Rational Protein Modification Leading to Resistance of Enzymes to

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Biomacromolecules 2004, 5, 1947-1955

1947

Rational Protein Modification Leading to Resistance of Enzymes to TiO2-UV Irradiation-Induced Inactivation Bhalchandra S. Lele† and Alan J. Russell*,‡ McGowan Institute for Regenerative Medicine and Departments of Bioengineering and Surgery, University of Pittsburgh, 100 Technology Drive, Pittsburgh, Pennsylvania 15219 Received May 5, 2004; Revised Manuscript Received July 12, 2004

Photoexcited TiO2 degrades biomolecules such as nucleic acids, cell membrane proteins, and enzymes. Stabilization of enzyme activity against the deactivation caused by the combination of TiO2-UV is essential if we are to develop novel hybrid materials exhibiting photocatalytic and biocatalytic activities useful for decontamination applications. In this paper we describe the stabilization of a model enzyme, chymotrypsin, against TiO2-UV-induced deactivation by conjugating the enzyme with UV-absorbing, carboxyl-terminated oligo[2-[3-(2H-benzotriazol-2-yl)-4-hydroxyphenyl]ethyl methacrylate] [oligo(HBMA)-COOH]. Chymotrypsin was completely deactivated within 3 h, whereas the chymotrypsin-oligo(HBMA) conjugate retained >50% activity even after 5 h of exposure to TiO2-UV (λmax 365 nm). The degree of enzyme stabilization induced by the conjugated UV absorber was 2-fold higher than that from the equivalent number of conjugated PEG chains. Spectroscopic characterizations revealed that chymotrypsin-oligo(HBMA) absorbs UV light and initially resists photoexcitation of TiO2. Modified chymotrypsin also exhibited resistance to changes in the secondary structure during the deactivation. This method of stabilizing enzymes against photodegradation could be also useful in photolithographic enzyme immobilizations for sensors and arrays or for stabilization of any UV-sensitive protein. Introduction The photocatalytic activity of titanium dioxide (TiO2) is finding novel applications in organic and inorganic toxic waste treatment,1 degradation of nerve agents,2 antibacterial/ antifungal surfaces,3 self-cleaning windows,4 steel plates,5 paints,6 and polyester fabrics.7 Anatase-type TiO2 absorbs ultraviolet radiation (UV) (from sunlight) having energy greater than its optical band gap of 3.2 eV, resulting in the formation of an electron-hole pair. The electron generated following the absorption of a photon is a reducing agent and the hole formed is a powerful oxidizing agent, which causes oxidative degradation of organic molecules. Also, photoexcited TiO2 generates hydroxyl and peroxide radicals which can react with organic molecules.8,9 Combining TiO2 photocatalysis with biocatalysis would be desirable for the development of novel inorganic-organic hybrid materials exhibiting a broad range of catalytic activities useful in decontamination of chemical and biological agents. Previously, we have reported a biocatalytic coating containing the nerve agent-degrading enzyme diisopropylfluorophosphatase.10 We are now incorporating photocatalytic activity in similar coatings. For sensing applications, various proteins have been adsorbed on the surface of TiO2 via electrostatic interactions.11a-c However, upon absorption of UV light, excited TiO2 rapidly * To whom correspondence should be addressed. † McGowan Institute for Regenerative Medicine and Department of Bioengineering. ‡ McGowan Institute for Regenerative Medicine and Department of Surgery, University of Pittsburgh.

deactivates enzymes such as glucose oxidase (70% activity loss within 3 h of irradiation).11d Moreover, photosensitized TiO2 causes oxidative damage to biomolecules such as RNA11e and cell membrane proteins.12 Deactivation of enzymes can also be caused by their exposure to UV alone. For example, glucose oxidase13 and horseradish peroxidase14 lost significant activities upon exposure to UV (λmax 365 nm) and γ radiations during enzyme immobilizations by photopolymerization with vinyl monomer. A 70% reduction in the activity of catalase was reported when the enzyme-monomer mixture was irradiated with a mercury lamp (450 W).15 Studies of UV-irradiated enzymes such as chymotrypsin,16 trypsin,16 and cutinase17 and proteins such as R-lactalbumin,18 wool keratin,19 and human eye lens20 and serum proteins21 have demonstrated that the excited tryptophan residues transfer electrons to the nearest disulfide linkages and cause their cleavage. Enzymes have been stabilized by poly(ethylene glycol)-,22 poly(N-vinyl-2-pyrrolidone)-,23 or poly(2-hydroxyethyl methacrylate)24-based membranes synthesized by UV irradiation of enzyme-monomer mixture at low temperature. However, to the best of our knowledge, there is no literature report on specifically designed enzymepolymer conjugates that are resistant to UV and/or TiO2UV irradiation. In this paper we describe the stabilization of a model enzyme, chymotrypsin, against degradation by TiO2-UV via conjugation with a UV-absorbing, carboxyl-terminated, oligo[2-[3-(2H-benzotriazol-2-yl)-4-hydroxyphenyl]ethyl methacrylate] [oligo(HBMA)-COOH]. Chymotrypsin-oligo(HBMA) retained activity via UV absorption and initial

