- Email: [email protected]
[21] Photochemical immobilization of enzymes and other biochemicals
280
IMMOBILIZATION TECHNIQUES
[21]
model-building, and some medical applications. When scaling-up, however, new limitations to these procedures appear: among these are poor mechanical properties of the support and, often, high cost. One advantage of chemical aggregation over binding of enzymes onto a carrier is that the carrier adds to the reactor a volume that can be reduced significantly by aggregation. Over encapsulation, one advantage is the possibility of greatly reducing diffusional constraints by particle division and superficial coating. The efficiency of these methods depends on the cross-linking conditions, on the availability of active functions (e.g., NH: of lysine, or SH of cysteine) at the surface of the enzyme molecule, on the interference of the functions that play a part at its active site, and on its adaptability to the substrate. Some enzymes give very poor activity yields by chemical aggregation. This seems to be the case with enzymes of higher molecular weight and with enzymes made up of subunits. This is not a general rule, and it may be due to the increased probability of molecular hindrances or to unfavorable regulation of these sophisticated molecules. Other techniques of immobilization should be preferred in such cases. Many difficulties in the use of aggregation procedures should be partly or completely overcome in the future by a more systematic choice of suitable procedures and by a deeper insight into the potential application of other bi- or multifunctional chemical agents. At present, a good knowledge of the technical mistakes to be avoided and of parameters to be taken into account, and also a certain degree of expertise in the use of one set of cross-linking conditions, help to solve many of the problems in the immobilization of a large variety of enzymes.
[21] P h o t o c h e m i c a l I m m o b i l i z a t i o n o f E n z y m e s and Other Biochemicals
By
PATRICK GUIRE
Electromagnetic radiation offers some distinct advantages over thermal energy for the activation of chemical reactions involving biological and other chemical compounds. Compared to most thermochemical reactions (activated by kinetic energy uptake), photochemical activation is a relatively rapid (essentially instantaneous) process, which is easily and quickly initiated and terminated and can produce a highly energetic and relatively homogeneous reactive species capable of the rapid formation of covalent bonds with target molecules with relatively little depen-
[21]
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dence upon the temperature and pH of the reaction mixture or even the chemical character of the target group. The quantum character of the interaction of electromagnetic energy with molecules or groups of atoms provides an additional advantage in the design and use of bifunctional or cross-linking agents for the covalent coupling of one molecule or functional group with another. Since cross-linking requires reaction with at least two groups, probably differing appreciably in chemical reactivity and/or spatial location, much better control over the reaction process is provided to the operator through the use of a stepwise cross-linking process. Such a time-controlled stepwise reaction process is available through the use of a reagent containing both a thermally activatible group and a light activatible (photochemical) group or a reagent with two or more photochemical groups with little overlap in their photoactivation (action) spectra. For the coupling of enzymes and most other biochemicals to each other or to relatively inert water-insoluble carrier materials, one would seek a photochemical group that is quite stable in the dark under most conditions that are compatible with the enzyme or other biochemical and stable under reaction conditions required for activating the thermochemical group. The photochemical group should be subject to activation by light of those wavelengths (e.g., visible light) that are harmless to most enzymes and other biochemicals. The photoactivated species thus produced should exhibit minimum dependence of its reactivity upon temperature, pH, target group character, and other reaction parameters of importance to the maintenance of the biological activity of the target molecules. In 1969 Knowles e t al. 1-3 demonstrated the 2-nitro-4-azidophenyl (ANP) group to exhibit these desired characteristics to a useful degree. Fluoro-2-nitro-4-azidobenzene (FNAB) is an example of a combined thermochemical-photochemical bifunctional reagent useful for the stepwise coupling of even quite fragile biochemical and other molecules or functional groups to one another or to water-insoluble carrier materials. The aryl azidc group is quite stable in the dark under physiological conditions and even under many harsher reaction conditions useful for the thermochemical replacement of the fluorine by nucleophilic groups on carrier derivatives or on soluble compounds containing other functional groups differing from the nitrophenyl fluoride in conditions of thermal activation and target specificity. The basic structure of the stepwise crosslinking reagents emphasized herein is presented in Fig. 1, where the R 1 G. W. J. Fleet, R. R. Porter, and J. R. K. Knowles, Nature (London) 224, 511 (1969). 2 G. W. J. Fleet, J. R. Knowles, and R. R. Porter, B~ochem. J. 128, 499 (1972). 3 Jeremy R. Knowles, Ace. Chem. Res. S, 155 (1972).
