Smart PEGylation of Trypsin - American Chemical Society


Jul 12, 2010 - Fraunhofer Institute for Applied Polymer Research. ‡ Max-Planck Institute of Molecular Plant Physiology. Biomacromolecules 2010, 11, ...
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Smart PEGylation of Trypsin Zoya Zarafshani,† Toshihiro Obata,‡ and Jean-Franc¸ois Lutz*,† Research Group Nanotechnology for Life Science, Fraunhofer Institute for Applied Polymer Research, Geiselbergstrasse 69, Potsdam-Golm 14476, Germany, and Max-Planck Institute of Molecular Plant Physiology, Potsdam-Golm 14476, Germany Received May 7, 2010; Revised Manuscript Received June 22, 2010

Thermoresponsive oligo(ethylene glycol)-based copolymers were investigated for trypsin conjugation. These copolymers have been synthesized by atom transfer radical polymerization of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) with oligo(ethylene glycol) methyl ether methacrylate (OEGMA475, Mn ) 475 g · mol-1) at 60 °C in the presence of copper(I) chloride and 2,2′-bipyridyl. Two different ATRP initiators, containing succinimidyl ester moieties, were tested, namely, N-succinimidyl-2-bromopropionate and N-succinimidyl-2bromoisobutyrate. In both cases, ATRP afforded well-defined polymers with a narrow molecular weight distribution and controlled chain-ends. However, the efficiency of initiation of the two initiators was lower than 1 and therefore the formed polymers exhibited a higher than expected mean degree of polymerization. Nevertheless, all types of polymers could be conjugated to trypsin. The conjugation reaction was performed in borax-HCl buffer. Sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE) indicated that polymer/enzyme conjugates were obtained in all cases. However, (co)polymers initiated by N-succinimidyl-2-bromopropionate led to the best conjugation results. The formed P(MEO2MA-co-OEGMA475)-trypsin conjugates were found to be thermoresponsive and moreover exhibited a higher enzymatic activity than unmodified trypsin.

Introduction The modification of proteins with synthetic polymer chains (i.e., polymer bioconjugation) is a topic of prime importance in polymer science and bioscience.1 Indeed, synthetic watersoluble polymer chains may confer additional properties to functional native proteins. An archetypal example of this is the modification of proteins with linear poly(ethylene glycol) (PEG) chains or so-called PEGylation.2 In this approach, short functional PEG chains are reacted with accessible functional groups (e.g., thiols or primary amines) located on the protein surface. The resulting PEGylated proteins exhibit generally an enhanced aqueous stability. Additionally, bioinert PEG chains protect proteins from degradation in vivo. Thus, PEGylation is extensively used nowadays in protein therapeutics.2 However, although very useful, standard linear PEG remains on the whole a passive macromolecule, in the sense that its properties are not really adjustable. In recent years, new approaches have been developed for grafting “smarter” polymers, such as thermoresponsive or pH-responsive polymers, on proteins.3,4 In particular, the pioneer works of Hoffman, Stayton, and co-workers have opened interesting perspectives in this area of research.5,6 For instance, these authors demonstrated that poly(N-isopropylacrylamide) (PNIPAM) is an appealing macromolecule for modifying proteins. This thermoresponsive polymer exhibit a lower critical solution temperature (LCST) at approximately 32 °C in aqueous medium and therefore its solubility can be conveniently switched in between room and body temperature.7 PNIPAM conjugation is, for example, particularly relevant in biocatalysis. For instance, PNIPAMenzyme conjugates can be easily recycled via thermoprecipitation.8 Alternatively, PNIPAM can be used to control the catalytic activity of some enzymes.9 Thus, following Hoffman * To whom correspondence should be addressed. E-mail: [email protected] † Fraunhofer Institute for Applied Polymer Research. ‡ Max-Planck Institute of Molecular Plant Physiology.