10.1021/bm049728o CCC: $27.50 © 2004 American Chemical Society Published on Web 08/21/2004

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resistance to photoexcitation of TiO2. The modified enzyme also resisted changes in the secondary structure during the deactivation. The conjugation of a UV absorber to an enzyme molecule reported in this paper is generally applicable where the stabilization of a biomolecule is needed against photodegradation. Experimental Section Materials. R-Chymotrypsin (from bovine pancreas), Nsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide, sodium deoxycholate, N-hydroxysuccinimide (NHS), sodium phosphate (Na2HPO4), bicinchoninic acid solution, copper(II) sulfate solution, and bovine serum albumin protein standards were purchased from Sigma Co. (St. Louis, MO). Monomethoxy poly(ethylene glycol) succinimidyl ester (MPEG-SPA, MW 5000) was purchased from Nektar Therapeutics (Huntsville, AL). 4,4′-Azobis(cyanovaleric acid), 3-mercaptopropionic acid, 2-[3-(2H-benzotriazol-2-yl)-4-hydroxyphenyl]ethyl methacrylate (HBMA), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane, n-hexane, dioxane, and 1,3-dicyclohexylcarbodiimide (DCC) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Centrifugal dialysisfiltration tubes (Centricon Plus-20) with 10 000 molecular weight cut off (MWCO) were purchased from Millipore Co. (Bedford, MA). TiO2 (Degussa P25) was obtained from Degussa AG, Frankfurt, Germany. Methods: NMR Spectroscopy. 1H NMR spectra were recorded on a Bruker spectrometer operating at 300 MHz. Molecular weight (Mn) of oligo(HBMA)-COOH was estimated from the ratio of number of aromatic protons to the carboxyl proton of the end group. MALDI-TOF Spectrometry. Conjugation of oligo(HBMA) to chymotrypsin was characterized by analyses performed on a Perseptive Biosystems Voyager Elite MALDITOF spectrometer. The acceleration voltage was set at 20 kV in a linear mode. Enzyme solution (0.5-1.0 mg/mL) was mixed with an equal volume of matrix (0.5 mL of water, 0.5 mL of acetonitrile, 2 µL of trifluoroacetic acid, and 8 mg of R-cyano-4-hydroxycinnamic acid), and 2 µL of the resulting mixture was spotted on the plate target. Spectra were recorded after solvent evaporation. Fluorescence Spectroscopy. At 60-min intervals, 1.5-mL aliquots of UV irradiated enzyme-TiO2 suspension (enzyme 0.8 mg/mL, TiO2 0.25 mg/mL) were collected and filtered through a 0.2-µm filter. The clear solution was placed in a quartz cuvette (path length 10 mm) inside a luminescence spectrometer (Perkin-Elmer, Shelton, CT, model LS 5B). Excitation and emission band gaps were set at 3 nm. Samples were excited at 295 nm and emission spectra were recorded from 300 to 410 nm. CD Spectroscopy. At 60-min intervals, clear solutions were obtained from UV-irradiated enzyme-TiO2 suspensions. Protein solutions were diluted to obtain concentrations of 0.1 mg/mL. Aliquots (400 µL) of the sample (0.1 mg/ mL) were placed in a quartz cuvette (path length 1 mm) inside an Aviv CD spectrometer (model 202). Each spectrum was accumulated by averaging 10 scans between 195 and 260 nm. All spectra were corrected for background signals

Lele and Russell

of the buffer. Mean residual ellipticity ([Θ]λ in degrees‚ centimeter2‚decimole-1) values were obtained from Θobsd: [Θ]λ ) ΘobsdMW/10lcn

