Enzyme Immobilization on Heparin V. P. TORCHILIN, E. V. IL’INA, Z. A. STRELTSOVA, V. N. SMIRNOV, and E. I. CHAZOV, National Research Center of Cardiology, AMS USSR, Moscow, U S S R
Summary We describe the preparation and some of the properties of heparin-bound a-chymotrypsin that were obtained via activation of heparin with water-solublecarbodiimide. Immobilized enzyme has unchanged kinetic characteristics toward low-molecularweight and macromolecular substrates. The heparin-bound enzymes could have a wide range of medical applications.
INTRODUCTION Water-soluble and water-insoluble immobilized enzymes for different applications have been prepared utilizing mainly polysaccharides, synthetic polymers, and copolymers as carriers.14 A l b ~ m i n , ~ collagen: and synthetic polypeptides7have also been used for enzyme immobilization. Regardless of the existence of a great number of different carriers for immobilization, only a small part of them can be used for biomedical applications. In the latter case, the carriers used should meet definite requirements: they should be chemically inert, have no antigenic activity, and be easily degradable in the living organism. These requirements narrow the range of carriers that are of potential use in medical enzyme immobilization. The use of substances which are typical for a living organism (fibrins) to prepare a conjugate with an active enzyme is of great interest but has received little attention. It is even more interesting when a carrier by itself has useful biological activity and can intensify the expected action of the bound enzyme. The use of heparin as the carrier for binding of thrombolytic enzymes should have definite advantages because heparinization is often used during the thrombolytic enzyme therapy. Journal of Biomedical Materials, Research, Vol. 12,585-590 (1978) 0021-9304/78/0012-0585$01.00 01978 John Wiley & Sons, Inc.
586
TORCHILIN ET kL.
This report is a summary of our study of covalent compounds between heparin and a model enzyme, a-chymotrypsin.
MATERIALS A N D METHODS Crystalline bovine a-chymotrypsin (E. C. 3.4.4.5) was the product of the Olaine chemical reagents plant (USSR) and had a specific activity of 60%, determined by spectrophotometric titration with p nitrophenyl trimethylacetate according to Ref. 9. Heparin and specific substrate for a-chymotrypsin, N-acetyl-L-tyrosine ethyl ester (ATEE), were obtained from Koch-Light (England), 1-ethyl-3(3dimethylaminopropy1)-carbodiimide (EDC) was bought from Sigma (USA). Sephadex G-75 was the product of Pharmacia (Sweden). All other reagents used were provided by Reachim (USSR). Binding of a-chymotrypsin with heparin was made using coupling agent EDC. For this purpose, 25 mg of heparin were dissolved in 10 ml0.05M phosphate buffer, pH = 7.0. Then, 25 mg of EDC were added to solution, followed by the addition of 20 mg a-chymotrypsin. The reaction was carried out a t 4OC for 10 hr. The separation of bound and native a-chymotrypsin was performed by a gel chromatography Sephadex G-75 column (50 X 2.5 cm). Elution was done with phosphate buffer, pH = 8.25, at the rate of 1ml/min. High ionic strength was used (0.025M phosphate buffer in 1M NaC1) in order to destroy electrostatic complex between the enzyme and heparin in which the enzyme can be bound noncovalently.lOJ1 Gel filtration was monitored spectrophotometrically by measuring the eluant absorbance at 280 nm. The measurements were made on Turner uv spectrophotometer (USA). The kinetic studies of immobilized and native a-chymotrypsin were carried out on pH-state TTT-Ic (Radiometer, Denmark) using the specific substrate ATEE at pH 7.5 and a cell temperature of 25"C, and starting at a concentration of ATEE of 5 X low3M. Temperature inactivation of native and immobilized enzymes was studied at Eo = 0.3 X M, at 37"C, in phosphate buffer, pH 8.2, by periodic measurements of the primary rate of substrate hydrolysis under the conditions described. The inhibition of immobilized a-chymotrypsin by soybean trypsin pH = 7, at 25"C, using protein inhibitor was studied at [El0 = ATEE as a substrate a t concentration M in 0.1 M KC1. Mea-
ENZYME IMMOBILIZATION ON HEPARIN
587
surements were made by pH-state technique. Ki was calculated in Linweaver-Berk coordinates.
RESULTS AND DISCUSSION The structure of heparin molecule is shown below:
-oi+-r9!