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IMMOBILIZATION TECHNIQUES
R Scheme
I ~
Scheme II
NO2 Ns
Step I (Thermochemical) Carrier
Step 2 (Photochemical) Enzyme
~'~
k
A S
Enzyme
-'- ANP-Carrier ~
ANP-Enzyme-
hv
[21]
Enzyme-ANPoCarrier
Carrier
FI~. 1. Alternative schemes for the stepwise thermophotochemical coupling of enzymes with carrier materials. ANP, azidonitrophenyl group. group represents in our use fluorine or other thermochemically activated groups with better activation conditions a n d / o r target specificities. The dark-stable aryl azide (ANP) group is activated by visible light to generate N2 and the aryl nitrene diradical, which is a short-lived species capable of insertion into even carbon-hydrogen bonds 3 with little dependence upon the temperature or p H of the reaction mixture over the ranges useful for enzyme couplings. 4 I f the thermochemical group is on a side chain substituted for the fluorine of FNAB, the thermochemical and the photochemical activations m a y be performed with essentially complete independence of each other. While this allows the operator to choose either time sequence for the two reaction steps, the thermochemical stability and low level of target specificity of the photochemical group recommends it for the second or final step of the cross-linking process for most immobilization systems. Therefore, we outline in Fig. 1 alternative schemes for the stepwise immobilization of enzymes with thermophotochemical bifunctional reagents, in both of which the d a r k reaction is executed first. Whereas the potential value of Scheme I I is great and is currently being demonstrated in several laboratories TM for the coupling of hor4p. Guire, M. Yaqub, C. Chirpich, R. Blake, and E. Podrebarac. Manuscript in preparation. 5F. Richards, J. Lifter, C. L. Hew, M. Yoshioka, and W. H. Konigsberg, Biochemistry 13, 3572 (1974). 6C. A. Converse and F. F. Richards, Biochemistry 8, 4431 (1969). I. Schwartz and J. Ofengand, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2042 (1975). s W. G. Hanstein, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2126 (1975). ' G. Rudnick, R. Weil, and H. R. Kabaek, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 1525 (1975). ~oD. Levy and T. Trosper, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2404 (1975). 11D. Levy, Biochim. Biophys. Acta 332, 329 (1973). I~B. E. Haley, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2250 (1975). 1~L. W. Yielding, W. E. White, and K. L. Yielding, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2084 (1975).
[21]
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mones, haptens, and other biologically active ligands to their receptor sites on maeromolecules, subcellular organelles, and intact cells and tissues, we shall describe only our experience with Scheme I in this treatise on immobilization techniques. We have demonstrated the usefulness of FNAB and various related stepwise cross-linking reagents containing the nitrophenylazide photoreactive group for the thermochemical preparation of dark-stable photoreactive derivatives of various carrier materials. These photoreactive carrier derivatives may be prepared, washed, and stored wet or dry at ambient temperatures in the dark. The user need merely choose the preferable carrier derivative, mix it in water with his subject enzyme at the temperature and pH chosen for optimizing the enzyme stability and/or interaction with the carrier, and execute the covalent coupling by mere illumination with visible light. The photoimmobiliz~d enzyme product may then be processed and used by the usual procedures. Materials
Essentially all the hydrophilic carrier materials currently being used for enzyme immobilization are expected to be compatible with the photoimmobilization process. Suitable azidonitrophenyl (ANP) derivatives may be coupled to these carriers, in most cases by the same thermochemical reactions used to couple these carriers to enzymes. Approximately 15 ANP derivatives differing in the length, chemical character, and mode of coupling of the extender arm between the polymer backbone and the ANP group have been prepared and demonstrated to be useful for the photoimmobilization of enzymes. These derivatives are based on five basic materials, which are commercially available: controlled-pore glass (aminoalkyl, with and without zirconium), cellulose (aminoethyl and carboxymethyl), Sephadex (carboxymethyl), agarose (aminopropyl, aminohexyl, and aminoethylaminopropyl), and polyacrylamide (aminoethyl). Most other natural and synthetic polymers useful as enzyme carriers are expected to be compatible with this Scheme I process. Scheme II is expected to be preferable for the coupling of enzymes to carriers that are effective competitors to the enzymes as targets for the photochemical aryl nitrene coupling (e.g., insoluble proteins, such as collagen, and carriers with a high aromatic content, such as polystyrene). Fluoro-2-nitro-4-azidobenzene ( F N A B ) . This thermophotochemical bifunctional reagent is now commercially available (e.g., FNPA from ~4H. Kiefer, J. Lindstrom, E. S. Lennox, and S. J. Singer, Proc. Natl. Acad. Sci. U.S.A. 67, 1688 (1970). 15B. A. Winter and A. Goldstein, Mol. Pharmacol. 6, 601 (1972). le U. Das Gupta and J. S. Rieske, Biochem. Biophys. Res. Commun. 54, 1247 (1973). !, j. A. Katzenellenbogen, Annu. Rep. Med. Chem. 9, 222 (1974).
