Incorporation of Poly(ethylene glycol)-Proteins into Polymers - ACS

Chapter 9

Incorporation of Poly(ethylene glycol)-Proteins into Polymers

Downloaded by PURDUE UNIV on August 31, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch009

Janice L. Panza, Keith E. LeJeune, Srikanth Venkatasubramanian, and Alan J. Russell Department of Chemical Engineering and Center for Biotechnology and Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261 Combining polymer chemistry and enzyme catalysis can be achieved by using enzymes as co-monomers in polymerization reactions. This approach requires the protection of the enzyme from the polymerization environment, which is often organic rather than aqueous. In this chapter we describe how the modification of proteins with polyethylene glycol derivatives can prepare an enzyme for covalent incorporation into a polymer during the synthesis of that polymer. First, the polyethylene glycol serves to solubilize the enzyme in the polymerization environment. Second, the use of heterofunctional polyethylene glycols containing monomeric functionalities serves to involve the protein in the polymerization itself. Thus far, subtilisin (a key industrial enzyme used in biological detergents), thermolysin (the enzyme used for biocatalytic synthesis of aspartame) and chymotrypsin (one of the most studied enzymes commercially available) have been incorporated successfully into acrylic based materials. Enzymes are proteins which catalyze a diverse range of reactions. Enzyme catalysis is widely studied because of the substrate selectivity and catalytic efficiency inherent to enzymatic processes. These attributes, along with regioselectivity and stereoselectivity have made enzymes attractive catalysts in synthetic chemistry (7), having a broad range of potential applications. In spite of the potential of biocatalysis, short catalytic lifetime and catalyst thermal sensitivity can limit process productivity. Catalyst recovery and re-use is often not straightforward due to the solubility of enzymes in predominately aqueous reaction media. Organic solvents have been examined as potential media for enzymatic processes in order to minimize these drawbacks; however, altered specificity and significant decreases in activity are often observed (2). The most important factor in the successful application of an enzyme is to immobilize the enzyme to an insoluble solid support. Enzyme immobilization techniques are being continually developed and refined for applications in a wide variety of areas including the food, chemical synthesis, agricultural, pharmaceutical, and polymer industries (7). The benefits of enzyme immobilization include increased enzyme stability, decreased thermal sensitivity, and enhanced process versatility (3). For an immobilization technique to be practical, the immobilized enzyme should bind irreversibly (covalently) to the support matrix, 134

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retain a significant degree of native specific activity, and maintain structural stability in the presence of substrate, product, and reaction solvent. Enzyme immobilization has been performed on a wide variety of support materials, including alumina pellets (4), trityl agarose (5), and glass/silica beads (6). Organic polymers also make excellent enzyme support materials due to their structural flexibility and solvent resiliency. Numerous polymers, including nylons (7,8), acrylates (9), and several copolymer blends (10,11) have been utilized as effective supports. While the attachment of proteins to polymers is commonplace, it is still limited by the need for an accessible polymer architecture. Consider, for example, how one could immobilize a protein onto an ultrafiltration membrane where the pore size of the membrane was smaller than that of the protein. In such an instance, the only way to introduce a protein into the membrane would be during the polymerization process itself. Protein immobilization during polymer synthesis is also attractive because it enhances the possibility for the formation of multiple bonds between the protein and the growing polymeric support. Such multi-point attachment can dramatically stabilize many proteins (12,13). While there are many advantages associated with a single step immobilization and polymer synthesis, there is also a major limitation. Proteins are generally not soluble in the organic solvents which are necessary for effective polymer synthesis. Thus, if we are to take advantage of the opportunities which arise from multi-point attachment, we must address the question of how to solubilize proteins in organic media, and how to facilitate a reaction between the growing polymer chain in the solvent and the surface of the protein. The low solubility of proteins in organic solvents can be overcome by covalently attaching a long chain amphiphilic molecule to the surface of the protein. Polyethylene glycol (PEG) has been shown to be an ideal molecule for such surface modification strategies (14). PEG-modified enzymes demonstrate dramatically enhanced enzyme solubility in organic solvents, close to native activity and specificity, and enhanced thermal stability (14). Confusion can arise about immunogenic effects because the addition of PEG tails to small hapten molecules increases immunogenicity by increasing molecular weight. In fact, there is a delicate balance between the degree of PEG modification, and the effect that the alteration in structure will have on the protein's properties. Since a PEG-protein can be dissolved in an organic solvent while retaining its native structure, if the enzyme were also reactive (as a co-monomer) in a polymerization reaction, the enzyme would potentially become intrinsically coupled to the polymeric material. Undoubtedly, the number of cross-links would be related to the number of reactive sites on the soluble protein. A "PEGylated" enzyme would not normally be reactive in a polymerization reaction. The most accessible and reactive groups on the protein surface will probably have reacted with the PEG itself, and the P E G will undoubtedly reduce access of many molecules to the surface of the protein. Indeed, it is this very ability of PEG to "protect" the surface of the protein that makes it so attractive an agent for increasing enzyme solubility in non-aqueous media. In order to react PEGylated proteins with monomers in organic solution a heterofunctional P E G must be employed. One end of the derivatizing PEG must be designed to couple to a protein, while the other should be a reactive group able to couple to a polymer. This approach is summarized in Figure 1, where the reactive PEG is capped with an NHS-ester (to react with the protein), and an allyl group (to incorporate the resulting PEGylated protein into an acrylic polymer). This example is particularly interesting because it demonstrates the additional opportunity to "self polymerize" the PEG-protein in the absence of additional monomers (15). There are many approaches to modifying proteins with PEG, and most would be applicable to use with a heterofunctional PEG designed for application in Figure 1. Particularly popular approaches to P E G modification are outlined throughout this book, and include, cyanuric chloride activation of PEG hydroxyl groups (16,17), hydroxyl activation through use of phenylchloroformates, nitrophenylcarbonate (18), In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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and succinimidylsuccinates (79). Some approaches have drawbacks, such as the inactivation of enzymes with thiol functionalities in close proximity to the active site by cyanuric chloride (18). For the most part, all these strategies react a PEG chain with the free amino group of lysine residues on accessible surfaces of the protein. Once again, the chemistry and methods for such reactions are detailed elsewhere in this text. In this chapter we will review the growing body of literature concerning the synthesis, activity, and stability of PEG-polymer-protein materials.