The presence of free carboxilic groups in heparin molecule makes it possible to bind it with the c-amino groups of the enzyme via activation by EDC according to the following scheme:
0
0
I
Hep-C-
,NHR,
-c\\
NR2
+ E-NH2
I
-Hep-C-NH-E
+ NHR,--C-NHR, II
0
The polyelectrolytic nature of heparin gives the opportunity of binding large quantities of the enzyme due to a prereaction electrostatic complex formation between oppositely charged enzyme and carrier under certain reaction conditi0ns.~~-13 The results of gel-filtration chromatography of the reaction mixture shown on Fig. 1 demonstrate that part of the enzyme binds with heparin: after treating the enzyme with EDC in the presence of heparin, we obtained two fractions with noticeable enzymatic activity. One of them has an elution volume characteristic of the native enzyme; and the other, a much lower elution volume. The comparison of results obtained at different ionic strengths shows that increasing the ionic strength (upto 1M NaC1) increases the free enzyme portion, probably due to destruction of the electrostatic complex between the enzyme and heparin. Spectrophotometric study of the fraction with
TORCHILIN ET AL.
588
50
100
I50
200
volume , ml Fig. 1. Gel chromatography of native chymotrypsin (1) and heparin-bound achymotrypsin (2,3) on Sephadex G-75 column. curve 2: ionic strength of eluant, 0.025 M curve 3: ionic strength of eluant, 1 M.
low elution volume shows that this fraction contains about 100 mg of a-chymotrypsin per 1g of heparin. The enzymatic activity of this fraction is about 90% from calculated. The fact that this elution peak contains covalently bound heparin-enzyme conjugate is proved by the same elution volume of noncovalent electrostatic a-chymotrypsin-heparin complex, which was described in Refs. 10 and 11. Besides the sulphur content in lyophilized preparation from earlier elution peak depending on reaction conditions was up to 9-11% (sometimes less) and corresponded enzyme-heparin ratios of 1:15 to 1:4. We have studied the catalytic properties of heparin-bound a chymotrypsin following hydrolysis of specific substrate ATEE. These data are given in Table I. It is seen that no significant changes in enzyme properties were found after modification. K , values are practically identical for native and heparin-bound a-chymotrypsin (the slight increase in K , value for heparin-bound enzyme may be a result of steric hindrances). The decrease in Kcatfor heparin-bound enzyme is explained by local decrease in p H of the medium in the
ENZYME IMMOBILIZATION ON HEPARIN
589
TABLE I The Parameters of Catalytic ATEE Hydrolysis
KU
Kcat
(X103 M )
Catalyst Native cu-chymotrypsin Heparin-bound a-chymotrypsin
1.00 1.30
(sec-l) 160 80
pHopt 8.0 9.0
vicinity of active center of the enzyme due to high concentration of heparin carboxylic groups. The shift of pH optimum to the basic pH range has the same explanation-the action of the electrostatic field of the carrier molecule. It was very interesting to observe the ability of heparin-bound enzyme to interact with macromolecular substrates. This property is extremely important for the system under investigation because macromolecular fibrin is the native substrate for thrombolytic enzymes. To answer this question, we have also studied the inhibition of immobilized enzyme by trypsin protein inhibitor from soybean as a model reaction. It was found that it is possible to observe 100% inhibition at inhibitor concentrations 2-2.5-fold greater than that of native enzyme. At the same time, the inhibition constant, Ki, remains practically unchanged for native and bound enzyme, that is, about
ZI
.-z0 4-
0
0
2
4
6
24
time ,hours Fig. 2. Thermal inactivation of heparin-bound or-chymotrypsin (1) and native or-chymotrypsin (2) under similar conditions.
590
TORCHILIN E T AL.