(1)

where MW is the molecular weight of chymotrypsin, l is the path length (0.1 cm), n is the total number of amino acid residues in chymotrypsin (241), and c is the concentration (in grams per milliliter). Exposure of Enzymes to UV-Irradiated TiO2. Enzyme (0.8 mg of protein/mL, total 10 mL in 25 mM phosphate buffer, pH 7.5) was placed in an open scintillation vial. TiO2 fine powder (0.25 mg/mL) was added to the protein solution and the suspension was stirred gently at room temperature (25 °C) with a magnetic stir bar placed inside the vial. The enzyme-TiO2 suspension was placed under a Blak-Ray longwave UV lamp (model B-100AP, UVP, San Gabriel, CA). The distance between the UV lamp and the vial was 18 cm. At this distance, the UV irradiance at 365 nm (λmax) was 8 mW/cm2 (determined with a Blak-Ray UV meter, model J-221). It was also verified that there was no thermal denaturation of the enzyme during irradiation and the temperature of the enzyme-TiO2 suspension remained constant (25 ( 2 °C) throughout. Determination of Residual Enzyme Activity. Measurable loss in enzyme activities were observed at >30 min intervals, once the deactivation started. At 30-min intervals, a 100-µL aliquot was removed from the irradiated enzyme-TiO2 suspension and added to 1.2 mL of N-succinyl-Ala-Ala-ProPhe-p-nitroanilide solution (0.5 mg/mL in 25 mM phosphate buffer, pH 7.5). The ratio enzyme:substrate was 3.4 µM: 800 µM. After 1 min, the rate of reaction remained constant, indicating we were within the initial rate regime. At this point, the TiO2-enzyme-substrate suspension was filtered through a 0.2-µm filter and the absorbance of hydrolyzed p-nitroaniline was measured at 412 nm on a Perkin-Elmer spectrophotometer (model Lambda 45). Hydrolysis of the substrate by the buffer was negligible during the assay time. Original activities of native and modified chymotrypsins were also determined as described above. It was also confirmed that TiO2 alone did not cause hydrolysis of the substrate. Dye Discoloration by TiO2-UV in the Presence or Absence of the UV Absorber. This experiment was performed to determine the ability of HBMA to resist photoexcitation of TiO2 via absorption of UV light. Bromophenol blue sodium salt (0.1 mg) and a water-soluble copolymer of 40% (w/w) 2-acrylamido-2-methylpropanesulfonic acid (AMPS) and 60% (w/w) HBMA [poly(AMPSco-HBMA)] (1 mg) were dissolved in 10 mL of phosphate buffer (25 mM, pH 7.5). TiO2 fine powder (0.25 mg/mL) was added to the dye solution, which was stirred gently at room temperature (25 °C). The suspension was irradiated with UV light (λmax 365 nm) as described above. At 60-min intervals, 1.2-mL aliquots were removed from the irradiating suspension and filtered through a 0.2-µm filter. Discoloration of bromophenol blue (due to the photooxidation) was monitored by measuring the absorbance at 591 nm. We confirmed that addition of poly(AMPS) had no effect on the