and Stayton’s work, a number of authors studied routes for grafting PNIPAM on enzymes or on globular proteins. In particular, it was demonstrated that controlled radical polymerization techniques such as atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer polymerization (RAFT) are very relevant methods for PNIPAM conjugation.10,11 These methods allow, for example, the synthesis of defined heterotelechelic polymers, which can be reacted with the proteins.12-16 Alternatively, NIPAM can be directly polymerized from the protein surface.17,18 Still, PNIPAM has some drawbacks. For instance, in the specific context of protein technology, it should be noted that the amide functions of PNIPAM may form nonspecific interactions with proteins.19,20 Hence, some alternatives to PNIPAM have been proposed in recent years. To date, the most studied candidates are elastin-like polypeptides,21 poly(alkyl oxazolines),22,23 and poly(meth)acrylates with short oligo(ethylene glycol) side chains.24-27 Our group investigated extensively the latter class of polymers. In particular, it was demonstrated that the thermoresponsive behavior of these polymers can be precisely tuned via a controlled copolymerization process. For example, we described that the atom transfer radical copolymerization of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) with oligo(ethylene glycol) methyl ether methacrylates (e.g., OEGMA475, Mn ) 475 g · mol-1 or OEGMA300, Mn ) 300 g · mol-1) leads to the formation of well-defined thermoresponsive copolymers with sharp reversible phase transitions in water.28-31 Moreover, these polymers are bioinert and biocompatible.32-34 Thus, these polymers have been already used for developing a broad range of stimuli-responsive materials such as hydrogels,35-37 microgels,38,39 micellar assemblies,40-42 modified inorganic nano-objects,43,44 and switchable surfaces.45-48 However, these thermoresponsive polymers have been barely tested so far for protein conjugation. Theato and co-workers described the synthesis of PMEO2MA-protein conjugates.49,50 Nevertheless, the thermoresponsiveness of the formed biocon-

10.1021/bm1005036  2010 American Chemical Society Published on Web 07/12/2010

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Table 1. Properties of the Copolymers of MEO2MA and OEGMA475 Prepared by ATRPa

P1 P2 P3 P4 P5

I

I/MEO2MA/OEGMA475

conv.b

Mnc[g · mol-1]

Mw/Mnc

Mn,thd[g · mol-1]

fe

cloud pointf[°C]

1 1 1 2 2

1:25:0 1:24.25:0.75 1:22.5:2.5 1:22.5:2.5 1:45:5

0.70 0.62 0.80 0.99 0.86

7400 7080 7700 8100 13400

1.24 1.15 1.19 1.15 1.19

3290 3050 4330 5360 9320

0.44 0.43 0.56 0.66 0.69

30 33 43 41 40

a Experimental conditions: 60 °C; overnight; in ethanol solution (monomer/ethanol ) 1:1.25 (v/v)); [I]0/[CuCl]0/[Bipy] ) 1/1/2, I stands for initiator. Overall monomer conversion measured by 1H NMR from the crude reaction products. c Measured by SEC in THF. d Mn,th ) conversion (188 [MEO2MA]0 + 475 [OEGMA]0)/[I]0. e Initiator efficiency estimated from the SEC data. f Measured in pure deionized water at a concentration of 3 mg · mL-1. b