MA. It is believed that the immobilization of enzyme by the 2X method described does not change its reactivity with low-molecular-weight and macromolecular substrates. The study of thermal inactivation of heparin-bound a-chymotrypsin demonstrated the differences in the rates of enzyme inactivation for native and bound a-chymotrypsin. In similar conditions (Fig. 2), native enzyme completely loses its activity in 6 hr. At the same time, heparin-bound a-chymotrypsin preserves up to 80%of its activity for 24 hr. This fact may be explained by a significant decrease in the rate of autolysis due to steric hindrances in the case of the bound enzyme. This mechanism is very important in preserving the biological activity of bound enzymes in the living organism, where exogenous enzymes lose their activity in a short time due to the action of endogeneous proteases. In conclusion, it is suggested that heparin can be effectively used as a carrier for enzyme immobilization. It is possible to obtain a heparin-enzyme preparation in which the enzyme has an unchanged biological activity. References 1. R. Goldman, L. Goldstein, and E. Katchalsky, in G. R. Stark, Ed., Biochemical Aspects of Reactions on Solid Supports, Academic, New York, 1971. 2. G. J. H. Melrose, Reu. Pure Appl. Chem., 21,83 (1971). 3. R. Axen, J. Porath, and S. Ernback, Nature, 214,1302 (1967). 4. E. K. Bauman, L. H. Goodson, G. G. Guilbault, and D. N. Kramer, Anal. Chem., 37,1378 (1965). 5. S. Avrameas and T. Ternynck, Immunochemistry, 6,53 (1969). 6. S. Suzuki, I. Karuhe, and I. Watanabe, Int. Farm. Symp. Kyoto, February 3-5, 1970, p. 70. 7. S. Avrameas and T. Ternynck, J. Biol. Chem., 242,1651 (1967). 8. J. G. Dillon, M. R. Begue-Canton, R. L. Bleakeley, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. Kazdy, J. V. Killheffer, T. H. Marshall, C. G. Miller, R. W. Roeske, and J. R. Stoops, J. Am. Chem. Soc., 88,5890 (1964). 10. 2. A. Streltsova, E. E. Braudo, and V. B. Tolstoguzov, Bioorgan. Khim., 1, 267 (1975). 11. 2. A. Streltsova, V. K. Shwedas, A. V. Maksimenko, A. A. Kliosov, E. E. Braudo, V. B. Tolstoguzov, and I. V. Berezin, Bioorgan. Khim., 1,1469 (1975). 12. V. P. Torchilin, V. S. Goldmacher, A. M. Klibanov, K. Martinek, E. G . Tischenko, V. N. Smirnov, 1. V. Berezin, and E. I. Chazov, USSR-USA Symposium on Chemistry and Physics of Proteins, Riga, 1976, p. 116 (abstract). 13. V. P. Torchilin, I. L. Reiser, E. G. Tischenko, E. V. Il’ina, V. N. Smirnov, and E. I. Chazov, Bioorgan. Khim., 2,1687 (1976).
Received April 4,1977 Revised July 20,1977
Summary We describe the preparation and some of the properties of heparin-bound a-chymotrypsin that were obtained via activation of heparin with water-solublecarbodiimide. Immobilized enzyme has unchanged kinetic characteristics toward low-molecularweight and macromolecular substrates. The heparin-bound enzymes could have a wide range of medical applications.
INTRODUCTION Water-soluble and water-insoluble immobilized enzymes for different applications have been prepared utilizing mainly polysaccharides, synthetic polymers, and copolymers as carriers.14 A l b ~ m i n , ~ collagen: and synthetic polypeptides7have also been used for enzyme immobilization. Regardless of the existence of a great number of different carriers for immobilization, only a small part of them can be used for biomedical applications. In the latter case, the carriers used should meet definite requirements: they should be chemically inert, have no antigenic activity, and be easily degradable in the living organism. These requirements narrow the range of carriers that are of potential use in medical enzyme immobilization. The use of substances which are typical for a living organism (fibrins) to prepare a conjugate with an active enzyme is of great interest but has received little attention. It is even more interesting when a carrier by itself has useful biological activity and can intensify the expected action of the bound enzyme. The use of heparin as the carrier for binding of thrombolytic enzymes should have definite advantages because heparinization is often used during the thrombolytic enzyme therapy. Journal of Biomedical Materials, Research, Vol. 12,585-590 (1978) 0021-9304/78/0012-0585$01.00 01978 John Wiley & Sons, Inc.
586
TORCHILIN ET kL.
This report is a summary of our study of covalent compounds between heparin and a model enzyme, a-chymotrypsin.