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[21]
Pierce Cat. No. 23700, or ICN-K&K Cat. No. 28064). It may be prepared from 4-fluoro-3-nitroaniline by the diazotization-azide substitution methodology described by Fleet, Knowles, and PorterY 4-Azido-2-nitrophenyl-aminoalkylcarboxyl derivatives may be prepared by allowing FNAB to react with the chosen aminocarboxylic acid compound (e.g., glycine, fl-alanine) in dimethyl sulfoxide according to Levy, 11 in aqueous ethanol with Na~CO3 according to Fleet, ~ or in aqueous ethanol with Ca (OH)2 according to the procedure used in our laboratory. 4 ANP-aminoalkylamine derivatives may be obtained by allowing FNAB to react with equimolar or excess diamines (e.g., ethylenediamine,
1,3-diaminopropanol).4 Other thermochemically functional groups more useful for coupling to the particular carrier material of choice may be expected to be substituted onto the photoreactive ANP group by these or other known ¢hermochemical reaction procedures. Procedures In the limited space available for this introduction to photoimmobilization methodology, the use of a commercially available thermophotochemical bifunctional reagent (FNAB) with commercially available aminoalkyl carrier materials will be described. Only Scheme I, the preparation and use of dark-stable, photoreactive carrier derivatives for the gentle and facile photoimmobilization of enzymes, will be described in detail. Step 1. Dark Reaction. Thermally stable photoreactive derivatives of aminoalkyl matrix or carrier materials (ANP-AAM) may be prepared by allowing FNAB to react in the dark or in dim light with a suspension of an aminoalkyl matrix (AAM) or carrier material in alkaline aqueous ethanol. FNAB exhibits tbermochemical reactivity properties for fluoride displacement similar to those of Sanger's reagent (fluorodinitrobenzene), the latter being significantly more reactive. The azide group of FNAB appears to be thermochemically stable in many solvents at temperatures up to at least 50 ° and pH up to at least 10.5. The useful conditions (except for low light intensity) for the FNAB -~ AAM reaction, and subsequent processing and storage of the ANP-AAM products, are expected to be subject to wide variation. A specific set of process conditions that are expected to be useful for a large number of aminoalkyl carrier products is described below. The aminoalkyl carrier material (e.g., aminoethyl cellulose, aminoalkyl agarose, aminoalkyl glass) is suspended in and equilibrated with an aqueous alkaline buffer (e.g., 0.5 M borate, pH 9.5--10). The washed
[21]
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equilibrated material is recovered as a packed bed of gel or a slurry of particles. FNAB of sufficient quantity to give the desired molar ratio over carrier amino groups (a range of 1--3 is usually satisfactory) is dissolved in ethanol of two times the volume of the aqueous carrier suspension or gel and the two are mixed together. This and subsequent exposure of the photochemical materials prior to photolysis (step 2) is executed in dim daylight or darker. The reaction flask is stoppered and the contents mixed at 37°-40 ° for 16-64 hr. The reaction mixture solid is then separated by filtration or centrifugation, washed sequentially with 95% ethanol until the washings are colorless, with 1 M NaC1 in H20 or a buffer of the pH desired for the planned photoimmobilization use, then with this buffer or with deionized water. If the material is to be dried for storage before use, the fibrous or particulate materials should be washed finally with H20, the gels with ethanol. These washed derivatives may then be dried under vacuum at room temperature and stored in the dark at ambient temperature until used. Step 2. Photoimmobilization Procedure. The reaction conditions useful with these photoreactive carrier materials are subject to large variation without harm to the photochemical reaction. Such variables as pH, temperature, and ionic strength may be chosen for the optimum activity stability of the enzyme to be immobilized. The reaction rate depends mostly upon the intensity of illumination with visible light. Satisfactory illumination, mixing, temperature control, and measurement of the N2 evolved may be obtained in an illuminated Warburg apparatus. Although the photochemical immobilization reaction can be activated to completion in several hours by a regular 200 or 300 W tungsten filament light bulb, or a focused microscope illuminator, still better light intensity at the target and improved light-to-heat ratio can be obtained with parallel-beam tungsten-halide projector lamp bulbs [e.g., Eumig 711R (EU 1172) 12 V, 100 W, reflector lamp bulb] mounted on the manometer-flask carrier for movement with the target flask during the motion for sample stirring {Fig. 2). tl/2 of N2 evolution in photoimmobilizations with this illuminated Warburg setup, operated at 10 V on the lamp, usually fell in the range of 20-40 min. The photoreactive carrier material is equilibrated to the pH chosen for the enzyme and washed with water to remove organic buffer ions if any have been used. The carrier material is suspended to form a slurry thin enough for efficient mixing, with water or aqueous inorganic buffer containing enzyme up to 10% the dry weight of the carrier material. The system is then flushed with N~ before illumination as a precaution against possible O2 interference with the desired nitrene reactions.