jugates was not studied in these works. The modification of peptides and proteins with poly(meth)acrylates having long oligo(ethylene glycol) side chains has also been reported.51-58 Yet, these polymers are permanently hydrophilic (i.e., LCST is generally above 85 °C) and therefore behave more or less like conventional linear PEG (i.e., passive PEGylation).24,59,60 In this context, the modification of enzymes or globular proteins with oligo(ethylene glycol)-based thermoresponsive polymers could be very interesting. Indeed, this bioconjugation approach could combine the advantages of both PEG (i.e., hydrophilicity, protection and biorepellency) and PNIPAM conjugation (i.e., controllable thermosensitivity). Such an example of “smart PEGylation” is described in the present article. Thermoresponsive copolymers P(MEO2MA-co-OEGMA475) were grafted on trypsin. This enzyme was selected as a model for this study because its polymer conjugation is particularly important.61 Trypsin is a digestive enzyme, which cleaves protein chains.62 Thus, outside its native environment, trypsin is an unstable macromolecule with a relatively high tendency toward selfdigestion. In laboratory conditions, trypsin should be stored at low temperatures (i.e., -20 °C) and in optimized media. In this context, the modification of trypsin with “smart” biocompatible polymers may offer multiple advantages: (i) water-soluble polymers may enhance the solubility, thermal stability, and the catalytic efficiency of the enzyme, (ii) a controlled thermoprecipitation can be used to recycle the enzyme, and (iii) the thermoresponsive behavior can also be used to control biocatalysis and therefore to prevent self-digestion. For instance, it was already demonstrated that both PEG and PNIPAM conjugation strongly enhance the in vitro behavior of trypsin.63-70 Herein, a series of well-defined copolymers with variable chain lengths and compositions were synthesized and tested for trypsin bioconjugation. The formed conjugates were characterized using SDS-PAGE, turbidimetry, and enzyme activity tests.

Experimental Section Chemicals. 2-(2-Methoxyethoxy)ethyl methacrylate (Aldrich, 95%), oligo(ethylene glycol) methyl ether methacrylate (Aldrich, Mn ) 475 g · mol-1), 2,2′ bipyridyl (Bipy; Fluka, 98%), N-hydroxy-succinimide (Aldrich, 98%), 2-bromopropionyl bromide, 2-bromoisobutyric acid (Aldrich, 98%), N,N′-dicyclohexylcarbodiimide (DCC; Fluka, g99%), buffer solution (pH 9.0 at 20 °C, Borax/hydrochloric acid, Fluka), trypsin from bovine pancreas (type I, Aldrich, protein 90-100%), and NR-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA; Aldrich, g99%) were used as received. Copper(I) chloride (Acros, 95%) was washed with glacial acetic acid to remove any soluble oxidized species, filtered, washed with ethanol, and dried. Synthesis of ATRP Initiator 1 (N-Succinimidyl-2-bromopropionate). This procedure was adapted from the literature.71,72 NHydroxysuccinimide (5.33 g, 46.3 mmol) and triethylamine (92.6 mmol) were dissolved under an argon atmosphere in anhydrous dichloromethane (200 mL) in a three-neck round-bottom flask equipped with a condenser. The flask was cooled to 0 °C and 2-bromopropionyl