Protein Polymer Synthesis Protein polymer synthesis can be performed by two methods. The first is a two step process where the enzyme is modified with PEG containing a polymerizable functional group at one terminus, followed by polymerization of the PEG-modified enzyme with other polymerizable monomers. This results in a polymer with a low percentage of incorporated protein (20). An example of this procedure is shown in Figure 2. Both subtilisin Carlsberg and thermolysin have been modified by this method (9,20). The PEG-protein conjugate is soluble in a variety of solvents (up to 5 mg/ml in chloroform, methylene chloride, 1,1,1-trichloroethane, carbon tetrachloride, toluene, benzene, dimethyl sulfoxide, tetrahydrofuran, dioxane, or acetonitrile), including the ones used for the polymer synthesis (chloroform, carbon tetrachloride). For modified subtilisin to remain in solution in tetrahydrofuran and acetonitrile, slight heating is required. The modified enzyme is not soluble in solvents such as hexane, acetone, ethyl ether and isoamyl alcohol. The second method is a one step process where both the enzyme and the P E G molecule contain polymerizable functional groups which can be used to directly polymerize the protein and the PEG molecule together. This results in a hydrogel of PEG and protein (27). The enzyme α-chymotrypsin has been modified by this method (27,22). PEG-Enzyme Reactions Many studies have been performed on PEG-modification of proteins due its diverse biotechnological applications (14). In the case of PEG-protein-polymer synthesis, PEG is being used in order to increase the solubility of a protein in organic solvents (23,24). Other polymers, such as polystyrene, have also been used to increase the solubility of proteins in organic solvents (25). In general, PEG itself is first modified with a monomeric functional group at one end of the molecule to form an activated PEG. This functional group is a reactive terminal group that can react with a functional group on a protein. The reaction will most likely occur with an ε-amino group of a lysine residue on the protein (26). One example of an activated PEG is PEG-aldehyde. Yang et. al. used PEGaldehyde to modify the enzyme subtilisin Carlsberg (20). Subtilisin Carlsberg is a serine protease, best known for its activity in biological detergents. The annual market for sales of this enzyme in the US is approximately $300 million, and it has been extensively studied for many years. In this study, PEG-monomethacrylate was first converted to an aldehyde by a procedure adapted from Wirth (27). The P E G molecule was then converted to a difunctional PEG, with an aldehyde group at one end and a methacrylate group at the other. The PEG-aldehyde was then used to modify subtilisin by the reaction of the aldehyde with a lysine residue on subtilisin by reductive alkylation. In a second study by Yang et al., both subtilisin and thermolysin were modified with P E G (9). Thermolysin is a metalloendopeptidase. PEG-A, an N hydroxysuccinimide activated polyethylene glycol acrylate, which contained an aery late group at one terminus and an active ester at the other terminus, was used in the modification of the enzymes. PEG-Α reacted with the enzymes by nucleophilic substitution on the lysine residues of the enzymes. In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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Of course, other functional groups can be used to modify the P E G molecule. For example, cyanuric chloride and p-nitrophenol chloroformate have been used to activate the hydroxyl group of the PEG molecule via nucleophilic substitution (18). Both trypsin (28) and subtilisin (26) have been modified with cyanuric chlorideactivated PEG (CC-PEG) andp-nitrophenol carbonate-PEG (NPC-PEG). Many different techniques exist that can be used to verify that a protein has been modified with PEG. Depending on the technique chosen, it can be determined whether, where, and how modification has occurred. A simple test to ascertain modification of a protein with PEG is solubility data. As mentioned before, proteins have low solubility in organic solvents, attachment of PEG molecules increases the solubility of proteins in organic solvents. Therefore, if a PEG-modified enzyme is soluble in an organic solvent in which the unmodified protein was not, modification has been established. The solubilization of the PEGmodified protein can be used to purify the unmodified protein from the modified protein. Khan has speculated that the PEG-modified enzymes are not actually soluble in organic solvents, but they just form aggregates in the solvents that appear optically transparent (29). This does not change the fact that this property can be used to confirm PEG-modification. High pressure liquid chromatography (HPLC) is another useful technique to substantiate PEG-modification. In addition, HPLC analysis can elucidate the degree of modification (what fraction of the protein molecules protein is modified with PEG). The retention times for native, singly-modified, and doubly-modified fractions of protein will vary. A comparison of the peaks for each fraction will indicate the degree of modification of the protein. HPLC can, however, give misleading results under some circumstances (33, 34, 35). Both matrix assisted laser desorption time of flight (MALDI TOF-MS) and electrospray mass spectrometry (ES-MS) are also useful tools with which to determine the degree and yield of a modification strategy. Figure 3 shows the dramatic and detailed information that ES-MS and M A L D I - M S in practice can provide. These methods cannot, however, distinguish where on the protein the modification has taken place, and as with all analysis tools they must be used judiciously and in combination with all the tools available for characterization (such as capillary electrophoresis) (36,37,38). The modification could take place at any lysine residue or the amino terminus of the protein (20). It is very unlikely that the modification occurs at the same position on each molecule. Because there is no convenient method to separate modified protein molecules based on where the modification is located, the properties of PEG-modified proteins are actually properties of a heterogeneous mixture. Protein staining techniques such as the Bradford method, SDS-polyacrylamide gel electrophoresis, gel filtration chromatography, and amino acid analysis are also used in the analysis of the PEG-proteins. The Bradford method can determine the amount of protein in a sample (30). This assay involves the binding of a dye to the protein causing a shift in absorption which can be measured spectrophotometrically. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography can be used to separate the modified fraction of protein based on size. Activity and Stability Activity and stability studies have been performed on many PEG-modified enzymes, prior to utilization in a given system or polymer. The primary purpose of these studies has been to investigate the stability of the enzyme against both temperature and pH before and after PEG modification, and to assess the role of the chemistry of modification on the eventual properties of the modified material. For example, activity studies were performed on PEG-modified subtilisin in aqueous solutions, organic solutions, and a mixture of the two (20,26). It was found that although PEGmodified enzymes showed less activity than the native subtilisin in the aqueous environments, the modified enzymes still retained substantial activity. In addition, In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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the PEG-modified subtilisin was active in organic solvents, thus the modified enzyme should remain active when solubilized in the organic solvents necessary to incorporate it into a polymer (20). The stability of some PEG-modified enzymes, particularly PEG-subtilisin, have also been shown to be markedly higher than the native forms of the enzymes with time and against temperature and pH (20,26,28). For subtilisin, one hypothesis which explains this increase in stability in buffer is a reduction in the rate of autolysis of the subtilisin via steric hindrance caused by the large P E G molecules (20,26). Alternatively, intrinsic changes in the stability of the enzyme may occur upon modification (26). A third hypothesis is that the stability of PEG-modified proteins may result from a highly hydrogen-bonded structure around the enzyme caused by the long PEG chains (28). Also, by attaching PEG molecules to an enzyme, the enzyme may become less flexible in aqueous solution which would make the enzyme less likely to unfold (31). The stability of PEG-modified enzymes is significant because the stability of the polymerized enzyme will most likely be more similar to that of the modified enzyme than the native enzyme (20). Polymerization Once a protein has been modified with PEG, it is now soluble in many organic solvents in which it was not soluble in its native state. In addition, an enzyme modified with PEG retains its activity in water, organic solvents, and some mixtures of the two. Usually an enzyme would have to be incorporated into a polymer through a polymerization in an aqueous solution due to the limited solubility of the protein in organic solvents. However, modification with PEG allows the choice of many new polymer systems since the enzyme is now soluble in organic solvents. Synthesis Enzymes have been incorporated into polymers by two methods. The first involves polymerizing the enzyme modified with PEG molecules containing polymerizable functional groups at the PEG terminus with other monomers and crosslinkers. A n example of this is the polymerization of PEG-modified subtilisin with methyl methacrylate (9,20). The PEG molecules contained a terminal monomethacrylate that could undergo polymerization in the presence of monomers of methyl methacrylate, a crosslinker solution of trimethylolpropane trimethacrylate, and an initiator. Free radical polymerization of the mixture resulted in the incorporation of PEG-modified subtilisin in an acrylate polymer. An alternative method uses PEG and proteins which have been modified with polymerizable functional groups, enabling co-polymerization. Fulcrand et. al. used this method to cross-link PEG chains and α-chymotrypsin (21,22). First, both P E G and α-chymotrypsin were separately modified with acryloyl chloride to give acrylated derivatives of both PEG and α-chymotrypsin. These two reactants were allowed to co-polymerize along with bisacryloyl PEG as a crosslinker, the monomethylether of monoacrylated PEG as the matrix agent, and free radicals. The result was achymotrypsin combined with PEG in a polymeric hydrogel. Although both procedures led to an enzyme being incorporated in a polymer network, there is a distinct difference between the two polymerizations. Polymerization of subtilisin into the acrylate polymer took place in an organic solution of carbon tetrachloride and chloroform. The polymerization of the achymotrypsin into the PEG hydrogel took place in a aqueous solution of borate buffer. One of the major benefits of incorporating PEG-modified enzymes into polymers, as mentioned before, is the ability to have to reaction occur in organic solvent. This enables solvent engineering of polymer properties in order to design a polymer with appropriate properties that fit a given application. By keeping the protein content at less than 2 % by weight, the resulting polymer will have the same In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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properties as the polymer without incorporated protein (20). For example, if diffusion limitations of a substrate are a concern, a polymer system that will yield a highly porous material can be used to incorporate the enzyme. In addition, a variety of shapes and morphologies of the polymers can be fabricated, including beads, fibers, or membranes (20). There are three different ways that a protein can be immobilized in a polymer: covalent attachment, adsorption to the surface, or entrapment within pores (9). Since covalent attachment of the PEG-modified enzyme to polymer during synthesis is desired, the biopolymers are usually rinsed extensively after polymerization to remove adsorbed or trapped protein. The polymer may also be crushed or broken before rinsing. By assaying the rinsates for residual activity, the efficiency of the incorporation can be determined. Downloaded by PURDUE UNIV on August 31, 2014 | http://pubs.acs.org Publication Date: August 5, 1997 | doi: 10.1021/bk-1997-0680.ch009

