Enzymatic Activity of Alkaline Phosphatase Inside Protein and Polymer

Biomacromolecules 2004, 5, 572-579

572

Enzymatic Activity of Alkaline Phosphatase Inside Protein and Polymer Structures Fabricated via Multiphoton Excitation Swarna Basu and Paul J. Campagnola* University of Connecticut Health Center, Department of Cell Biology and Center for Biomedical Imaging Technology, Farmington, Connecticut 06030 Received October 20, 2003; Revised Manuscript Received November 20, 2003

We demonstrate micron scale control of bioactivity through the use of multiphoton excited photochemistry, where this technique has been used to cross-link three-dimensional matrixes of alkaline phosphatase, bovine serum albumin, and polyacrylamide and combinations therein. Using a fluorescence-based assay (ELF-97), the enzymatic activity has been studied using a Michaelis-Menten analysis, and we have measured the specificity constants kcat/KM for alkaline phosphatase in both the protein and polymer matrixes to be on the order of 105-106 M-1 s-1and are comparable to known literature values in other environments. It is found that the enzyme is simply entrapped in the polymer matrix, whereas it is completely covalently bound in the protein structures. The relative reaction rate of alkaline phosphatase bound to BSA with the ELF substrate was measured as a function of cross-link density and was found to decrease in the more tightly formed matrixes, indicating a decrease in the diffusion in the matrix. Introduction Biological systems are known to fundamentally function at the nanoscale of proteins and bio-macromolecules and at the micron scale of individual cells and cellular components. If biomolecules are to be used in engineered devices such as chip-based diagnostics, bio-microelectromechanical machines (bioMEMs),1-3 and tissue scaffolds, the technology should be organized as close as possible to the size scales of these biomolecules and cells (in three dimensions). In this work, we are interested in probing biological activity within micron scale fabricated protein and polymer matrixes. Such structures could ultimately prove useful for several biomedical applications, including the preparation of nanoscale gradient gels for separations or in the control of diffusion coefficients of compounds from gels to provide highly localized delivery of pharmaceutics and other bioactive compounds into cells and tissues. Photochemistry is an ideal approach to fabricate biomimetic structures because, unlike bulk chemistry, this provides for spatial control of the desired reaction. However, traditional methods utilizing linear excitation (one-photon) provide essentially only two-dimensional lateral capability, because, even with the use of focused light, little control over the axial dimension is possible. Thus 3D structures can be created only by “writing on top of” previously created 2D layers, with potentially altering effects on the preexisting layers. By contrast, the use of multiphoton excitation (MPE) provides intrinsic three-dimensionality,4 because the desired photochemistry can be exclusively confined to the plane of * To whom correspondence should be addressed. (P.J.C.) University of Connecticut Health Center, Department of Cell Biology, Center for Biomedical Imaging Technology, MC-1507, 263 Farmington Avenue, Farmington, CT 06030. Phone: 860-679-4354. Fax: 860-679-1039. E-mail: [email protected].

focus through precise control of the laser power. In the last several years, this method has emerged as a powerful new fabrication tool, with potential applications in 3D data storage, photonics, and photolithography already being demonstrated.5-12 Most of this work has involved the use of synthetic polymers, including acrylates and urethanes, to create structures such as waveguides and readout and display devices. In our previous work, using microscope-like optics, we carried out multiphoton excited fabrication within aqueous solutions as this enables facile 3D fabrication with proteins, other biomolecules, and hydrogels in a biocompatible environment.13-15 We have used traditional photochemistries (e.g., xanthenes) for the polymerization and developed new photoactivators based on xanthene and benzophenone chromophores for cross-linking collagens.15 An important aspect to be considered is whether bioactivity of enzymes or other bioactive molecules is maintained following exposure to the intense laser intensities that are required for efficient multiphoton excitation of the photoactivator. This is because it is conceivable that proteins can become denatured by undesired two and three-photon absorption, as well as other highly nonlinear optical processes (e.g., damaging ionization from plasma formation), resulting from these high peak powers. It is further possible that the cross-linking will block the active site. In a purely qualitative manner, we previously showed that the enzyme alkaline phosphatase (EC 3.1.3.1 or AP) displayed phosphatase activity within cross-linked matrixes of bovine serum albumin (BSA) and polyacrylamide.13 Here we extend this work to quantitatively measure the activity of alkaline phosphatase in different cross-linked proteins and polymer environments using the Michaelis-Menten kinetic analysis to determine specificity constants kcat/KM. We examine the enzyme kinetics for three distinct cases of 3D structures