MATERIALS A N D METHODS Crystalline bovine a-chymotrypsin (E. C. 3.4.4.5) was the product of the Olaine chemical reagents plant (USSR) and had a specific activity of 60%, determined by spectrophotometric titration with p nitrophenyl trimethylacetate according to Ref. 9. Heparin and specific substrate for a-chymotrypsin, N-acetyl-L-tyrosine ethyl ester (ATEE), were obtained from Koch-Light (England), 1-ethyl-3(3dimethylaminopropy1)-carbodiimide (EDC) was bought from Sigma (USA). Sephadex G-75 was the product of Pharmacia (Sweden). All other reagents used were provided by Reachim (USSR). Binding of a-chymotrypsin with heparin was made using coupling agent EDC. For this purpose, 25 mg of heparin were dissolved in 10 ml0.05M phosphate buffer, pH = 7.0. Then, 25 mg of EDC were added to solution, followed by the addition of 20 mg a-chymotrypsin. The reaction was carried out a t 4OC for 10 hr. The separation of bound and native a-chymotrypsin was performed by a gel chromatography Sephadex G-75 column (50 X 2.5 cm). Elution was done with phosphate buffer, pH = 8.25, at the rate of 1ml/min. High ionic strength was used (0.025M phosphate buffer in 1M NaC1) in order to destroy electrostatic complex between the enzyme and heparin in which the enzyme can be bound noncovalently.lOJ1 Gel filtration was monitored spectrophotometrically by measuring the eluant absorbance at 280 nm. The measurements were made on Turner uv spectrophotometer (USA). The kinetic studies of immobilized and native a-chymotrypsin were carried out on pH-state TTT-Ic (Radiometer, Denmark) using the specific substrate ATEE at pH 7.5 and a cell temperature of 25"C, and starting at a concentration of ATEE of 5 X low3M. Temperature inactivation of native and immobilized enzymes was studied at Eo = 0.3 X M, at 37"C, in phosphate buffer, pH 8.2, by periodic measurements of the primary rate of substrate hydrolysis under the conditions described. The inhibition of immobilized a-chymotrypsin by soybean trypsin pH = 7, at 25"C, using protein inhibitor was studied at [El0 = ATEE as a substrate a t concentration M in 0.1 M KC1. Mea-
ENZYME IMMOBILIZATION ON HEPARIN
587
surements were made by pH-state technique. Ki was calculated in Linweaver-Berk coordinates.
RESULTS AND DISCUSSION The structure of heparin molecule is shown below:
-oi+-r9!
The presence of free carboxilic groups in heparin molecule makes it possible to bind it with the c-amino groups of the enzyme via activation by EDC according to the following scheme:
0
0
I
Hep-C-
,NHR,
-c\\
NR2
+ E-NH2
I
-Hep-C-NH-E
+ NHR,--C-NHR, II
0
The polyelectrolytic nature of heparin gives the opportunity of binding large quantities of the enzyme due to a prereaction electrostatic complex formation between oppositely charged enzyme and carrier under certain reaction conditi0ns.~~-13 The results of gel-filtration chromatography of the reaction mixture shown on Fig. 1 demonstrate that part of the enzyme binds with heparin: after treating the enzyme with EDC in the presence of heparin, we obtained two fractions with noticeable enzymatic activity. One of them has an elution volume characteristic of the native enzyme; and the other, a much lower elution volume. The comparison of results obtained at different ionic strengths shows that increasing the ionic strength (upto 1M NaC1) increases the free enzyme portion, probably due to destruction of the electrostatic complex between the enzyme and heparin. Spectrophotometric study of the fraction with
TORCHILIN ET AL.
588
50
100
I50
200
volume , ml Fig. 1. Gel chromatography of native chymotrypsin (1) and heparin-bound achymotrypsin (2,3) on Sephadex G-75 column. curve 2: ionic strength of eluant, 0.025 M curve 3: ionic strength of eluant, 1 M.