Characterization The protein content in the polymer can be determined using the Bradford method as mentioned previously (30). Transmission electron microscopy or scanning electron microscopy can be used to confirm the size and shape of the pores within the polymer and morphology differences between polymers with incorporated proteins and polymers without proteins. Finally, BET analysis can be used to estimate the surface area of the polymers. Activity and Stability of Biopolymers Once PEG-modified enzymes have been incorporated in a polymer system, the biopolymer must be tested for activity and stability of the enzyme. It has been shown the PEG-modified enzymes retain activity and stability, but incorporation of the PEGmodified enzymes into polymers requires the enzyme to be exposed to harsh environments. For the most part, PEG-modified enzymes retain considerable activity when incorporated into polymers. Yang compared the activity of native enzyme to the immobilized enzyme, and although reduced, the immobilized enzyme did exhibit approximately 12 % activity retention (activity reported as kcat/Km) (9). It is not surprising that the activity of the biopolymer was reduced in respect to the native enzyme because immobilization may decrease the accessibility of the substrate to the active site. In addition, the hydrophobicity of the polymer may attract hydrophobic substrates into hydrophobic areas of the polymer, away from the active sites of the immobilized enzymes (9). Fulcrand used the biopolymer hydrogel to access esterase activity in water, demonstrating that α-chymotrypsin retains significant activity in aqueous solutions (21). Stability In order to assess the stability of the biopolymer in the presence of substrate, Yang designed and constructed a continuous flow reactor (9,20). The flow cell apparatus consisted of substrate being first pumped into the reference cell of spectrophotometer; second, to a stirred reactor containing the biopolymer; third, to the sample cell of the spectrophotometer; and finally to a waste collection vessel. A schematic of the flow cell apparatus is shown in Figure 4. They tested the polymer-incorporated subtilisin in the system and found that the biopolymer remained active for over 100 days, at which time the experiment was ended. This established that enzymes covalently immobilized in a polymer have increased stability. The enhanced stability of the enzyme is likely the result of a number of factors. By incorporating the enzyme into a polymer, the enzyme molecules are probably more rigid (9). That is, the enzyme is less likely to be denatured by unfolding since it is held in place by the covalent immobilization. Guisân has shown that stability of In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.