Enzyme Resistance to TiO2-UV-Induced Inactivation

rate of bromophenol blue photooxidation. Also, there was no change in the absorbance at 591 nm due to TiO2 or UV alone. Adsorption of Enzyme on TiO2 Particles. Ability of native or modified chymotrypsin to bind to TiO2 particles was measured as follows. Enzyme (0.8 mg of protein/mL, total 10 mL in 25 mM phosphate buffer, pH 7.5) was placed in an open scintillation vial. TiO2 fine powder (0.25 mg/ mL) was added to the protein solution and the suspension was stirred gently at room temperature (25 °C) with a magnetic stir bar placed inside the vial. The suspension was irradiated as described above. At 30-min intervals, a 300 µL aliquot was removed and filtered through a 0.2-µm filter. The amount of protein in the filtrate was estimated by bicinchoninic acid protein assay. Data were corrected by subtracting negligible nonspecific protein adsorption by the syringe filters. Synthesis of Oligo(HBMA)-COOH. In a three-necked round-bottom flask equipped with a reflux condenser, 4 g of HBMA (12 mmol), 0.34 g of 4,4′-azobis(cyanovaleric acid) (1.2 mmol), and 0.065 g of 3-mercaptopropionic acid (0.6 mmol) were dissolved in 40 mL of DMF. Nitrogen gas was purged through the DMF solution for 30 min at room temperature. Polymerization was conducted at 80 °C for 12 h under the continuous purging of nitrogen. Oligo(HBMA)COOH was isolated by precipitation of the DMF solution into 1 L of distilled water (pH 1.5). The product was purified by reprecipitation from dichloromethane into n-hexane. Yield 2 g (50%). 1 H NMR (CDCl3): δ 1.0-2.0 (br m, -CH2-C-CH3 of polymer backbone), 3.0 (s, benzyl -CH2- of HBMA), 4.1 (s, -COO-CH2-CH2-O- of hydroxyethyl spacer in HBMA), 7.08.7 (m, aromatic protons of HBMA), 11.2 (s, phenolic -OH of HBMA). Mn ) 700 (from 1H NMR). Synthesis of Oligo(HBMA)-COONHS. Oligo(HBMA)COOH (1 g) was dissolved in dichloromethane (20 mL) and 5-fold molar excesses of NHS and DCC were added to the dichloromethane solution. The reaction mixture was stirred for 16 h at 25 °C and filtered to remove dicyclohexyl urea. The clear solution was poured into 500 mL of n-hexane under stirring to precipitate oligo(HBMA)-COONHS. The product was purified by reprecipitation from dichloromethane into n-hexane [yield 0.6 g (60%)]. 1 H NMR (CDCl3): δ 1.0-2.0 (br m, -CH2-C-CH3 of polymer backbone), 3.0 (s, benzyl -CH2- of HBMA), 3.6 (s, -CH2-CO- of NHS protons), 4.1 (s, -COO-CH2-CH2- of hydroxyethyl spacer in HBMA), 7.0-8.7 (m, aromatic protons of HBMA), 11.2 (s, phenolic -OH of HBMA). Synthesis of Chymotrypsin-Oligo(HBMA) Conjugates. The conjugates were synthesized according to the procedure reported by Baszkin et al.25 R-Chymotrypsin (100 mg) was dissolved in phosphate buffer (100 mL of 160 mM, pH 8.8) containing 0.6% (w/w) sodium deoxycholate. Oligo(HBMA)COONHS (70 mg) was dissolved in anhydrous dioxane (2 mL) and added to the chymotrypsin solution under stirring. The reaction mixture was stirred at 25 °C for 2 h and filtered through a 0.45-µm filter to remove the precipitated oligo(HBMA)-COOH. The clear solution (∼1 mg of protein/mL) was placed in the centrifugal dialysis-filtration tubes (Cen-

Biomacromolecules, Vol. 5, No. 5, 2004 1949 Table 1. Synthesis and MALDI-TOF Characterization Data for Chymotrypsin-Oligo(HBMA) with Increasing Degree of Conjugation no.

oligo(HBMA)COONHS (g)a

conjugate

mol wtb

1 2 3

0.07 0.21 0.63

chymotrypsin-oligo(HBMA_1) chymotrypsin-oligo(HBMA_2) chymotrypsin-oligo(HBMA_3)

26 040 26 730 27 100

a One hundred milligrams of chymotrypsin was used in each conjugation reaction. b Native chymotrypsin m/z ) 25 187. Molecular weights of the conjugates were determined by MALDI-TOF spectrometry as described in the text.

