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[22] Nonaqueous synthesis of polystyryl lysozyme
288
IMMOBILIZATION TECHNIQUES
[221
the enzyme-carrier couple formed by established thermochemical processes.~,ls,l' Although the low level of target specificity exhibited in the nitrene coupling reaction provides significant advantages for the immobilization of enzymes and other biological macromolecules, this may present a limitation for the photochemical immobilization of smaller biologically active molecules in which target group specificity for the coupling reaction may be necessary for the maintenance of biological activity. In addition the susceptibility of the carrier material itself to reaction with the nitrene may limit the Scheme I (Fig. 1) process to those carriers containing little aromatic or other hydrophobic character. One must be cautious also in the use of organic solvents or organic buffer ions in the photolysis mixture, since these may provide competitive targets for the nitrene reactions. On the basis of experience with the photoimmobilization of approximately 10 enzymes with approximately 15 different ANP-carrier derivatives, 4 one would expect an ANP-carrier containing 50-100 ~moles of ANP per dry gram to photoimmobilize 20-70 mg of enzyme with a free enzyme activity equivalent of 5-60 mg of enzyme per dry gram. Appreciably higher values may be expected from a carrier with larger exposed surface area and improved carrier-enzyme compatibility for the immobilization step (e.g., ionic attraction for concentrating the enzyme at the carrier surface) and subsequent catalytic function. ,8 M. Yaqub and P. Guire, J. Biomed. Mater. Res. 8, 291 (1974). ,Dp. Guire, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2674 (1975).
[2 2] N o n a q u e o u s S y n t h e s i s o f P o l y s t y r y l L y s o z y m e B y H. D. BROWN and S. K. CHATTOPADHYAY
Reaction utilizng organic solvation in enzyme polymer complex syntheses have a number of potential advantages over methods limited to aqueous conditions: increase of available reactive moieties of the protein; increase in the available polymeric matrices (by virtue of appropriate solubility as well as available alternative reactive groups); use of the solvent system to control the configurational properties of the enzyme. A representative technique is lysozyme binding to polystyrene under anhydrous conditions using N,N'-carbonyldiimidazole as an activating agent. Enzyme complexes synthesized by this technique have enhanced thermal
[22]
NONAQUEOUSSYNTHESIS OF POLYSTYRYL LYSOZYME
289
and chemical stability and modified pH and substrate optima; the protein moiety may have an altered configuration. 1
Synthesis Copoly(styrene-4-vinylbenzoic acid) lysozyme can be prepared under anhydrous conditions, in N,N-dimethylformamide, using N,N-carbonyldiimidazole as an activating agent.
Reagents Benzoyl peroxide N,N'-Carbonyldiimidazole 4-Chlorostyrene N,N-Dimethyl formamide Lysozyme (EC 3.2.1.17) Maleate buffer, 0.1 M, pH 6.2 Styrene 4-Vinylbenzoic acid 2
Preparation of the Copolymer. A polymerization tube is charged with 12.4 g (0.119 mole) of styrene and 5.90 g (0.039 mole) of 4-vinylbenzoic acid. The mixture is treated with 250 mg of benzoyl peroxide and stirred until solution of the catalyst is complete. The tube is sealed and immersed in an oil bath at 80 ° for 24 hr. A clear, colorless polymer, removed from the polymerization vessel, is ground to a fine powder in a mortar. The product (18.1 g) is characterized by IR (KBr) ; 1680 cm-1 (C=O) 1420, 1280, 1175, 1017, and 825 cm -1. Preparation of Lysozyme Polymer Complex. A 50-ml flask equipped for magnetic stirring and fitted with a drying tube is charged with 200 mg of copolymer and 5 ml of N,N-dimethylformamide. The mixture is stirred to dissolve the polymer, and 70 mg (0.432 mmole) of N,NP-carbonyl diimidazole are added. Carbon dioxide is evolved immediately, and the mixture is stirred for 30 min to complete the conversion to the imidazolide. Twenty milligrams of crystalline lysozyme are added in one portion, and the two-phase reaction mixture is stirred for 18 hr at 25 °. At the end of the stirring period unreacted lysozyme is removed by centrifugation, and the clear, colorless organic supernatant is decanted. The dimethylformamide solution of the polymer-enzyme complex is slowly poured into 50 ml of 0.1 M, pH 6.2, maleate buffer. A fine, white precipitate separates immediately and is removed by centrifugation. This product is washed with fresh buffer and suspended in 10 ml of buffer for assay. 1H. D. Brown, G. J. Bartling, and S. K. Chattopadhyay, in "Enzyme Engineering" (E. K. Pye and L. B. Wingard, Jr., eds.), Vol. 2, p. 83. Plenum, New York, 1974. 2j. R. Leebrick and H. E. Ramsden, J. Org. Chem. 23, 935 (1958).