10.1021/bm0344194 CCC: $27.50 © 2004 American Chemical Society Published on Web 01/13/2004

Enzymatic Activity of Alkaline Phosphatase

created via two-photon excited cross-linking and polymerization: (1) matrixes of cross-linked AP, (2) BSA/AP crosslinked mixed matrixes, and (3) AP entrapped within polyacrylamide. The first case is used to interrogate the activity of AP in the absence of other species and examine if the laser exposure and cross-linking affects the reactivity. The mixed protein and polyacrylamide matrixes are model systems for microscale delivery systems, as both BSA and polyacrylamide (in the polymeric form) provide a biocompatible environment. We show that in all cases the observed specificity constants are in good agreement with known literature values for alkaline phosphatase, indicating that its activity is retained. We further characterize the matrix properties in terms of cross-link densities and cross-linked protein concentration. Additionally, through salt extractions, we show that the enzyme is simply entrapped in the polymer matrix, whereas it is essentially all covalently bound in the protein structures. Experimental Section (i) Materials. Bovine serum albumin (Calbiochem), Rose Bengal (Sigma), calf-intestinal alkaline phosphatase (3000 units/mg, New England BioLabs), Texas Red-labeled bovine serum albumin, Rhodamine-Dextran (10 kDa), ELF-97 and ELF-97 alcohol (Molecular Probes, Eugene, OR), 30:1 acrylamide-bis acrylamide (Sigma), ascorbic acid (Aldrich), 1,4-diazabicyclo[2,2,2]-octane (DABCO), and sodium azide (Aldrich) were all used without further purification. Crosslinked protein structures involving BSA were fabricated directly on glass microscope slides that were soaked in concentrated BSA solution in order to minimize the nonspecific adsorption of AP to the glass substrate. (ii) Fabrication Apparatus. The fabrication instrument is a home-built laser scanning nonlinear optical microscope, capable of two-photon fluorescence and second harmonic generation imaging, where the design and performance have been previously described.16 The multiphoton excitation is achieved through a femtosecond near-infrared titaniumsapphire oscillator (Mira 900-F, Coherent, Santa Clara, CA) that is pumped by a 5 W Verdi Nd:YVO4 (Coherent). The repetition rate of the laser is 76 MHz, and the pulse width is approximately 100 fs. The laser is coupled to an upright microscope (Axioskop, Zeiss, Jena, Germany), equipped with bright field and fluorescence optics. A 20×, 0.75 numerical aperture (NA) objective lens (Zeiss) was used for the fabrication and resulted in lateral and axial resolutions of approximately 0.75 and 2.5 microns, respectively, as determined by post-fabrication optical and SEM imaging of crosslinked BSA structures.16 Two-photon excited fluorescence is detected in a nondescanned epi-illumination configuration, as this leads to greatly improved sensitivity over confocal detection (as a pinhole is not needed for nonlinear excitation). The fluorescence is separated from the laser by a 525 nm long pass dichroic mirror and two BG39 color glass filters (8 OD blocking) and detected in single photon counting mode. This path was also used to measure the relative twophoton absorption cross sections of Rose Bengal at 750 and 806 nm using the well-known fluorescence method.17 The