low elution volume shows that this fraction contains about 100 mg of a-chymotrypsin per 1g of heparin. The enzymatic activity of this fraction is about 90% from calculated. The fact that this elution peak contains covalently bound heparin-enzyme conjugate is proved by the same elution volume of noncovalent electrostatic a-chymotrypsin-heparin complex, which was described in Refs. 10 and 11. Besides the sulphur content in lyophilized preparation from earlier elution peak depending on reaction conditions was up to 9-11% (sometimes less) and corresponded enzyme-heparin ratios of 1:15 to 1:4. We have studied the catalytic properties of heparin-bound a chymotrypsin following hydrolysis of specific substrate ATEE. These data are given in Table I. It is seen that no significant changes in enzyme properties were found after modification. K , values are practically identical for native and heparin-bound a-chymotrypsin (the slight increase in K , value for heparin-bound enzyme may be a result of steric hindrances). The decrease in Kcatfor heparin-bound enzyme is explained by local decrease in p H of the medium in the
ENZYME IMMOBILIZATION ON HEPARIN
589
TABLE I The Parameters of Catalytic ATEE Hydrolysis
KU
Kcat
(X103 M )
Catalyst Native cu-chymotrypsin Heparin-bound a-chymotrypsin
1.00 1.30
(sec-l) 160 80
pHopt 8.0 9.0
vicinity of active center of the enzyme due to high concentration of heparin carboxylic groups. The shift of pH optimum to the basic pH range has the same explanation-the action of the electrostatic field of the carrier molecule. It was very interesting to observe the ability of heparin-bound enzyme to interact with macromolecular substrates. This property is extremely important for the system under investigation because macromolecular fibrin is the native substrate for thrombolytic enzymes. To answer this question, we have also studied the inhibition of immobilized enzyme by trypsin protein inhibitor from soybean as a model reaction. It was found that it is possible to observe 100% inhibition at inhibitor concentrations 2-2.5-fold greater than that of native enzyme. At the same time, the inhibition constant, Ki, remains practically unchanged for native and bound enzyme, that is, about
ZI
.-z0 4-
0
0
2
4
6
24
time ,hours Fig. 2. Thermal inactivation of heparin-bound or-chymotrypsin (1) and native or-chymotrypsin (2) under similar conditions.
590
TORCHILIN E T AL.
MA. It is believed that the immobilization of enzyme by the 2X method described does not change its reactivity with low-molecular-weight and macromolecular substrates. The study of thermal inactivation of heparin-bound a-chymotrypsin demonstrated the differences in the rates of enzyme inactivation for native and bound a-chymotrypsin. In similar conditions (Fig. 2), native enzyme completely loses its activity in 6 hr. At the same time, heparin-bound a-chymotrypsin preserves up to 80%of its activity for 24 hr. This fact may be explained by a significant decrease in the rate of autolysis due to steric hindrances in the case of the bound enzyme. This mechanism is very important in preserving the biological activity of bound enzymes in the living organism, where exogenous enzymes lose their activity in a short time due to the action of endogeneous proteases. In conclusion, it is suggested that heparin can be effectively used as a carrier for enzyme immobilization. It is possible to obtain a heparin-enzyme preparation in which the enzyme has an unchanged biological activity. References 1. R. Goldman, L. Goldstein, and E. Katchalsky, in G. R. Stark, Ed., Biochemical Aspects of Reactions on Solid Supports, Academic, New York, 1971. 2. G. J. H. Melrose, Reu. Pure Appl. Chem., 21,83 (1971). 3. R. Axen, J. Porath, and S. Ernback, Nature, 214,1302 (1967). 4. E. K. Bauman, L. H. Goodson, G. G. Guilbault, and D. N. Kramer, Anal. Chem., 37,1378 (1965). 5. S. Avrameas and T. Ternynck, Immunochemistry, 6,53 (1969). 6. S. Suzuki, I. Karuhe, and I. Watanabe, Int. Farm. Symp. Kyoto, February 3-5, 1970, p. 70. 7. S. Avrameas and T. Ternynck, J. Biol. Chem., 242,1651 (1967). 8. J. G. Dillon, M. R. Begue-Canton, R. L. Bleakeley, L. J. Brubacher, J. Feder, C. R. Gunter, F. J. Kazdy, J. V. Killheffer, T. H. Marshall, C. G. Miller, R. W. Roeske, and J. R. Stoops, J. Am. Chem. Soc., 88,5890 (1964). 10. 2. A. Streltsova, E. E. Braudo, and V. B. Tolstoguzov, Bioorgan. Khim., 1, 267 (1975). 11. 2. A. Streltsova, V. K. Shwedas, A. V. Maksimenko, A. A. Kliosov, E. E. Braudo, V. B. Tolstoguzov, and I. V. Berezin, Bioorgan. Khim., 1,1469 (1975). 12. V. P. Torchilin, V. S. Goldmacher, A. M. Klibanov, K. Martinek, E. G . Tischenko, V. N. Smirnov, 1. V. Berezin, and E. I. Chazov, USSR-USA Symposium on Chemistry and Physics of Proteins, Riga, 1976, p. 116 (abstract). 13. V. P. Torchilin, I. L. Reiser, E. G. Tischenko, E. V. Il’ina, V. N. Smirnov, and E. I. Chazov, Bioorgan. Khim., 2,1687 (1976).
Received April 4,1977 Revised July 20,1977