290
IMMOBILIZATION TECHNIQUES
[22]
Assay The enzymic hydrolysis of N-acetylhexosaminidic linkages of chitinderived oligomers is the basis of the assay described here. Activity is determined by colorimetric measurement of the formation of reducing groups (Park and Johnson, 3 modified). Hydrolysis of the fl (1-->4) linkage in linear poly-N-acetylglucosamine of M i c r o c o c c u s cell walls, measured densitometrically, 4 is also useful though less appropriate to the immobilized enzyme. Reagents
Maleate buffer, 0.1 M, pH 6.2. Hexa- (N-acetylglucosamine) 5 Ferricyanide solution, 50 mg potassium ferricyanide in 100 ml of water. Store in brown bottle Carbonate-cyanide solution, 530 mg of sodium carbonate and 65 mg of potassium cyanide (KCN) in 100 ml of water Ferric iron solution, 150 mg of ferric ammonium sulfate and 100 mg of sodium lauryl sulfate in 100 ml of 0.05 N sulfuric acid Procedure. The reaction mixtures consist of 0.12 mg of bound protein and 1.0 mg of hexa-(N-acetylglucosamine) in a total of 2.0 ml of 0.1 M maleate buffer, p H 6.2. Appropriate controls are also used. Samples are incubated at 37 ° with constant stirring for 30 min. The reaction is stopped by immersion into boiling water. After cooling to 25 ° the polymer-enzyme complex is removed by centrifugation and a 0.2-ml aliquot is removed from each tube. Each aliquot is diluted with 0.3 ml of water; 0.5 ml of carbonate-cyanide solution and 0.5 ml of ferricyanide solution are added. Assay tubes are placed in a boiling water bath for 5 rain and then cooled in an ice bath. One milliliter of ferricyanide solution and 5.0 ml of ferric iron solution are added. After 15 min the absorbency of the solution is measured against a reagent blank at 690 nm. Reducing groups equivalent is calculated from a standard curve using glucose 6 as reference. Bound protein is determined by the method of Lowry et al. 7 using lysozyme as standard.
s j. T. Park and M. J. Johnson, J. Biol. Chem. 181, 149 (1949). 4D. Shugar, Biochlm. Biophys. Acta 8, 302 (1952). 5From chitin; J. A. Rupley, L. Butler, M. Gerring, F. J. l=Iardegen, and R. Pecoraro, Proc. Natl. Acad. Sci. U,q_4.57, 1088 (1967). 6A unit of activity is defined as the production of 1 /zg of glucose per minute per milligram of protein at 37°. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 26S (1951).
IMMOBILIZATION TECHNIQUES
[221
the enzyme-carrier couple formed by established thermochemical processes.~,ls,l' Although the low level of target specificity exhibited in the nitrene coupling reaction provides significant advantages for the immobilization of enzymes and other biological macromolecules, this may present a limitation for the photochemical immobilization of smaller biologically active molecules in which target group specificity for the coupling reaction may be necessary for the maintenance of biological activity. In addition the susceptibility of the carrier material itself to reaction with the nitrene may limit the Scheme I (Fig. 1) process to those carriers containing little aromatic or other hydrophobic character. One must be cautious also in the use of organic solvents or organic buffer ions in the photolysis mixture, since these may provide competitive targets for the nitrene reactions. On the basis of experience with the photoimmobilization of approximately 10 enzymes with approximately 15 different ANP-carrier derivatives, 4 one would expect an ANP-carrier containing 50-100 ~moles of ANP per dry gram to photoimmobilize 20-70 mg of enzyme with a free enzyme activity equivalent of 5-60 mg of enzyme per dry gram. Appreciably higher values may be expected from a carrier with larger exposed surface area and improved carrier-enzyme compatibility for the immobilization step (e.g., ionic attraction for concentrating the enzyme at the carrier surface) and subsequent catalytic function. ,8 M. Yaqub and P. Guire, J. Biomed. Mater. Res. 8, 291 (1974). ,Dp. Guire, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2674 (1975).
[2 2] N o n a q u e o u s S y n t h e s i s o f P o l y s t y r y l L y s o z y m e B y H. D. BROWN and S. K. CHATTOPADHYAY
Reaction utilizng organic solvation in enzyme polymer complex syntheses have a number of potential advantages over methods limited to aqueous conditions: increase of available reactive moieties of the protein; increase in the available polymeric matrices (by virtue of appropriate solubility as well as available alternative reactive groups); use of the solvent system to control the configurational properties of the enzyme. A representative technique is lysozyme binding to polystyrene under anhydrous conditions using N,N'-carbonyldiimidazole as an activating agent. Enzyme complexes synthesized by this technique have enhanced thermal
[22]
NONAQUEOUSSYNTHESIS OF POLYSTYRYL LYSOZYME
289
and chemical stability and modified pH and substrate optima; the protein moiety may have an altered configuration. 1
Synthesis Copoly(styrene-4-vinylbenzoic acid) lysozyme can be prepared under anhydrous conditions, in N,N-dimethylformamide, using N,N-carbonyldiimidazole as an activating agent.