Biomacromolecules, Vol. 5, No. 2, 2004 573

code for operating this instrument for both the scan control and simultaneous data acquisition was written entirely using LabVIEW 6.1 in the form of a graphical user interface code and is freely available on our website at: http://www. cbit.uchc.edu/faculty_nv/campagnola/fabrication.html. (iii) Fabrication and Kinetic Measurements. The wavelengths used for two-photon excitation of Rose Bengal for cross-linking and polymerization were 750 and 806 nm. Typical average powers at the sample were approximately 5 mW or 100 pJ/pulse. The activity of the AP is determined through the reaction of the ELF-97 substrate (5′-chloro-2phosphoryloxyphenyl)-6-chloro-4(3H)-quinazoline (Molecular Probes), whose reaction with phosphatase is shown in Figure 1a. The one-photon absorption band for the substrate is between 300 and 400 nm, and here we use 750 nm for the two-photon excitation. Before cleavage of the phosphate group, the substrate emits only dim blue fluorescence; however, the cleaved product, ELF-97 alcohol, displays an intense fluorescence spectrum between 500 and 600 nm. Although the ELF reaction could be measured by ordinary fluorescence, we use two-photon excited fluorescence detection because this assay can be performed by imaging the MPE cross-linked matrix on the same microscope as used for the fabrication. Preparations are created on a glass microscope slide, glass spacers, and a coverslip, such that fluid can flow through the chamber and the enclosed volume is approximately 100 µL. Three-dimensional rectangular structures (160 µm × 100 µm × 6 µm) of proteins (AP and AP/BSA mixture) and polyacrylamide were fabricated for all of the measurements in this work, where the photochemistry and diagnostics have been described before.13,14 For the enzyme kinetics experiments, the fabricated samples were soaked in distilled water for 15 min to wash the residual Rose Bengal and then airdried. An aqueous solution of ELF-97 (concentration range of 5 × 10-5 to 0.5 mM) was added, and the increase in fluorescence intensity was measured across the center of the sample by line scan imaging and averaging the total intensity over several scans. These measurements were taken at intervals over a 60 min time course and the resulting rates were then plotted against the ELF-97 concentration to determine the Michaelis-Menten kinetic parameters. Results and Discussion (i) Cross-Linking Mechanism of Proteins Using TwoPhoton Excitation of Rose Bengal. In the present work, we utilize Rose Bengal (RB) photochemistries in conjunction with two-photon excitation. There are two generally accepted mechanisms for protein cross-linking with photoactivators such as Rose Bengal, both proceeding through the first triplet state: reaction with singlet oxygen or via hydrogen atom abstraction.18-23 Here we directly investigated the singlet oxygen pathway by examining the quenching effects of wellknown free radical scavengers: the azide anion,24 ascorbic acid, and 1,4-diazabicyclo[2,2,2]-octane (DABCO). We attempted to cross-link BSA in solutions that were saturated with each of these three reagents and no cross-linked matrixes were formed after prolonged exposure to two-photon excita-

574

Biomacromolecules, Vol. 5, No. 2, 2004

Basu and Campagnola

Figure 1. (a) Enzyme-catalyzed hydrolysis of ELF-97, resulting in the formation of ELF-97 alcohol. The substrate emission changes from dim blue to bright green upon cleavage of the phosphate group. (b) Power dependence curve of ELF-97 alcohol (1 mM in DMSO) measured at 750 nm, as a log-log plot of fluorescence intensity vs average power. The slope was 1.92, verifying the two-photon character of the absorption. (c) Plot of fluorescence intensity as a function of concentration of ELF-97 alcohol in DMSO, when excited by two-photon absorption at 750 nm. The response is linear up to approximately 0.5 mM.

tion (much in excess of that normally required for crosslinking), indicating that the singlet oxygen was indeed required for the reaction. The analogous experiment was also carried out using the pure enzyme solution of AP (2.5 × 10-6 M) and RB (2.5 × 10-4 M) with saturated azide, and the same lack of cross-linking was observed. A much lower concentration of ascorbic acid (∼1 mM) was found to decrease the cross-linking rate by approximately 2-fold. The singlet oxygen mechanism is indeed expected to be the favored mechanism primarily because RB has singlet oxygen yields of 75-85% (depending on solution conditions)25 and the cross-linking reactions were not taking place in an oxygen-free environment. In a previous report, the singlet oxygen pathway was not directly investigated, but we suggested that the hydrogen atom pathway was involved13 because the Rose Bengal appeared to be consumed. However, this dependence may actually have been due to bleaching of the RB by reaction with singlet oxygen, and hydrogen abstraction may have accounted for only a small portion of the observed cross-linking.