bromide (20 g, 92.6 mmol) was added dropwise. The mixture was stirred, allowed to reach room temperature, and then stirred overnight at room temperature. Then the reaction mixture was extracted three times with water, and the organic phase was evaporated under reduced pressure to give a brownish solid. This solid was dissolved in isopropanol at 70-80 °C and a few drops of dichloromethane were added. By cooling the solution with liquid nitrogen, a white product was recrystallized from the solution. The process of recrystallization was repeated three times and a white pure product was formed. 1H NMR (CDCl3) δ (ppm) 1.95 (d, 3H, CH3), 2.83 (s, 4H, CH2), 4.60 (q, 1H, CH). Synthesis of ATRP Initiator 2 (N-Succinimidyl 2-Bromo-bromoisobutyrate). This initiator was synthesized according to a procedure reported by Pan and co-workers.73 2-Bromoisobutyric acid (6.68 g, 40 mmol) and N-hydroxysuccinimide (5.52 g, 48 mmol) were dissolved in 200 mL of anhydrous dichloromethane. DCC was added into the solution. The reaction mixture was stirred at room temperature for 24 h. A white byproduct was separated by filtration. The filtrate was washed with distilled water three times for removal of the unreacted Nhydroxysuccinimide and then dried over anhydrous sodium sulfate overnight. After removal of the solvent under reduced pressure, the residue was crystallized from hexane. 1H NMR (CDCl3) δ (ppm) 2.05 (s, 6H, CH3), 2.83 (s, 4H, CH2). General Procedure for the Atom Transfer Radical (Co)polymerization of MEO2MA and OEGMA475. Copper(I) chloride and 2,2′bipyridyl were added to a Schlenk tube sealed with a septum. The tube was purged with dry argon for a few minutes. Then, a degassed mixture of oligo(ethylene glycol) methyl ether methacrylate (OEGMA), 2-(2methoxyethoxy) ethyl methacrylate (MEO2MA), initiator, and ethanol was added through the septum with a degassed syringe. The mixture was heated at 60 °C in an oil bath for 1 day. The experiment was stopped by opening the flask and exposing the catalyst to air. The solution was diluted with demonized water and subsequently purified by dialysis against water (Roth, ZelluTrans membrane, molecular weight cutoff: 4000-6000). Last, water was removed by rotary evaporation. General Procedure for the Polymer Conjugation of Trypsin. The conjugation procedure was adapted from the literature.66 NHSfunctionalized copolymer (P1-P5 in Table 1) was dissolved in a small amount of DMF (polymer concentration ∼ 200 mg · mL-1). Trypsin was dissolved in buffer (borax/HCl, pH ) 9 at 20 °C) at 4 °C (trypsin concentration ∼ 40 mg · mL-1). The two solutions were then mixed and gently shaken at 4 °C for 4 h and then at room temperature for 2 h to allow the conjugation to proceed. Various molar ratios polymer/ trypsin were tested ranging from 3/1 to 11/1. After reaction, the conjugation solution was dialyzed in Tris/HCl buffer (pH 8.2, 0.05 M Tris containing 0.02 M CaCl2) for 2 weeks at 4 °C. Dialysis membranes with a molecular weight cutoff of 25.000 g · mol-1 were used to selectively separate unreacted polymer (7080 < Mn < 13400) and unreacted trypsin (23.8 kDa) from the formed conjugates. The buffer solution was renewed two times a day. It was previously demonstrated that trypsin conjugates should be stable for approximately 30 days in these dialysis conditions.68 After dialysis, different isolation procedures were used depending on the type of polymer used for conjugation. For polymers having a low LCST in buffer (i.e., cloud point around 30 °C), the dialysis solution was heated up to 33 °C and centrifuged for 30 min at 14000 rpm speed. This step allows separation of the

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Scheme 1. General Strategy for Preparing P(MEO2MA-co-OEGMA475)-Trypsin Conjugatesa

a

Experimental conditions: (i) ethanol, 60 °C, CuCl, Bipy; (ii) 4 °C and RT in borax-HCl buffer, pH ) 9.