Reagents Benzoyl peroxide N,N'-Carbonyldiimidazole 4-Chlorostyrene N,N-Dimethyl formamide Lysozyme (EC 3.2.1.17) Maleate buffer, 0.1 M, pH 6.2 Styrene 4-Vinylbenzoic acid 2
Preparation of the Copolymer. A polymerization tube is charged with 12.4 g (0.119 mole) of styrene and 5.90 g (0.039 mole) of 4-vinylbenzoic acid. The mixture is treated with 250 mg of benzoyl peroxide and stirred until solution of the catalyst is complete. The tube is sealed and immersed in an oil bath at 80 ° for 24 hr. A clear, colorless polymer, removed from the polymerization vessel, is ground to a fine powder in a mortar. The product (18.1 g) is characterized by IR (KBr) ; 1680 cm-1 (C=O) 1420, 1280, 1175, 1017, and 825 cm -1. Preparation of Lysozyme Polymer Complex. A 50-ml flask equipped for magnetic stirring and fitted with a drying tube is charged with 200 mg of copolymer and 5 ml of N,N-dimethylformamide. The mixture is stirred to dissolve the polymer, and 70 mg (0.432 mmole) of N,NP-carbonyl diimidazole are added. Carbon dioxide is evolved immediately, and the mixture is stirred for 30 min to complete the conversion to the imidazolide. Twenty milligrams of crystalline lysozyme are added in one portion, and the two-phase reaction mixture is stirred for 18 hr at 25 °. At the end of the stirring period unreacted lysozyme is removed by centrifugation, and the clear, colorless organic supernatant is decanted. The dimethylformamide solution of the polymer-enzyme complex is slowly poured into 50 ml of 0.1 M, pH 6.2, maleate buffer. A fine, white precipitate separates immediately and is removed by centrifugation. This product is washed with fresh buffer and suspended in 10 ml of buffer for assay. 1H. D. Brown, G. J. Bartling, and S. K. Chattopadhyay, in "Enzyme Engineering" (E. K. Pye and L. B. Wingard, Jr., eds.), Vol. 2, p. 83. Plenum, New York, 1974. 2j. R. Leebrick and H. E. Ramsden, J. Org. Chem. 23, 935 (1958).
290
IMMOBILIZATION TECHNIQUES
[22]
Assay The enzymic hydrolysis of N-acetylhexosaminidic linkages of chitinderived oligomers is the basis of the assay described here. Activity is determined by colorimetric measurement of the formation of reducing groups (Park and Johnson, 3 modified). Hydrolysis of the fl (1-->4) linkage in linear poly-N-acetylglucosamine of M i c r o c o c c u s cell walls, measured densitometrically, 4 is also useful though less appropriate to the immobilized enzyme. Reagents
Maleate buffer, 0.1 M, pH 6.2. Hexa- (N-acetylglucosamine) 5 Ferricyanide solution, 50 mg potassium ferricyanide in 100 ml of water. Store in brown bottle Carbonate-cyanide solution, 530 mg of sodium carbonate and 65 mg of potassium cyanide (KCN) in 100 ml of water Ferric iron solution, 150 mg of ferric ammonium sulfate and 100 mg of sodium lauryl sulfate in 100 ml of 0.05 N sulfuric acid Procedure. The reaction mixtures consist of 0.12 mg of bound protein and 1.0 mg of hexa-(N-acetylglucosamine) in a total of 2.0 ml of 0.1 M maleate buffer, p H 6.2. Appropriate controls are also used. Samples are incubated at 37 ° with constant stirring for 30 min. The reaction is stopped by immersion into boiling water. After cooling to 25 ° the polymer-enzyme complex is removed by centrifugation and a 0.2-ml aliquot is removed from each tube. Each aliquot is diluted with 0.3 ml of water; 0.5 ml of carbonate-cyanide solution and 0.5 ml of ferricyanide solution are added. Assay tubes are placed in a boiling water bath for 5 rain and then cooled in an ice bath. One milliliter of ferricyanide solution and 5.0 ml of ferric iron solution are added. After 15 min the absorbency of the solution is measured against a reagent blank at 690 nm. Reducing groups equivalent is calculated from a standard curve using glucose 6 as reference. Bound protein is determined by the method of Lowry et al. 7 using lysozyme as standard.
s j. T. Park and M. J. Johnson, J. Biol. Chem. 181, 149 (1949). 4D. Shugar, Biochlm. Biophys. Acta 8, 302 (1952). 5From chitin; J. A. Rupley, L. Butler, M. Gerring, F. J. l=Iardegen, and R. Pecoraro, Proc. Natl. Acad. Sci. U,q_4.57, 1088 (1967). 6A unit of activity is defined as the production of 1 /zg of glucose per minute per milligram of protein at 37°. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 26S (1951).