(ii) Fluorescence Characteristics of ELF-97. Our prior report on the use of alkaline phosphatase in cross-linked BSA and polyacrylamide matrixes was purely qualitative, and no kinetic measurements were attempted. To accurately quantify the bioactivity of the enzyme, the appropriate range of optical parameters has to be determined. First, by varying the laser power, the quadratic nature of the two-photon excitation of the ELF substrate was established, and the power range was chosen in order to avoid chromophore saturation. Figure 1b shows the resulting data as a log-log plot of fluorescence intensity of the fluorescent ELF alcohol vs laser power whose slope yields the order of the absorption. The fluorescence intensity shows a roughly quadratic dependence (slope ) 1.92) on laser power up to approximately 300 mW (measured before the microscope, which has approximately 2-fold further losses). Higher powers led to decreasing values of the absorption order, indicating that chromophore saturation was occurring. All experiments were done within the quadratic regime. The linear response range of the fluorescence intensity as a function of the ELF concentration was


also established using the ELF alcohol solution at constant laser power for two-photon excitation at 750 nm (Figure 1c). The fluorescence intensity was linear up to ∼0.5 mM and became sublinear at higher concentrations, indicating some fluorescence quenching was occurring. The MichaelisMenten parameters were determined within the linear concentration range of the fluorescence response. It should be noted that, although the enzymatic reaction is being carried out in an aqueous medium, the ELF alcohol product is insoluble and collects as a solid inside the protein or polymer matrix. The quantum yield of the solid product is not known; therefore, we measure the fluorescence of the product across a wide area via repetitive line scan imaging to obtain a spatial average. This is valid because this product stays localized, and therefore, the intensity measurements are self-consistent and reproducible. We did observe that, immediately following the addition of the ELF substrate, the rate of increase of fluorescence at early time has been found to be 4-fold higher along the edges of the matrix than near the center. This sharp increase, however, occurs only in the first few seconds of the monitoring process, compared to the 60 min observation time. Still, the fluorescence was measured away from the matrix borders in order to avoid this complication. (iii) Matrix Characterizations. An ultimate goal of this work is the creation of micro devices and it is therefore important to characterize the essential physical properties of the cross-linked protein and polymer matrixes. (a) Cross-Link Density and RelatiVe AP Reaction Rates. A fundamental physical property of these fabricated structures is the cross-link density, and it is important to understand how this quantity affects the diffusion and the relative rates of enzyme reaction with the ELF substrate. Conventional swelling experiments have often been used to measure this parameter in polymer gels but cannot be used on the micron scale structures, because this procedure requires weighing of the swelled product. Cross-link density has also been reported in terms of integrated laser exposure dose or some indirectly measured physical characteristic such as a decrease in amino acid (tryptophan, tyrosine) fluorescence following cross-linking.26 In previous publications, we have reporting cross-link density in terms of integrated laser exposure (in µJ/cm2) or number of scans carried out at fixed average power.13-15 As this method is self-consistent, we also use this approach here. Figure 2a shows the relative reaction rate of AP in a BSA matrix with constant ELF substrate concentration (4.0 × 10-4 M) as a function of exposure dose over a 3-fold range. As expected, a sharp decrease in the relative reaction rate is observed due to the decreased diffusion from the increased cross-linking. An asymptotic behavior is seen at the higher integrated doses (∼300 µJ/cm2), indicating the cross-link density had reached its terminal value, which we quantitatively address below. The analogous experiment was also carried out at 750 nm. We chose this wavelength because the π f π* transitions of aromatic residues such as tyrosine and phenylalanine give rise to absorption between 250 and 260 nm (accessible via three photon absorption at 750 nm) and may be possibly damaging. To compare the data at these wavelengths, the


Figure 2. Relative reaction rates of ELF-97 as a function of protein cross-link density after the photopolymerization of BSA-AP was carried out at (a) 806 nm and (b) 750 nm. Higher cross-link densities yield slower reaction rates due to decreased diffusion and become asymptotic when the available cross-linking sites on the proteins have reacted. The relative rates at the two wavelengths are also comparable, indicating little denaturing at the higher photon energy.