286
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[21]
Fia. 2. Modified Warburg apparatus for photochemical coupling reactions. The parallel-beam tungsten-halide projector lamps are mounted on the manometer platform of a Bronwill Warburg Apparatus Model UV so that the light beams may be directed continuously through the clear Plexiglas temperature control bath container into the Warburg flasks during reciprocal motion in the water bath. An example of a useful experimental protocol follows: 20-25 mg dry of A N P carrier material previously equilibrated to p H 4.5-5.0 is placed in an approximately 5-ml Warburg flask without a center well. H~O (usually 0.5-2 ml, depending upon the type of carrier material being used) containing 2-3 mg of invertase (fl-fructofuranosidase from bakers' yeast) is added, and the flask is attached to the Warburg manometer. The sample is then placed on the W a r b u r g apparatus with the flask immersed
[21]
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287
in ice-water. The manometer-flask system is flushed with a stream of N2 for a few minutes while temperature equilibration is occurring. Work up to this point is done in dim daylight. The flask-manometer system is then closed, a zero-time manometer reading is taken, and illumination with mixing motion is initiated and continued until N~ evolution ceases, as indicated by the periodic manometer readings. When the parallel-beam 12 V, 100 W projector bulb is operated at 10 V, the reaction is usually continued from 2 to 5 hr illumination time. Further processing of the immobilized enzyme need be no different from that of enzyme immobilized by other methods. Satisfactory photoimmobilization without measurement of the rate or extent of N._, evolution may be obtained by placing such an enzyme ~ANP-AAM reaction mixture in an approximately 5-ml round-bottom flask blown from glass tubing. The mouth is covered with a gum rubber dropper bulb, and the flask is flushed with N._,or other inert gas. The flask is then placed in a bath for temperature control and stirred magnetically for about 16 hr illumination with a focused 8 V, 5 amp microscope illuminator light passing through about 0.5 cm of 1 M aqueous sodium nitrite in a petri dish. Discussion
Many advantages are obvious in the use of stepwise cross-linking reagents for coupling carrier materials to enzymes and other biochemicals. The high level of reaction independence of the two reactive groups of certain thermophotochemical bifunctional reagents allows the chemist to prepare dark-stable photoreactive derivatives of his chosen carrier material and to further "tailor" it (e.g., modify its ionic character, add ligands, and/or otherwise modify the "microenvironment" of the carrier surface) for the particular enzyme system(s) to be immobilized. These dark-stable photoreactive carrier materials can be removed from storage and used in the facile covalent coupling of enzymes by merely mixing them together in an aqueous slurry at the temperature and pH chosen for the enzyme and illuminating the mixture with visible light. Since most enzymes are transparent to light of wavelengths required to activate the nitrophenyl azide, and the aryl nitrene thus produced exhibits a relatively low level of specificity for the target amino acid, the enzyme molecule is expected to be covalentty immobilized with exceptionally little stress by this process. The photoimmobilized enzyme product is thus expected to exhibit relatively high levels of catalytic capacity and specific activity. Otherwise its properties are found to be quite similar to those of
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
IMMOBILIZATION TECHNIQUES
[21]
model-building, and some medical applications. When scaling-up, however, new limitations to these procedures appear: among these are poor mechanical properties of the support and, often, high cost. One advantage of chemical aggregation over binding of enzymes onto a carrier is that the carrier adds to the reactor a volume that can be reduced significantly by aggregation. Over encapsulation, one advantage is the possibility of greatly reducing diffusional constraints by particle division and superficial coating. The efficiency of these methods depends on the cross-linking conditions, on the availability of active functions (e.g., NH: of lysine, or SH of cysteine) at the surface of the enzyme molecule, on the interference of the functions that play a part at its active site, and on its adaptability to the substrate. Some enzymes give very