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Figure 4. Flow cell apparatus for determination of the activity and stability of protein-polymers (Adapted from ref. 20.).


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enzyme bound to supports is enhanced by multi-point enzyme-support attachments (52). In addition, improved stability may be due to decreased autolysis of the enzyme because of the steric hindrance of the PEG molecules and polymer network (9).


Organic Solutions PEG-modified enzymes are soluble and display activity in organic solvents. Incorporating the PEG-modified enzymes into polymers may lead to new applications. One of the advantages of PEG-modified enzyme incorporation into polymers is its activity in organic solutions. When polymerized with P E G in a hydrogel, achymotrypsin can be used to catalyze the synthesis of peptides in organic solvents (21,22). In addition, PEG-modified subtilisin has displayed activity in organic solvents (9,20). It should be noted that both enzymes required some water in the organic solvents in order for the enzymes to become active (9,22). The polymer may protect the enzyme from denaturation by the solvent due to the covalent attachment of the enzyme to the polymer, thereby allowing the enzyme to remain active. In addition to remaining active in organic solvents, PEG-modified enzymes incorporated into polymers also show increased stability in organic solvents over their native forms. Even under very harsh conditions, polymer-incorporated subtilisin retained its activity after many cycles of exposure (9). Since the biopolymer was able to withstand the harsh conditions of the recycling experiments, the biopolymers will probably be able to function in most organic solvents. Conclusions PEG-modified proteins have been incorporated into polymers. One method first modifies an enzyme with a heterofunctional PEG molecule, that contains an activating group at one terminus to covalently attach to the enzyme and another functional group at the other terminus that is capable of polymerizing. The next step is to polymerize the PEG-modified protein with monomer and crosslinkers to form the polymer. A second method modifies both the enzyme and P E G molecules separately with polymerizable functional groups, then initiates a co-polymerization between them. Both methods result in biopolymers that are active and stable in aqueous and organic solutions. As a result of the greatly enhanced stability of the enzymes, the productivity of the biocatalysts is dramatically enhanced. Literature Cited 1. Margolin, A. L. Chemtech 1991, March, 160-167. 2. Dordick, J. S. Applied Catalysis 1991, 1, 1-51. 3. Martinek, K.; Klibanov, A. M.; Goldmacher, V. S.; Berezin, I. V. Biochim. Biophys. Acta 1977, 485, 1-12. 4. Vasudevan, P. T.; Thakun, D. S. Appl. Biochem. Biotech. 1994, 49, 173-189. 5. Caldwell, S. R.; Raushel, F. M. Biotechnol. Bioeng. 1991, 37, 103-109. 6. Vasudevan, P. T.; Weiland, R. H. Biotechnol. Bioeng. 1993, 41, 231-236. 7. Chellapandian, M.; Sastry, C. A. Bioprocess Engineering 1994, 11, 17-21. 8. Caldwell, S. R.; Raushel, F. M. Appl. Biochem. Biotech. 