tricon Plus-20; 30 000 MWCO) and centrifuged at 4000 rpm for 15 min. The concentrated retentate was diluted to 20 mL with phosphate buffer (25 mM, pH 7.5) and dial-filtered again as described above. The amount of conjugate obtained was estimated by bicinchoninic acid protein assay. Yield 2030%. MALDI-TOF analytic data are listed in Table 1. Synthesis of Chymotrypsin-PEG1. R-Chymotrypsin (100 mg) was dissolved in borate buffer (20 mL of 50 mM, pH 8.5). MPEG-SPA (100 mg of MW 5000) was added to the enzyme solution and stirred for 1 h at 25 °C. The conjugate was purified first by use of Centricon Plus-20 (MWCO ) 30 000) centrifugal dialysis-filtration tubes and then by dialysis in a Spectra/Por dialysis membrane (MWCO ) 12 000, Spectrum Laboratories Inc., Rancho Dominguez, CA). Yield 40-60%. MALDI-TOF analysis: m/z ) 30 00040 000 (three peaks, attachment of 1-3 PEG chains). Synthesis of Chymotrypsin-PEG2. R-Chymotrypsin (100 mg) was dissolved in borate buffer (20 mL of 50 mM, pH 8.5). MPEG-SPA (500 mg of MW 5000) was added to the enzyme solution and stirred for 1 h at 25 °C. The conjugate was purified first by use of Centricon Plus-20 (MWCO ) 30 000) centrifugal dialysis-filtration tubes and then by dialysis in a Spectra/Por dialysis membrane having MWCO of 12 000. Yield 40-60%. MALDI-TOF analysis: m/z ) 40 000-60 000 (five peaks, attachment of 3-7 PEG chains). Results and Discussion In hybrid catalytic coatings, a biocatalyst and photocatalyst must be simultaneously active. Given the known propensity for enzymes to be inactivated by photocatalysts, we developed a strategy to modify the enzyme covalently with a UV absorber that has been used as a “sunblock” in plastics. We hypothesized that since proteins are known to bind to TiO2, the UV absorption by the modified enzyme could locally reduce the degree of photoexcitation of TiO2 and thereby protect the enzyme. 2-(2-Hydroxyphenyl)-2H-benzotriazoles are attractive UV stabilizers due to their ability to absorb photoexcitation energy and dissipate this electronic energy into vibrational energy via intramolecular proton transfer.26,27 2-[3-(2H-Benzotriazol-2-yl)-4-hydroxyphenyl]ethyl methacrylate (HBMA) is a commercially available and radically polymerizable monomer. We chose to synthesize short-chain poly(HBMA)-COOH that could be reacted with available and reactive -NH2 groups of lysine residues on the surface of a model enzyme, chymotrypsin.

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Figure 1. Reaction scheme used to synthesize oligo(HBMA)-COOH and its conjugates with R-chymotrypsin.

Oligo(HBMA)-COONHS. Our initial experiments to react poly(HBMA)-COONHS (MW ∼ 5000) with chymotrypsin in an aqueous buffer solution resulted in immediate precipitation of the polymer from dioxane. This subsequently resulted in an unmodified chymotrypsin. We therefore synthesized low molecular weight oligo(HBMA)-COOH using high amounts of carboxyl-terminated initiator and chain transfer agent in the free radical polymerization of HBMA. The oligomer retained partial miscibility in binary solvent mixtures of dioxane and water. From 1H NMR measurements, the molecular weight (Mn) of oligo(HBMA)-COOH was estimated to be 700-900 (dimer/trimer of HBMA). Carboxyl end groups of the oligomer were activated via DCC-mediated condensation with NHS. The activation was confirmed from the 1H NMR spectrum of oligo(HBMA)COONHS showing the presence of a peak at δ 3.6, which is characteristic of NHS protons. Chymotrypsin-Oligo(HBMA). The conjugation of UVabsorbing oligomers to -NH2 groups of lysine residues in the enzyme is schematically shown in Figure 1. Table 1 lists

the ratios of chymotrypsin to oligo(HBMA)-COONHS used to synthesize the three conjugates described in this paper. Modification of native chymotrypsin was confirmed by mass spectrometry. Figure 2a shows the MALDI-TOF spectrum of native chymotrypsin with a molecular ion peak at m/z ) 25 187. Figure 2b shows the MALDI-TOF spectrum of chymotrypsin-oligo(HBMA_1) with a molecular ion peak at m/z ) 26 040. The increase of ∼800 Da in the mass of the modified enzyme indicates the conjugation of one molecule of oligo(HBMA) per molecule of chymotrypsin. With further increases in the amount of reactant, we obtained conjugation of two or three molecules of oligo(HBMA) per molecule of the enzyme (Table 1). The conjugation reaction was also characterized by UV spectroscopy. The UV-vis spectrum of chymotrypsinoligo(HBMA) exhibited a peak at 340 nm, characteristic of the HBMA moiety (data not shown). All conjugates retained >90% activity of native chymotrypsin as measured from their hydrolytic activity against the specific substrate N-succinylAla-Ala-Pro-Phe-p-nitroanilide. Higher amounts (>2 g) of

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Figure 3. Effect of number of covalently attached oligo(HBMA) chains on chymotrypsin activity after exposure to UV-irradiated TiO2 (enzyme concentration ) 0.8 mg/mL, TiO2 concentration ) 0.25 mg/mL). The enzyme activity was monitored at 30-min intervals. Data reported are the average of duplicate experiments.