poor activity yields by chemical aggregation. This seems to be the case with enzymes of higher molecular weight and with enzymes made up of subunits. This is not a general rule, and it may be due to the increased probability of molecular hindrances or to unfavorable regulation of these sophisticated molecules. Other techniques of immobilization should be preferred in such cases. Many difficulties in the use of aggregation procedures should be partly or completely overcome in the future by a more systematic choice of suitable procedures and by a deeper insight into the potential application of other bi- or multifunctional chemical agents. At present, a good knowledge of the technical mistakes to be avoided and of parameters to be taken into account, and also a certain degree of expertise in the use of one set of cross-linking conditions, help to solve many of the problems in the immobilization of a large variety of enzymes.
[21] P h o t o c h e m i c a l I m m o b i l i z a t i o n o f E n z y m e s and Other Biochemicals
By
PATRICK GUIRE
Electromagnetic radiation offers some distinct advantages over thermal energy for the activation of chemical reactions involving biological and other chemical compounds. Compared to most thermochemical reactions (activated by kinetic energy uptake), photochemical activation is a relatively rapid (essentially instantaneous) process, which is easily and quickly initiated and terminated and can produce a highly energetic and relatively homogeneous reactive species capable of the rapid formation of covalent bonds with target molecules with relatively little depen-
[21]
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281
dence upon the temperature and pH of the reaction mixture or even the chemical character of the target group. The quantum character of the interaction of electromagnetic energy with molecules or groups of atoms provides an additional advantage in the design and use of bifunctional or cross-linking agents for the covalent coupling of one molecule or functional group with another. Since cross-linking requires reaction with at least two groups, probably differing appreciably in chemical reactivity and/or spatial location, much better control over the reaction process is provided to the operator through the use of a stepwise cross-linking process. Such a time-controlled stepwise reaction process is available through the use of a reagent containing both a thermally activatible group and a light activatible (photochemical) group or a reagent with two or more photochemical groups with little overlap in their photoactivation (action) spectra. For the coupling of enzymes and most other biochemicals to each other or to relatively inert water-insoluble carrier materials, one would seek a photochemical group that is quite stable in the dark under most conditions that are compatible with the enzyme or other biochemical and stable under reaction conditions required for activating the thermochemical group. The photochemical group should be subject to activation by light of those wavelengths (e.g., visible light) that are harmless to most enzymes and other biochemicals. The photoactivated species thus produced should exhibit minimum dependence of its reactivity upon temperature, pH, target group character, and other reaction parameters of importance to the maintenance of the biological activity of the target molecules. In 1969 Knowles e t al. 1-3 demonstrated the 2-nitro-4-azidophenyl (ANP) group to exhibit these desired characteristics to a useful degree. Fluoro-2-nitro-4-azidobenzene (FNAB) is an example of a combined thermochemical-photochemical bifunctional reagent useful for the stepwise coupling of even quite fragile biochemical and other molecules or functional groups to one another or to water-insoluble carrier materials. The aryl azidc group is quite stable in the dark under physiological conditions and even under many harsher reaction conditions useful for the thermochemical replacement of the fluorine by nucleophilic groups on carrier derivatives or on soluble compounds containing other functional groups differing from the nitrophenyl fluoride in conditions of thermal activation and target specificity. The basic structure of the stepwise crosslinking reagents emphasized herein is presented in Fig. 1, where the R 1 G. W. J. Fleet, R. R. Porter, and J. R. K. Knowles, Nature (London) 224, 511 (1969). 2 G. W. J. Fleet, J. R. Knowles, and R. R. Porter, B~ochem. J. 128, 499 (1972). 3 Jeremy R. Knowles, Ace. Chem. Res. S, 155 (1972).