1991, 31, 59-73. 9. Yang, Z.; Mesiano, A. J.; Venkatasubramian, S.; Gross, S. H.; Harris, J. M.; Russell, A. J. J. Am. Chem. Soc. 1995, 117, 4843-4850. 10. Nguyen, A. L.; Luong, J. Biotechnol. Bioeng. 1989, 34, 1186-1190. 11. Hoshino, K.; Taniguchi, M.; Netsu, Y.; Fujii, M. J. Chem. Engr. Japan 1989, 22, 54-59. 12. LeJeune, Κ. E.; Russell, A. J. Biotechnol. Bioeng. 1996, 51, 450-457. 13. LeJeune, K. E.; Frazier, D. S.; Caranto, G. R.; Maxwell, D. M.; Amitai, G.; Russell, A. J.; Doctor, B. P. Proc. Med. Def. Biosc. Rev. 1996, June, 1-8. In Poly(ethylene glycol); Harris, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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14. Harris, J. M. In: Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenium Press: New York, 1992. 15. Andreopoulos, F. M.; Deible, C. R.; Stauffer, M. T.; Weber, S. G.; Wagner, W. R.; Beckman, E. J.; Russell, A. J. J. Am. Chem. Soc. 1996, 118, 62356240. 16. Abuchowski, Α.; Van Es, T.; Palczuk, Ν. C.; Davis, F. F. J. Biol. Chem. 1977, 252, 3578. 17. Imoto, T.; Yamada, H. In Protein Function: A Practical Approach; Creighton, T. E., Ed.; IRL Press: Oxford, 1989, 247-277. 18. Veronese, F. M.; Boccu, R. L. E.; Benassi, C. Α.; Schiavon, O. Appl. Biochem. Biotech. 1985, 11, 141-152. 19. Buckamann, A. F.; Morr M.; Johansson, G. Makromol. Chem. 1982, 182, 1379. 20. Yang, Z.; Williams, D.; Russell, A. J. Biotechnol. Bioeng. 1995, 45, 10-17. 21. Fulcrand, V.; Jacquier, R.; Lazaro, R.; Viallefont, P. Tetrahedron 1990, 46, 3909-3920. 22. Fulcrand, V.; Jacquier, R.; Lazaro, R.; Viallefont, P. Int. J. Peptide Protein Res. 1990, 38, 273-277. 23. Inada, Y.; Takahashi, K.; Yoshimoto, T.; Ajima, Α.; Matsushima, Α.; Saito, Y. Trends Biotechnol. 1986, 6, 190-194. 24. Yoshinaga, K.; Ishida, H.; Hagawa, T; Ohkubo, K. In: Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenium Press: New York, 1992, 103-114. 25. Ito, Y.; Fujii, H.; Imanashi, Y. Biotechnol. Prog. 1993, 9, pp 128-130. 26. Yang, Z.; Domach, M.; Auger, R.; Yang, F. X.; Russell, A. J. Enzyme Microb. Technol. 1996, 18, 82-89. 27. Wirth, P.; Souppe, J.; Tritsch, D.; Biellmann, J. F. Biorg. Chem. 1991, 19, 133-142. 28. Gaertner, H. F.; Puigserver, A. J. Enzyme Microb. Technol. 1992, 14, 150155. 29. Khan, S. Α.; Hailing, P. J Enzyme Microb. Technol. 1992, 14, 96-100. 30. Bradford, M. M. Anal. Biochem. 1976, 72, 248-254. 31. Baillargeon, M. W.; Sonnet, P. E. Ann. NY Acad. Sci. 1988, 542, 244-249. 32. Guisán, J. M. Enzyme Microb. Technol. 1988, 10, 375-382. 33. McGoff, R.; Baziotis, A.C.; Maskiewicz, R. Chem. Pharm. Bull.,1988, 36, 30793091. 34. Kurfurst, M.M., Anal. Biochem., 1992, 200, 244-248. 35. Chowdhury, S.K.; Doleman, M.; Johnston, D.J., J. Am. Mass. Spectrom., 1995, 6, 478-487. 36. Montaudo, G.; Montaudo, M.S.; Puglisi, C.; Samperi, F., Rapid Commun. Mass Spectrom., 1995, 9, 453-460. 37. Cunico, R.L.; Gruhn, V.; Kresin, L.; Nitecki, D.E.; Wiktorowicz, J.E., J. Chromatogr., 1991, 559, 467-477. 38. Bullock, J.; Chowdhury, S.; Johnston, D., Anal. Chem., 1996, 68, 3258-3264.


Incorporation of Poly(ethylene glycol)-Proteins into Polymers - ACS

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