Figure 2. MALDI-TOF spectra of (a) native R-chymotrypsin and (b) chymotrypsin-oligo(HBMA_1).

oligo(HBMA)-COONHS were required to further increase the loading on the enzyme. This increased the percentage of dioxane to 10-15% in the enzyme solution and significantly decreased the activity of resulting conjugates. Stability of Chymotrypsin-Oligo(HBMA) against TiO2UV. As expected, the stability of the modified chymotrypsins against TiO2-UV increased with an increase in the number of conjugated oligo(HBMA) chains (Figure 3). We also verified that 16 h of UV irradiation alone or contact with TiO2 alone caused no loss in the activities of native and modified chymotrypsins. Moreover, increasing concentration of TiO2 in irradiating enzyme solution increased the rate of enzyme deactivation (Figure 4). Thus enzyme deactivation was caused primarily by TiO2-UV. We then determined the significance of the presence of oligo(HBMA) in the microenvironment of chymotrypsin. Since oligo(HBMA)-COOH is insoluble in water, we synthesized a water-soluble copolymer of HBMA (60% w/w) with 2-acrylamido-2-methylpropanesulfonic acid (AMPS) (40% w/w) (data not shown). Poly(AMPS-co-HBMA) (MW 50 000) was mixed with native chymotrypsin in a ratio of 1:1 or 10:1-fold excess of HBMA over that present in chymotrypsin-oligo(HBMA_3). All protein solutions (0.8 mg/mL) were irradiated under the UV lamp after addition of TiO2 (0.25 mg/mL). Data in Figure 5 show that native chymotrypsin and its mixtures with poly(AMPS-co-HBMA)

Figure 4. Effect of TiO2 concentration on residual activity of chymotrypsin-oligo(HBMA_2) (enzyme concentration ) 0.8 mg/mL). The enzyme activity was monitored at 30-min intervals. Data reported are the average of duplicate experiments.

Figure 5. Effect of conjugation of the UV-absorber on the stability of chymotrypsin after exposure to UV-irradiated TiO2 (enzyme concentration ) 0.8 mg/mL, TiO2 concentration ) 0.25 mg/mL). The enzyme activity was monitored at 30-min intervals. Data reported are the average of duplicate experiments.

rapidly lost activity. Only when oligo(HBMA) was conjugated covalently to chymotrypsin was activity retained. We also characterized UVA absorption spectra of enzyme solutions being exposed to TiO2-UV. In the case of modified chymotrypsins, a 3-fold increase in the absorbance in UVA region was observed with an increase occurring over time

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Table 2. Adsorption of Chymotrypsin-Oligo(HBMA_3) on Irradiated TiO2 Particlesa time (min)

adsorption (%)

0 30 60 120

0 27 74 80

a Chymotrypsin-oligo(HBMA_3) (0.8 mg/mL) was stirred with TiO 2 (0.25 mg/mL) under UV irradiation. The amount of protein adsorbed was determined as described in the text. Data reported are averages of duplicate experiments.

(data not shown). Thus, the absorption of UV light by modified enzymes was correlated to their resistance to deactivation by TiO2-UV. Residual enzyme activity data in Figures 3-5 show that the major effect of oligo(HBMA) conjugation was to prolong the onset of deactivation rather than alter the eventual rate of that inactivation. These kinetic trends imply that the deactivation of the enzyme begins after degradation of HBMA conjugates by strong oxidizing effects of TiO2-UV. Retention of enzyme activity before inactivation begins implies efficient UV absorption by conjugated HBMA moieties and thereby resistance to the photoexcitation of TiO2. Mechanism of Enzyme Protection by HBMA against TiO2-UV. At present, there is little understanding of the mechanism of deactivation of biomolecules by photoexcited TiO2. Various changes (conformational as well as compositional) in the protein structure are possible due to redox and radical addition reactions catalyzed by TiO2-UV. As mentioned earlier, enzymes adsorb on the surface of TiO2 particles via electrostatic interactions and deactivate upon UV irradiation of the particles.11 In our work, we observed 80% adsorption of chymotrypsin on TiO2 particles within 2 h of stirring the chymotrypsin-TiO2 suspension (Table 2). Thus, in the system we studied we are sure that the UVabsorbing HBMA moieties that are conjugated to the enzyme are arrayed at the surface of the particles, thereby allowing HBMA to effectively compete with TiO2 for the UV light in the region of the HBMA. One could imagine, therefore, that the enzyme attached to the HBMA will experience a lower concentration of inactivating species because less UV light will be reaching the TiO2 surface close to the enzyme. The photocatalytic activity of TiO2 when exposed to UV light can be monitored by measuring the rate of photooxidative discoloration of a dye.1d Unfortunately, a number of often-used dyes exhibited binding with native and modified chymotrypsins and protection from the photooxidation (probably due to steric stabilization by high molecular weight proteins). Therefore, we measured the rates of discoloration of bromophenol blue dye (λmax 591 nm) in the presence or absence of water-soluble HBMA [poly(AMPS-co-HBMA) (0.1 mg/mL)]. Data in Figure 6 show that discoloration of bromophenol blue by irradiated TiO2 (0.25 mg/mL) is significantly inhibited in the presence of a small amount of UV-absorbing HBMA [∼0.06 mg/mL, equivalent to that present in chymotrypsin-oligo(HBMA_3)]. In combination, our results indicate that the most likely mechanism of stabilization is that HBMA, which we know has the ability to decrease the “activity” of TiO2, protects the enzyme and