thermoresponsive conjugate from the rest of the unreacted trypsin. After 30 min, the supernatant was removed and the white precipitate was stored at -18 °C. For polymers with a higher LCST (i.e., cloud point above 33 °C), a saturated aqueous solution of ammonium chloride was added to the dialysis solution to reduce the LCST of the solution to 30 °C. Indeed, at temperatures above 33 °C, the risk of autodigestion and denaturation of trypsin is non-negligible. The modified solution was then heated up at 30 °C and centrifuged for 30 min at a speed of 14000 rpm. After 30 min, the supernatant was removed and the white precipitate was stored at -18 °C. Measurements and Analysis. Size Exclusion Chromatography (SEC). Molecular weights and molecular weight distributions were determined by SEC performed at 25 °C in THF (flow rate 1 mL · min-1), using four 5 µ-SDV columns (one guard column and three columns of 4 × 103, 3 × 105, 2 × 106 Å). The detection was performed with a RI (DN-1000, WGE Dr. Bures) and a UV/vis detector (UV 2000; 260 nm). For calibration, linear polystyrene, or linear poly(methyl methacrylate) standards were used. Cloud Point Measurements. The cloud points of the polymer solutions in water were measured on a Tepper TP1 photometer (Mainz, Germany). Transmittance of polymer solutions in deionized water at 670 nm was monitored as a function of temperature (cell path length: 12 mm; one heating/cooling cycle at rate of 1 °C · min-1). 1 H NMR. 1H NMR spectra were recorded in CDCl3 on a Bruker DPX-400 operating at 400.1 MHz. Monomer conversions were calculated from 1H NMR spectra by comparing the integrations of the vinyl protons of the remaining monomers (5.56 and 6.12 ppm) to the overall integration of the region 3.90-4.40 ppm where two protons of the remaining monomers and two protons of the formed polymers resonate. An overall monomer conversion was calculated, as both monomers MEO2MA and OEGMA475 were assumed to exhibit comparable reactivities. Sodium Dodecyl Sulfate Poly(acrylamide) Gel Electrophoresis (SDS-PAGE). SDS-PAGE analysis was performed according to Laemmli with a 12.5% separating gel.74 Protein concentrations were determined by the method of Bradford using Bio-Rad Protein Assay kit (Bio-Rad, CA) with bovine serum albumin as a standard.75 Samples were loaded into each well to get 30 µg of protein. Protein solution was mixed with SDS-PAGE sample buffer (250 mM Tris pH6.8, 40% glycerol, 8% SDS, 20% 2-mercaptoehanol, and 0.01% bromophenol blue) with a ratio of 3:1 and heated at 95 °C for 10 min. The slab gel was composed of 2 cm of concentrating gel (5% acrylamide, 62.5 mM Tris pH6.8, 0.1% SDS) and 6 cm of separating gel (12.5% acrylamide, 375 mM Tris pH8.8, 0.1% SDS) with 1.5 mm thickness. Slab gel electrophoresis was performed by SE 250 mini vertical unit (Hoefer, Holliston, MA) for about 100 min with maximum current and voltage of 20 mA and 150 V, respectively. Proteins were visualized by bluesilver staining.76 Gels were incubated in staining solution (0.12% Coomassie brilliant blue G250, 10% ammonium sulfate,10% phosphoric acid, and 20% methanol) overnight with gentle shaking, and washed once with methanol. Gels were further incubated in water for several days to remove background. Different gel and protein concentrations were tested. In all measurements, raw conjugation product (i.e., immediately after reaction), conjugate solutions after dialysis, and conjugates redissolved in buffer after thermally induced precipitation

were compared. P(MEO2MA-co-OEGMA) copolymers were also loaded as a negative control to get 750 µg in a lane but did not give any band (data not shown). Enzyme ActiVity Tests. The enzyme activity tests were recorded on a UVIKON 941 PLUS spectrophotometer using tungsten halogen lamp (λ range: 290-900 nm) as a light source and a photomultiplier R 446 detector. The model substrate NR-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) was used. The enzymatic reaction of trypsin with BAPNA is monitored by measuring spectrophotometrically the amount of produced p-nitroaniline (absorption at 410 nm, extinction coefficient ) 8800). The tests were performed with a solution of polymer/trypsin conjugates in Tris/HCl buffer (pH ) 8.2).77 Prior to each measurement, the exact concentration of protein in the solution was determined by a Bradford assay.75 For the enzyme kinetics investigation, a series of tubes containing graded concentrations of BAPNA was prepared. At time zero, a fixed amount of the polymer/ enzyme conjugate was added in the tubes. The absorption/concentration of p-nitroaniline formed was measured in the early stages of the enzymatic reaction (i.e., 2-3 min after addition of the enzyme) and the change in absorbance was plotted against time. From the initial slope of these plots the initial reaction rate (Vi) for different substrate dilutions was determined. These data were then used to draw a Lineweaver-Burk plot, from which the maximum velocity (Vmax) and the Michaelis-Menten constant (Km) were extracted.78