282
IMMOBILIZATION TECHNIQUES
R Scheme
I ~
Scheme II
NO2 Ns
Step I (Thermochemical) Carrier
Step 2 (Photochemical) Enzyme
~'~
k
A S
Enzyme
-'- ANP-Carrier ~
ANP-Enzyme-
hv
[21]
Enzyme-ANPoCarrier
Carrier
FI~. 1. Alternative schemes for the stepwise thermophotochemical coupling of enzymes with carrier materials. ANP, azidonitrophenyl group. group represents in our use fluorine or other thermochemically activated groups with better activation conditions a n d / o r target specificities. The dark-stable aryl azide (ANP) group is activated by visible light to generate N2 and the aryl nitrene diradical, which is a short-lived species capable of insertion into even carbon-hydrogen bonds 3 with little dependence upon the temperature or p H of the reaction mixture over the ranges useful for enzyme couplings. 4 I f the thermochemical group is on a side chain substituted for the fluorine of FNAB, the thermochemical and the photochemical activations m a y be performed with essentially complete independence of each other. While this allows the operator to choose either time sequence for the two reaction steps, the thermochemical stability and low level of target specificity of the photochemical group recommends it for the second or final step of the cross-linking process for most immobilization systems. Therefore, we outline in Fig. 1 alternative schemes for the stepwise immobilization of enzymes with thermophotochemical bifunctional reagents, in both of which the d a r k reaction is executed first. Whereas the potential value of Scheme I I is great and is currently being demonstrated in several laboratories TM for the coupling of hor4p. Guire, M. Yaqub, C. Chirpich, R. Blake, and E. Podrebarac. Manuscript in preparation. 5F. Richards, J. Lifter, C. L. Hew, M. Yoshioka, and W. H. Konigsberg, Biochemistry 13, 3572 (1974). 6C. A. Converse and F. F. Richards, Biochemistry 8, 4431 (1969). I. Schwartz and J. Ofengand, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2042 (1975). s W. G. Hanstein, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2126 (1975). ' G. Rudnick, R. Weil, and H. R. Kabaek, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 1525 (1975). ~oD. Levy and T. Trosper, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2404 (1975). 11D. Levy, Biochim. Biophys. Acta 332, 329 (1973). I~B. E. Haley, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2250 (1975). 1~L. W. Yielding, W. E. White, and K. L. Yielding, Fed. Proc., Fed. Am. Soc. Exp. Biol. 34, Abst. No. 2084 (1975).
[21]
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283
mones, haptens, and other biologically active ligands to their receptor sites on maeromolecules, subcellular organelles, and intact cells and tissues, we shall describe only our experience with Scheme I in this treatise on immobilization techniques. We have demonstrated the usefulness of FNAB and various related stepwise cross-linking reagents containing the nitrophenylazide photoreactive group for the thermochemical preparation of dark-stable photoreactive derivatives of various carrier materials. These photoreactive carrier derivatives may be prepared, washed, and stored wet or dry at ambient temperatures in the dark. The user need merely choose the preferable carrier derivative, mix it in water with his subject enzyme at the temperature and pH chosen for optimizing the enzyme stability and/or interaction with the carrier, and execute the covalent coupling by mere illumination with visible light. The photoimmobiliz~d enzyme product may then be processed and used by the usual procedures. Materials
Essentially all the hydrophilic carrier materials currently being used for enzyme immobilization are expected to be compatible with the photoimmobilization process. Suitable azidonitrophenyl (ANP) derivatives may be coupled to these carriers, in most cases by the same thermochemical reactions used to couple these carriers to enzymes. Approximately 15 ANP derivatives differing in the length, chemical character, and mode of coupling of the extender arm between the polymer backbone and the ANP group have been prepared and demonstrated to be useful for the photoimmobilization of enzymes. These derivatives are based on five basic materials, which are commercially available: controlled-pore glass (aminoalkyl, with and without zirconium), cellulose (aminoethyl and carboxymethyl), Sephadex (carboxymethyl), agarose (aminopropyl, aminohexyl, and aminoethylaminopropyl), and polyacrylamide (aminoethyl). Most other natural and synthetic polymers useful as enzyme carriers are expected to be compatible with this Scheme I process. Scheme II is expected to be preferable for the coupling of enzymes to carriers that are effective competitors to the enzymes as targets for the photochemical aryl nitrene coupling (e.g., insoluble proteins, such as collagen, and carriers with a high aromatic content, such as polystyrene). Fluoro-2-nitro-4-azidobenzene ( F N A B ) . This thermophotochemical bifunctional reagent is now commercially available (e.g., FNPA from ~4H. Kiefer, J. Lindstrom, E. S. Lennox, and S. J. Singer, Proc. Natl. Acad. Sci. U.S.A. 67, 1688 (1970). 15B. A. Winter and A. Goldstein, Mol. Pharmacol. 6, 601 (1972). le U. Das Gupta and J. S. Rieske, Biochem. Biophys. Res. Commun. 54, 1247 (1973). !, j. A. Katzenellenbogen, Annu. Rep. Med. Chem. 9, 222 (1974).