Figure 6. Inhibition of TiO2-UV-induced photooxidation of bromophenol blue by HBMA. An aqueous solution of bromophenol blue (0.01 mg/mL) was mixed with TiO2 (0.25 mg/mL) and irradiated with UV light in the absence or presence of poly(AMPS-co-HBMA) (0.1 mg/mL) [∼0.06 mg HBMA, equivalent to that in chymotrypsin-oligo(HBMA_3)]. Data reported are the average of duplicate experiments.

Figure 7. Schematic representation of the mechanism of enzyme protection by conjugated oligo(HBMA) against deactivation by TiO2UV.

only does so when the enzyme and HBMA are colocated on the surface of TiO2 (Figure 7). Changes in the Secondary Structure of Enzymes. CD spectroscopy can yield important information about the conformational changes in the structure of chymotrypsin being deactivated by TiO2-UV. Figure 8a shows CD spectra of native chymotrypsin before and after exposure to TiO2UV. In the first 1 h of irradiation, although there is no loss in the enzyme activity, the CD spectra show a complete loss of the characteristic shoulder at 230 nm as well as a blue shift in the peak at 202 nm. This implies that although the “buried” active site of chymotrypsin is not yet exposed to photooxidation, the deformation of native protein structure starts immediately upon irradiation. It should also be noted that a 65% loss in enzyme activity takes place in next the hour of irradiation. Fischer et al.28 have also reported similar CD spectra of chymotrypsin inhibited by surfactant-loaded nanoparticles. CD spectra of the modified enzymes were obtained similarly. As an example, CD spectra of chymotrypsin-oligo(HBMA_3) are shown in Figure 8b. The modified and irradiated enzymes exhibit a gradual decrease in the characteristic shoulder at 230 nm. Unlike native

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Figure 9. Fluorescence emission spectra of native R-chymotrypsin obtained at 1-h intervals during exposure to TiO2-UV.

Figure 8. Circular dichroic spectra of (a) native R-chymotrypsin and (b) chymotrypsin-oligo(HBMA_3) after exposure to TiO2-UV.

chymotrypsin, a gradual decrease in the enzyme activity is observed for chymotrypsin-oligo(HBMA_3). Moreover, for the first 5 h of irradiation (65% activity retention), there is practically no change in the peak intensity and the peak position at 202 nm. These spectra imply that the modified enzymes resist changes in their secondary structures during the deactivation by TiO2-UV. The disappearance of the shoulder at 230 nm from the CD spectrum of chymotrypsin has been attributed to the exposure of tryptophan residues to a more polar environment.29 Perturbation and Degradation of Tryptophan Residues. Shifts in fluorescence maxima of tryptophan residues are indicators of their microenvironments.30 We recorded fluorescence emission spectra of all enzymes after UV irradiation in the presence or absence of TiO2. As expected, UV irradiation alone (for 5 h) did not cause any change in λmax emission or fluorescence intensity of tryptophan residues in native as well as modified chymotrypsins (data not shown). However, upon exposure to TiO2-UV for 1 h, λmax emission of tryptophan residues in native chymotrypsin shifted from 330 to 360 nm (Figure 9). Such a red shift of λmax emission indicates exposure of tryptophan residues to a more polar environment (due to changes in the secondary structure of the enzyme).31 Fischer et al.32 and Hong et al.33 observed similar red shifts in fluorescence emission spectra of chymotrypsin inhibited by surfactant-loaded nanoparticles. Moreover, Fisher et al.32 demonstrated that, upon recovery of the chymotrypsin activity, the λmax emission of tryptophan residues is restored to 330 nm. Figure 9 also shows that there