Results and Discussion Thermoresponsive P(MEO2MA-co-OEGMA475)-trypsin conjugates were synthesized via a grafting-onto strategy. In this approach, copolymers containing N-hydroxysuccinimide active ester R-chain-ends were reacted with the surface lysine residues of trypsin (Scheme 1).66 The oligo(ethylene glycol)-based thermoresponsive copolymers were prepared by atom transfer radical (co)polymerization of MEO2MA and OEGMA475 in ethanol solution. The polymerizations were performed at 60 °C in the presence of copper(I) chloride and 2,2′-bipyridyl. Two different ATRP initiators containing succinimidyl ester moieties were tested in the present study (1 and 2 in Scheme 1).71-73,79 Copolymers with variable chain-lengths and MEO2MA/OEGMA475 compositions were synthesized (Table 1). Typically, relatively short chain-lengths were targeted (i.e., 25 < DP < 50) because molecular weight play a significant role in grafting onto strategies. In addition, the comonomer composition was adjusted to get phase transitions in the range 30-35 °C in physiological buffer. Indeed, as mentioned in the Introduction, trypsin has a high tendency toward self digestion and thus cannot be handled at temperatures above 35 °C.66,68 After polymerization, the (co)polymers were purified by dialysis and analyzed by 1H NMR and SEC. The latter method indicated that well-defined macromolecules with a low molecular weight distribution were synthesized in all cases. However, the experimental molecular weights measured by SEC were found to be significantly higher than expected. These deviations

Smart PEGylation of Trypsin

Figure 1. 1H NMR spectra recorded in CDCl3 (zoom of the region 2.6-4.6 ppm) for an R-succinimidyl functional P(MEO2MA-coOEGMA475) copolymer (polymer P4 in Table 1) after ATRP synthesis and purification.

cannot be explained by sole SEC calibration. Indeed, it was demonstrated in previous papers that the molecular weight of P(MEO2MA-co-OEGMA) copolymers is fairly accurately estimated using polystyrene standards.28,31 Moreover, in the present case, comparable molecular weight values were obtained using polystyrene or poly(methyl methacrylate) standards (data not shown). Thus, the observed deviations are probably due to a low initiation efficiency f of 1 and 2. Haddleton and co-workers reported previously that these initiators lead to noticeable molecular weight deviations.71,72 For instance, they studied the ATRP of OEGMA475 in toluene solution and reported initiation efficiencies of 40-55% for 1 and 65% for 2.71 The present experimental data are in good agreement with the study of Haddleton and suggest initiating efficiencies of 43-56% and 66-69% for 1 and 2, respectively. Although slightly oversized, the formed macromolecules have a controlled molecular structure and could be used for trypsin bioconjugation. For instance, NMR measurements indicated that the purified polymers still contain succinimidyl ester end groups (i.e., the active ester moieties were not significantly hydrolyzed during the purification procedure). In all spectra, a clear chainend signal could be observed at 2.77 ppm (signal a in Figure 1). This signal corresponds to the four methylene protons of the succinimidyl ring. The average chain length of the formed copolymer chain was calculated by comparing the integration of this chain-end signal to the integration of some side-chain protons (e.g., protons b or d in Figure 1). These calculations matched the molecular weight deviations observed by SEC. This close agreement between SEC and NMR results suggests that all (co)polymer chains possess an active ester end group. Furthermore, all (co)polymers exhibited a LCST behavior in aqueous medium. Table 1 shows the cloud points measured in pure deionized water for this series of polymers. As previously reported, the phase transitions depended closely on the MEO2MA/ OEGMA composition of the (co)polymers.28,30,31 Thus, cloud points ranging from 30 to 43 °C were obtained. It should be noted, however, that the cloud points observed in pure water are usually about 3-4 °C higher than in physiological buffer.29,31,80 The bioconjugation of trypsin with polymers P1-P5 was performed at room temperature in borax-HCl buffer (Scheme 1) to synthesize the corresponding P(MEO2MA-co-OEGMA475)trypsin conjugates C1-C5. Trypsin possesses 15 lysine residues (including the N-terminus), which can potentially react with the succinimidyl end groups of the (co)polymers. However, it was previously reported that a maximum of 11 lysine sites can be