relative rates must be normalized by the relative two-photon absorption cross sections. By measuring the ratio of the fluorescence intensity of Rose Bengal at these two wavelengths at constant laser power and pulse width, we find the two-photon absorption cross section of Rose Bengal at 750 nm is approximately 2-fold larger than at 806 nm. The normalized curve for the relative rate at 750 nm is shown in Figure 2b and is similar to that at 806 nm and has approximately the same asymptotic value. This result indicates that even upon exposure to the higher energy photons the enzyme is still active and is consistent with results from the Michaelis-Menten kinetic analysis to be described below. This was not a foregone conclusion as excitation in this spectral range in live cells has been demonstrated to result in adverse biological effects such as abnormal cell division.27 Gratton27 suggested this occurred via destructive plasma formation, whereas Neher28 and Piston29 both demonstrated that photodamage and photobleaching in cells occurred with excitation in this spectral range with an absorption order of 2.5 or higher, indicating the damage proceeded, at least in part, via a three-photon or higher order process. This could be the result of breaking of covalent bonds as well as excitation of πfπ* transitions. This type of phototoxicity has been to shown to dramatically decrease at longer wavelengths (λ > 800 nm).27 (b) Terminal Cross-Link Density. We also need to determine if the apparent terminal cross-link density arises from using all the available reactive sites on the protein molecules

576


or simply because all of the available protein has been crosslinked. To investigate these scenarios, the diffusion coefficient of a fluorescent dye in BSA was measured after the initial fabrication process and again after separate addition of more Rose Bengal and BSA. A matrix of BSA was crosslinked at high laser exposure dose in the asymptotic region determined previously (approximately 300 µJ/cm2) and then soaked in a 1 mM 10 kD Rhodamine-Dextran solution. The diffusion coefficient was determined through the fluorescence recovery after the photobleaching (FRAP) method, using two-photon excitation.30 The bleach was performed at a single point, and the recovery was then monitored via line scan imaging with the laser power attenuated by 70%. Using an exponential fit, the recovery time, τD, was calculated to be approximately 4.4 s, corresponding to a diffusion coefficient D ) 1.0 × 10-10 cm2/s. To verify whether all available sites had formed cross-links, a solution of Rose Bengal was added and the structure exposed to full laser power for another 5 scans (∼33 µJ/cm2) and the FRAP experiment repeated. No increase in the τD of the Rhodamine-Dextran was observed (∼4.2 s), as would be expected had there been an increase in the cross-link density of the BSA matrix. This suggests that all available cross-linking sites had already reacted. The second related issue is whether further crosslinking can be promoted by the addition of more protein. To investigate this, we added a solution of BSA and Rose Bengal to the existing matrix and exposed it to the same initial laser flux. Similarly, a statistically similar value of τD (∼4.2 s) was obtained from the FRAP measurement. Thus, the asymptotes observed in Figure 2 of the relative reaction rate of AP indeed correspond to the limit of terminal crosslinking. This result could also in principle have been determined by measuring the tryptophan emission before and after cross-linking, where the reaction destroys the chromophore.26 However, the fabricated structures were too small to provide sufficient sensitivity in a conventional fluoremeter. It should be noted that the amount of laser exposure at which protein cross-link density reaches a terminal value varies from protein to protein and is the focus of our current work. A final consideration is whether the heights of cross-linked structures increase upon attempting to increase the crosslink density. Figure 3 shows the heights of two BSA structures, each fabricated with different laser exposure dose. These were measured by imaging the step height via Rose Bengal fluorescence at higher NA (1.3) than was used for the fabrication (0.75). The structures show no increase in height (∼6 µm) even when the total exposure dose (or number of scans at fixed power) is increased up to 60% or more. (c) Binding of AP in Protein and Polymer Matrixes. An important issue is the nature of the binding of the enzyme in the protein and polymer matrixes. For example, the enzyme can be simply entrapped or covalently bound. This can be determined by attempting to extract the enzyme out of the matrix using a concentrated salt solution. A mixed protein (BSA/AP) structure of high, asymptotic cross-link density (∼375 µJ/cm2 laser exposure dose) was fabricated and the reaction rate with the ELF substrate was measured as before. An identical structure was soaked for 24 h in 2 M

Basu and Campagnola

Figure 3. Heights of BSA matrixes following fabrication with exposure doses of approximately (a) 95 and (b) 155 µJ/cm2.