284
IMMOBILIZATION TECHNIQUES
[21]
Pierce Cat. No. 23700, or ICN-K&K Cat. No. 28064). It may be prepared from 4-fluoro-3-nitroaniline by the diazotization-azide substitution methodology described by Fleet, Knowles, and PorterY 4-Azido-2-nitrophenyl-aminoalkylcarboxyl derivatives may be prepared by allowing FNAB to react with the chosen aminocarboxylic acid compound (e.g., glycine, fl-alanine) in dimethyl sulfoxide according to Levy, 11 in aqueous ethanol with Na~CO3 according to Fleet, ~ or in aqueous ethanol with Ca (OH)2 according to the procedure used in our laboratory. 4 ANP-aminoalkylamine derivatives may be obtained by allowing FNAB to react with equimolar or excess diamines (e.g., ethylenediamine,
1,3-diaminopropanol).4 Other thermochemically functional groups more useful for coupling to the particular carrier material of choice may be expected to be substituted onto the photoreactive ANP group by these or other known ¢hermochemical reaction procedures. Procedures In the limited space available for this introduction to photoimmobilization methodology, the use of a commercially available thermophotochemical bifunctional reagent (FNAB) with commercially available aminoalkyl carrier materials will be described. Only Scheme I, the preparation and use of dark-stable, photoreactive carrier derivatives for the gentle and facile photoimmobilization of enzymes, will be described in detail. Step 1. Dark Reaction. Thermally stable photoreactive derivatives of aminoalkyl matrix or carrier materials (ANP-AAM) may be prepared by allowing FNAB to react in the dark or in dim light with a suspension of an aminoalkyl matrix (AAM) or carrier material in alkaline aqueous ethanol. FNAB exhibits tbermochemical reactivity properties for fluoride displacement similar to those of Sanger's reagent (fluorodinitrobenzene), the latter being significantly more reactive. The azide group of FNAB appears to be thermochemically stable in many solvents at temperatures up to at least 50 ° and pH up to at least 10.5. The useful conditions (except for low light intensity) for the FNAB -~ AAM reaction, and subsequent processing and storage of the ANP-AAM products, are expected to be subject to wide variation. A specific set of process conditions that are expected to be useful for a large number of aminoalkyl carrier products is described below. The aminoalkyl carrier material (e.g., aminoethyl cellulose, aminoalkyl agarose, aminoalkyl glass) is suspended in and equilibrated with an aqueous alkaline buffer (e.g., 0.5 M borate, pH 9.5--10). The washed
[21]
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equilibrated material is recovered as a packed bed of gel or a slurry of particles. FNAB of sufficient quantity to give the desired molar ratio over carrier amino groups (a range of 1--3 is usually satisfactory) is dissolved in ethanol of two times the volume of the aqueous carrier suspension or gel and the two are mixed together. This and subsequent exposure of the photochemical materials prior to photolysis (step 2) is executed in dim daylight or darker. The reaction flask is stoppered and the contents mixed at 37°-40 ° for 16-64 hr. The reaction mixture solid is then separated by filtration or centrifugation, washed sequentially with 95% ethanol until the washings are colorless, with 1 M NaC1 in H20 or a buffer of the pH desired for the planned photoimmobilization use, then with this buffer or with deionized water. If the material is to be dried for storage before use, the fibrous or particulate materials should be washed finally with H20, the gels with ethanol. These washed derivatives may then be dried under vacuum at room temperature and stored in the dark at ambient temperature until used. Step 2. Photoimmobilization Procedure. The reaction conditions useful with these photoreactive carrier materials are subject to large variation without harm to the photochemical reaction. Such variables as pH, temperature, and ionic strength may be chosen for the optimum activity stability of the enzyme to be immobilized. The reaction rate depends mostly upon the intensity of illumination with visible light. Satisfactory illumination, mixing, temperature control, and measurement of the N2 evolved may be obtained in an illuminated Warburg apparatus. Although the photochemical immobilization reaction can be activated to completion in several hours by a regular 200 or 300 W tungsten filament light bulb, or a focused microscope illuminator, still better light intensity at the target and improved light-to-heat ratio can be obtained with