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Figure 2. SDS-PAGE image obtained for unmodified trypsin (right) and various P(MEO2MA-co-OEGMA475)-trypsin conjugates. The conjugates C1 and C2 were obtained with polymers P1 and P2 (Table 1), respectively. A polymer/enzyme ratio of 6.5 was used in both cases. The samples C1′ and C2′ were isolated after conjugation and dialysis. The samples C1′′ and C2′′ are the final conjugates after reaction, dialysis, thermo-induced precipitation, and centrifugation. The acronym M represents protein molecular weight markers.

reacted with synthetic polymer chains.66 In the present case, a mild degree of bioconjugation (i.e., approximately five polymer chains per enzyme) was targeted. After reaction, the polymer/ enzyme conjugates were first purified by dialysis. The molecular weight cutoff of the dialysis membrane was carefully chosen to selectively separate unreacted polymer and trypsin from the formed bioconjugates. Figure 2 shows SDS-PAGE analysis of the bioconjugation mixtures C1′ and C2′ after this dialysis step. Although the contrast of these images is rather low, it is possible to see that high molecular weight bioconjugates were formed in both cases. Nevertheless, a small amount of unreacted trypsin is still noticeable. These results indicate that the dialysis procedure allows the complete removal of the unreacted polymers (the molecular weights of the (co)polymers are far below the cutoff of the membranes), but not of trypsin. Therefore, the samples were purified further. As highlighted in the introduction, one important advantage of thermoresponsive polymer/enzyme conjugates is the possibility to isolate them via thermoprecipitation. Yet, in the case of trypsin conjugates, the precipitation temperature should be under 35 °C to prevent autodigestion. The bioconjugation mixtures C1′ and C2′ were purified by thermoprecipitation, centrifuged, and analyzed by SDS-PAGE (samples C1′′ and C2′′ in Figure 2). The conjugate C1′′ could be easily precipitated out and isolated. Indeed, the LCST of the homopolymer P1 is low enough to allow a quantitative precipitation of the conjugates at mild temperature. On the other hand, the conjugate C2′′ could not be quantitatively precipitated in the same conditions. In that case, a saturated salt solution was used to lower the LCST of the polymer/enzyme conjugate. This approach was previously used to precipitate PNIPAM/trypsin conjugates.66,68 Figure 2 clearly indicates that the rest of the unreacted trypsin was removed during the centrifugation step. In addition, the SDS-PAGE images evidence the formation of P(MEO2MA-co-OEGMA475)-trypsin conjugates with a molecular weight of roughly 60.000 g · mol-1 (SDSPAGE molecular weight values are somewhat inaccurate for polymer bioconjugates and should therefore be considered with caution). This value suggests a conjugation degree of approximately five polymer chains per enzyme (a polymer/enzyme ratio of 6.5 was used in these two experiments). Hence, the reactive polymers P1 and P2 are efficient structures for trypsin modification. In comparison, the polymers P3-P5 were found to be less appropriate for bioconjugation. Overall, the LCST values of these polymers were too high for trypsin handling (see cloud points in Table 1). It should be also noted that

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Figure 3. Plots of transmittance as a function of temperature measured by turbidimetry (solid line, heating; dotted line, cooling) in aqueous solution (3 mg · mL-1) for copolymer P2 (Table 1) and the corresponding P(MEO2MA-co-OEGMA475)-trypsin conjugate C2.

Figure 4. Lineweaver-Burk plots obtained for BAPNA hydrolysis assays in the presence of unmodified trypsin (red dots and corresponding linear fit) or P(MEO2MA-co-OEGMA475)-trypsin conjugate C2 (blue dots and corresponding linear fit).