is a drastic decrease in the fluorescence intensity of tryptophan residues. This can be attributed to tryptophan degradation by photooxidation and/or reaction with hydroxyl and peroxide radicals generated from TiO2-UV. Continuing the irradiation after 1 h resulted in the loss of tryptophan fluorescence, indicating the complete degradation of tryptophan residues. Modified chymotrypsins also exhibited a decrease in tryptophan fluorescence intensity due to the degradation by photooxidation as mentioned above. However, there was no drastic change in the microenvironment of tryptophan residues in the modified chymotrypsins (no red shift in λmax emission). As an example, fluorescence emission spectra of chymotrypsin-oligo(HBMA_2) are presented in Figure 10. These data again indicate that modified chymotrypsins resist changes in their secondary structures during the deactivation by TiO2-UV. Comparing Chymotrypsin-Oligo(HBMA) and Chymotrypsin-PEG. Stabilization of many proteins against deactivation by heat, pH, and physiological environment has been achieved by their PEGylation.34 PEG chains form a hydrated cage around the modified protein molecule and sterically hinder the access of denaturing agents.35 On the basis of this hypothesized mechanism, we expected that PEGylated chymotrypsin could resist the deactivation by photoexcited TiO2 and we were interested in comparing the degree of protection relative to that derived by local absorption of UV at the TiO2-enzyme interface. We therefore synthesized conjugates of chymotrypsin with carboxylic acid end-functionalized PEG. MALDI-TOF spectrometry revealed that chymotrypsin-PEG1 had 1-3 chains and chymotrypsin-PEG2 had 3-7 chains of PEG (molecular weight 5000) conjugated per molecule of the enzyme. PEGylated chymotrypsins (0.8 mg/mL) were exposed to photoexcited TiO2 (0.25 mg/mL) and their stabilities were compared with that of the chymotrypsin-oligo(HBMA_3) (0.8 mg/mL). Data in Figure 11 show that chymotrypsinoligo(HBMA_3) exhibited significantly higher stability than chymotrypsin-PEG1. Also, the enzyme stabilization by three chains of oligo(HBMA) was equivalent to that by 3-7 chains of PEG (chymotrypsin-PEG2). At the molecular level, the number of ethylene oxide repeat units in PEG (MW 5000) is 113, whereas the number of repeat units in oligo(HBMA) is 2-3. Theoretically, conjugation of a high molecular weight

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photoexcitation of TiO2. The modified enzyme also resists changes in the secondary structure during the deactivation by TiO2-UV. Enzyme stabilization by the conjugated UV absorber was significantly higher than that by the equivalent number of conjugated PEG chains. The enzyme sunblock conjugates reported in this work should be useful in developing novel hybrid materials exhibiting photocatalytic and biocatalytic activities. This approach to stabilize UVsensitive enzymes against photodegradation would also be useful for photolithographic enzyme immobilizations on sensor electrodes and diagnostic arrays. Acknowledgment. We thank Dr. Mike Cascio, Department of Molecular Biology and Genetics, University of Pittsburgh, for permission to use the CD spectrometer and valuable advice in the data interpretation. We thank Dr. Mark Bier, Center for Molecular Analysis, Carnegie-Mellon University, Pittsburgh, PA, for permission to use the MALDITOF spectrometer and Dr. Jason A. Beberich, Agentase LLC, Pittsburgh, PA, for MALDI-TOF characterization of enzymes. This work was supported by the Department of Defense Multidisciplinary University Research Initiative (MURI) program administered by the Army Research Office under Grant DAAD 19-01-0619. References and Notes Figure 10. Fluorescence emission spectra of chymotrypsin-oligo(HBMA_2) obtained (a) before and after 1 h of exposure to TiO2UV and (b) at 1-h intervals during exposure to TiO2-UV.

Figure 11. Comparison of stability of chymotrypsins modified with PEG and oligo(HBMA) against TiO2-UV. Data reported are the average duplicate experiments.

poly(HBMA)-COOH to chymotrypsin could result in much higher stability. Thus, the UV absorber conjugate stabilizes the enzyme against photooxidation more efficiently than that of the steric stabilization provided by PEG conjugation. Conclusion We have stabilized a model enzyme, chymotrypsin, against photoexcited TiO2 by modifying the enzyme with a UVabsorbing oligomeric “sunblock”. The modified enzyme (adsorbed on TiO2) absorbs UV light and initially resists

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