parallel-beam tungsten-halide projector lamp bulbs [e.g., Eumig 711R (EU 1172) 12 V, 100 W, reflector lamp bulb] mounted on the manometer-flask carrier for movement with the target flask during the motion for sample stirring {Fig. 2). tl/2 of N2 evolution in photoimmobilizations with this illuminated Warburg setup, operated at 10 V on the lamp, usually fell in the range of 20-40 min. The photoreactive carrier material is equilibrated to the pH chosen for the enzyme and washed with water to remove organic buffer ions if any have been used. The carrier material is suspended to form a slurry thin enough for efficient mixing, with water or aqueous inorganic buffer containing enzyme up to 10% the dry weight of the carrier material. The system is then flushed with N~ before illumination as a precaution against possible O2 interference with the desired nitrene reactions.
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Fia. 2. Modified Warburg apparatus for photochemical coupling reactions. The parallel-beam tungsten-halide projector lamps are mounted on the manometer platform of a Bronwill Warburg Apparatus Model UV so that the light beams may be directed continuously through the clear Plexiglas temperature control bath container into the Warburg flasks during reciprocal motion in the water bath. An example of a useful experimental protocol follows: 20-25 mg dry of A N P carrier material previously equilibrated to p H 4.5-5.0 is placed in an approximately 5-ml Warburg flask without a center well. H~O (usually 0.5-2 ml, depending upon the type of carrier material being used) containing 2-3 mg of invertase (fl-fructofuranosidase from bakers' yeast) is added, and the flask is attached to the Warburg manometer. The sample is then placed on the W a r b u r g apparatus with the flask immersed
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in ice-water. The manometer-flask system is flushed with a stream of N2 for a few minutes while temperature equilibration is occurring. Work up to this point is done in dim daylight. The flask-manometer system is then closed, a zero-time manometer reading is taken, and illumination with mixing motion is initiated and continued until N~ evolution ceases, as indicated by the periodic manometer readings. When the parallel-beam 12 V, 100 W projector bulb is operated at 10 V, the reaction is usually continued from 2 to 5 hr illumination time. Further processing of the immobilized enzyme need be no different from that of enzyme immobilized by other methods. Satisfactory photoimmobilization without measurement of the rate or extent of N._, evolution may be obtained by placing such an enzyme ~ANP-AAM reaction mixture in an approximately 5-ml round-bottom flask blown from glass tubing. The mouth is covered with a gum rubber dropper bulb, and the flask is flushed with N._,or other inert gas. The flask is then placed in a bath for temperature control and stirred magnetically for about 16 hr illumination with a focused 8 V, 5 amp microscope illuminator light passing through about 0.5 cm of 1 M aqueous sodium nitrite in a petri dish. Discussion
Many advantages are obvious in the use of stepwise cross-linking reagents for coupling carrier materials to enzymes and other biochemicals. The high level of reaction independence of the two reactive groups of certain thermophotochemical bifunctional reagents allows the chemist to prepare dark-stable photoreactive derivatives of his chosen carrier material and to further "tailor" it (e.g., modify its ionic character, add ligands, and/or otherwise modify the "microenvironment" of the carrier surface) for the particular enzyme system(s) to be immobilized. These dark-stable photoreactive carrier materials can be removed from storage and used in the facile covalent coupling of enzymes by merely mixing them together in an aqueous slurry at the temperature and pH chosen for the enzyme and illuminating the mixture with visible light. Since most enzymes are transparent to light of wavelengths required to activate the nitrophenyl azide, and the aryl nitrene thus produced exhibits a relatively low level of specificity for the target amino acid, the enzyme molecule is expected to be covalentty immobilized with exceptionally little stress by this process. The photoimmobilized enzyme product is thus expected to exhibit relatively high levels of catalytic capacity and specific activity. Otherwise its properties are found to be quite similar to those of
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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