polymers prepared in the presence of initiator 2 are usually not very efficient for protein bioconjugation.52,71 For instance, in the present case, it was observed that polymers P4 and P5 led to relatively low degrees of conjugation (i.e., approximately one polymer chain per enzyme). The purified polymer/enzyme conjugates C1 and C2 were characterized further. Turbidimetry measurements indicated that both bioconjugates were thermoresponsive. The phase transitions of these biohybrids were roughly comparable to those of their thermoresponsive precursors. For example, Figure 3 compares the cloud point measurements obtained for the bioconjugate C2 and its parent copolymer P2. The cloud point of the conjugate was found to be approximately 2 °C higher than that of the polymer. Furthermore, the amidase activity of P(MEO2MA-coOEGMA475)-trypsin conjugates C1 and C2 was studied at 25 °C. NR-Benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) was chosen as a model substrate for these enzymatic tests. When hydrolyzed, this compound releases p-nitroaniline, which can be monitored spectrophotometrically (maximum absorbance at 410 nm). The enzymatic activity of C1 and C2 was compared to that of unmodified trypsin. For each structure, a series of solutions containing a fixed enzyme concentration and a gradual concentration of BAPNA was investigated. Figure 4 compares the Lineweaver-Burk plots obtained for unmodified trypsin and for the bioconjugate C2. The slopes of these graphs allow calculation of the Michaelis-Menten constant (Km), which reflects the enzyme-substrate affinity. Values of 1.34 and 0.3 were found for the enzyme and the conjugate, respectively. For the conjugate C1 (data not shown), a Km value of 0.29 was calculated. These results indicate that amidase activity of the conjugates is higher than that of unmodified trypsin (low Km values reflect a high enzyme-substrate affinity). It was previously reported that PNIPAM or PEG conjugation increases the enzymatic activity of trypsin.61,64,65,67,68 However, this interesting effect was never clearly explained. Nevertheless, it was proposed that the polymer modification may either influence the microenvironment of the enzymatic site or lead to allosteric changes in the enzyme conformation. The present data confirm that polymer conjugation has an apparent beneficial effect on the in vitro enzymatic behavior of trypsin.

Conclusion The modification of bovine pancreas trypsin with thermoresponsive oligo(ethylene glycol)-based (co)polymers was investigated. The polymers were synthesized by atom transfer radical copolymerization of 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methyl ether methacrylates (Mn ) 475 g · mol-1) in the presence of initiators containing succinimidyl ester moieties. This polymerization method afforded welldefined polymers with a narrow molecular weight distribution and defined chain ends. Yet, as previously described in the literature, a low apparent efficiency of initiation was observed for the succinimidyl-based initiators. Thus, the formed (co)polymers were slightly oversized. Nevertheless, these macromolecules exhibited controlled thermoresponsive properties and reactive succinimidyl end groups and could therefore be used for enzyme conjugation. The conjugation of the (co)polymers with trypsin was performed at room temperature in buffered milieu. The reactive end groups of the polymers were randomly reacted with primary amine functions of lysine residues of the enzyme. SDS-PAGE analysis indicated that polymer/enzyme conjugates were synthesized in all cases. The formed P(MEO2MA-co-OEGMA475)trypsin conjugates exhibited a thermoresponsive behavior and could be easily purified by thermoprecipitation. The phase transitions of the purified conjugates were on the whole comparable to those of the parent polymers. Furthermore, the amidase activity of the P(MEO2MA-co-OEGMA475)-trypsin conjugates was studied. Interestingly, it was observed that the polymer/enzyme conjugates exhibit a higher enzymatic activity than unmodified trypsin. Overall, these data indicate that thermoresponsive P(MEO2MA-co-OEGMA475) copolymers are promising structures for enzyme conjugation. The proof of principle described in this article can be extended to a broad variety of enzymes and globular proteins. Acknowledgment. The Fraunhofer Society and the German Research Foundation (DFG) are acknowledged for financial support (Sfb 448). Additionally, the authors thank Professor Andre´ Laschewsky (Universita¨t Potsdam) for fruitful discussions, Dr. Carola Fanter and Mirjam Mai (Fraunhofer IAP) for the spectrophotometric measurements, and Dr. Alisdair Fernie

Smart PEGylation of Trypsin

(MPIMP, Potsdam) for the access to the cold laboratory and gel electrophoresis.

References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42)

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