilide. The derivative best suited for isolation of SH-proteinases was the one containing 15% mercury derivative of methacrylanilide. The yield of the CPY isolated was 25%.
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Fig. 2. (A) Gly-Gly-p-aminobenzylsuccinic acid-Spheron: the column (5 ml) was equilibrated with 0.01 M sodium acetate (pH 5.0). The sample of autolysate (50 ml) was added and then the column was washed with 1 M sodium chloride--0.01 M sodium acetate (1 1), pH 4.3. The elution (arrow) was performed with 0.01 M phosphate buffer (pH 7). (B) Mercury-Spheron: the column was equilibrated with 0.1 M sodium acetate (pH 6). After the application of sample, elution (arrow) was performed with 0.05 M mercaptoethanol--0.1 M sodium acetate (pH 6.0). (C) Con A--Spheron: the column (10 ml) was washed with 0.1 M sodium acetate and 1 mM manganese chloride-calcium chloride-magnesium chloride and equilibrated with 0.4 M sodium chloride- 0.1 M sodium acetate (pH (pH 7). After the application of sample, elution (arrow) was performed with 0.1 M borate buffer (pH 6.5). A n o t h e r b i o s p e c f f i c a d s o r b e n t f o r CPY is i m m o b i l i z e d C o n A. Fig. 2C s h o w s t h e c h r o m a t o g r a m of CPY on a c o l u m n o f Con A - S p h e r o n . This specific a d s o r b e n t was p r e p a r e d b y the m o d i f i c a t i o n o f S p h e r o n with glucose [ 1 1 ] , to w h i c h C o n A was a t t a c h e d a f t e r p e r i o d a t e a c t i v a t i o n [ 1 2 ] . CPY was d e s o r b e d b y b o r a t e b u f f e r ; t h e y i e l d o f CPY isolated was 89%. T h e results o f b i o a f f i n i t y c h r o m a t o g r a p h y e x p e r i m e n t s s h o w t h a t t h e best yields o f active CPY w e r e o b t a i n e d with C o n A - - S p h e r o n . The h o m o g e n e i t y o f all t h e e n z y m e s isolated was assayed b y S D S - p o l y a c r y l a m i d e gel e l e c t r o phoresis ( P A G E ) (10% a c r y l a m i d e gel, p H 7.0). N o n e o f the e n z y m e s were completely pure. Fig. 3 s h o w s t h e c h r o m a t o g r a p h y p r o c e d u r e t h a t y i e l d e d t h e p u r e s t e n z y m e , n a m e l y i o n e x c h a n g e c h r o m a t o g r a p h y o f CPY o n D E A E - S p h e r o n . A g r a d i e n t o f s o d i u m c h l o r i d e was used f o r the e l u t i o n a n d t h e yield o f t h e e n z y m e i s o l a t e d was 72%. T h e e n z y m e was h o m o g e n e o u s w h e n a s s a y e d b y S D S - P A G E ; t h e specific a c t i v i t y was 100 U / m g . T h e f r a c t i o n o f C P Y isolated b y c h r o m a t o g r a p h y o n D E A E - - S p h e r o n was u s e d f o r irreversible i m m o b i l i z a t i o n o n Con A - - S p h e r o n a c c o r d i n g t o H s i a o and R o y e r [ 3 ] . CPY a f t e r a d s o r p t i o n o n Con A - - S p h e r o n was cross-linked w i t h C o n A b y g l u t a r a l d e h y d e . T h e a m o u n t o f i m m o b i l i z e d CPY d e t e r m i n e d by a m i n o acid analysis was 1.6 m g / g o f d r y s u p p o r t ; t h e p e p t i d a s e a c t i v i t y was 130 U / g of d r y derivative.
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Fig. 3. P u r i f i c a t i o n o f CPY o n D E A E - - S p h e r o n . T h e c o l u m n ( 1 0 m l ) was e q u i l i b r a t e d w i t h 0.1 M s o d i u m a c e t a t e ( p H 7.0). T h e flow-rate was 1 3 0 m l / h ; f r a c t i o n v o l u m e 3.5 ml; pressure 1 MPa. A g r a d i e n t o f s o d i u m c h l o r i d e was used f o r t h e e l u t i o n . I00-
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Fig. 4. T h e p H rate p r o f i l e s o f p e p t i d a s e activities o f i m m o b i l i z e d ( e ) a n d n a t i v e (o) CPY. T h e a c t i v i t y was m a s u r e d in 50 m M MES buffer.
321 T h e pH profile o f peptidase activity of native CPY and o f CPY a t t a c h e d to Con A - - S p h e r o n is s h o w n in Fig. 4. T h e pH o p t i m u m o f the b o u n d e n z y m e is shifted t o the acid side w i t h respect t o t h e o p t i m u m o f t h e native e n z y m e in the peptidase assay. F o r d e t e r m i n a t i o n o f the C-terminal s e q u e n c e of ribonuclease b y CPY a t t a c h e d to Con A - - S e p h a r o s e , an u l t r a f i l t r a t i o n cell was f o u n d very useful. Practically the same results as described b y Hsiao and R o y e r [3] were achieved. T h e rates at which a m i n o acids were released are in a g r e e m e n t with the k n o w n a m i n o acid s e q u e n c e o f t h e C-terminal p a r t o f t h e ribonuclease chain [ 1 3 ] . CONCLUSIONS
B i o a f f i n i t y c h r o m a t o g r a p h y can be used for e l a b o r a t i o n o f the c o n d i t i o n s f o r biospecific s o r p t i o n o f e n z y m e s t h a t can be used for their o r i e n t e d i m m o b i l i z a t i o n . T h e f o r m a t i o n o f biospecific c o m p l e x e s with d i f f e r e n t parts of the e n z y m e surface c o u l d be v e r y useful f o r the p r e p a r a t i o n o f i m m o b i l i z e d e n z y m e s f o r use as active catalysts. REFERENCES 1 M.Z. Atassi, Eur. J. Biochem., 145 (1984) 1. 2 H. Schott, Affinity Chromatography, Template Chromatography of Nucleic Acids and Proteins, Marcel Dekker, New York, 1984, pp. 1- 234. 3 H. Hsiao and G.P. Royer, Arch. Biochem. Biophys., 198 (1979) 379. 4 B. Solomon, R. Koppel and E. Katchalski-Katzir, Biotechnology, 2 (1984) 709. 5 K. Ikura, K. Okumura, M. Yoshikawa, R. Sasaki and H. Chiba, J. Appl. Biochem., 6 (1984) 222. 6 J. Turkov~, S. Vavreinov~,M. Kriv{kov~and J. Coupek, Biochim. Biophys. Acta, 386 (1975) 503. 7 J. Turkov~, K. Bl~ha, J. Hor~Sek, J. Vaj~ner, A. FrydryShov~ and J. Coupek, J. Chromatogr., 215 (1981) 165. 8 J.T. Johansen, K. Breddam and M. Ottesen, Carlsberg Res. Commun., 41 (1976) 1. 9 R. Hayashi, Methods Enzymol., 47 (1977) 86. 10 J. Turkov~i, Third European Congress on Biotechnology, Vol. 1, Verlag Chemic, Weinheim, 1984,~. 761. 11 K. Filka, J. Coupek and J. Kocourek, Biochim. Biophys. Acta, 539 (1978) 518. 12 D. VanSurov~i, J. Turkov~, J. ~oupek and A. Frydrychov~, Collect. Czech. Chem. Commun., 44 (1979) 3405. 13 D.G. Smyth, W.H. Stein and S. Moore, J. Biol. Chem., 238 (1963) 227.