NaCl, and the ELF substrate was then added after the extraction medium was removed. The relative reaction rates (as measured by the increase in ELF fluorescence) for the un-extracted and extracted matrixes were within 10% of each other, clearly indicating that the enzyme was not extracted and was covalently bound to the protein matrix. Repeating this experiment at lower cross-link densities yielded similar differences (5-10%) in relative rates. To further corroborate this finding, a UV absorption spectrum of the extracted solution was taken, and although this showed a small concentration of protein (absorption at 260 nm), there was no ELF activity of this solution. Although remote, we investigated the possibility that the enzyme was extracted but became denatured in the high salt solution. The control experiment of AP reacting with the ELF substrate in 2 M NaCl solution verified that the enzyme was still active under these conditions. Together, these findings allow us to place a lower bound of 90% of the AP being covalently linked in the BSA/AP matrix. Similar experiments were carried out using polyacrylamide gels, where, in this case, the enzyme is more likely to be entrapped rather than covalently bound. This is because, unlike proteins, the acrylamide needs an amine co-initiator for photopolymerization with Rose Bengal, and we have shown previously that the amine has a very large suppressive effect on protein cross-linking (at least 100 fold).13 Following fabrication, the polyacrylamide matrix was soaked in 2 M NaCl. The salt solution was then removed, and no enzymatic activity was observed in the polymer matrix following the addition of the ELF. This result proves that the hydrogel is indeed only trapping the enzyme. The UV absorption spectrum of the extracted solution showed the presence of


protein (at 260 nm) and also showed reactivity with the ELF substrate. Together, these findings show that enzyme is only entrapped (and in fact stays active following the extraction). In a later section, we use this approach to determine the exact concentration of enzyme present in the acrylamide gels. (d) Concentration of Protein in Cross-Linked Matrixes. To accurately measure the Michaelis-Menten kinetic parameters (namely kcat) for alkaline phosphatase, the exact enzyme concentration inside the matrix needs to be determined. Although the concentration of the enzyme in the initial solution is 2.5 × 10-6 M, the cross-linking may result in a higher concentration of the enzyme inside the matrix. To determine the actual protein concentration, we take a ratiometric approach by cross-linking fluorescently labeled BSA (Texas Red) and comparing the fluorescence in the starting solution to that in the cross-linked protein matrix. Because AP and BSA have similar molecular weights, it is reasonable to assume the same final ratio of initial and bound concentrations in the cross-linked matrixes for both these proteins. Several BSA matrixes were fabricated over a large range of cross-link density, and the fluorescence of the Texas Red-BSA conjugate was then measured using two-photon excitation at 850 nm. Because there is no appreciable Rose Bengal two-photon absorption at this wavelength, and the Texas Red fluorescence quantum yield is much greater than that of Rose Bengal (at least 50-fold), the measurement is essentially background free. At terminal cross-link density, as used in the subsequent Michaelis-Menten kinetic measurements, the fluorescence of the cross-linked protein was approximately 8 times that in free solution. We then use this ratio to determine the final concentration of AP in the protein matrixes, obtaining a value of 2.0 × 10-5 M. In the case of the polyacrylamide gels, we directly determined the AP concentration by extracting the enzyme with 2 M NaCl and measuring the AP concentration by UV absorption spectroscopy ( ) 87 000 M-1cm-1). The concentrations of alkaline phosphatase were determined to be 1.50 × 10-5 M in 15% acrylamide and 4.4 × 10-6 M in 25% acrylamide, both higher than the concentration of the enzyme in the initial solution (2.50 × 10-6 M). (iv) Activity of Alkaline Phosphatase in Protein and Polymer Matrixes. (a) ActiVity in Pure Alkaline Phosphatase Matrixes. In this section, using Michaelis-Menten kinetics analysis, we quantitatively determine the activity of the alkaline phosphatase (AP) in pure AP matrixes, in BSAAP mixed protein matrixes, and in polyacrylamide gels by evaluating the specificity constant kcat/KM. We first investigate the case of structures created exclusively from alkaline phosphatase. Matrixes consisting of AP were fabricated in the asymptotic limit of the cross-link density found earlier. Figure 4a shows a representative plot of the temporal evolution of the ELF fluorescence intensity ([S] ) 6 × 10-4 M), whose slope yields the reaction velocity, V. The velocity was analogously determined for many values of [S], and Figure 4b plots the rate vs substrate concentration and the expected hyperbolic form31 is observed. This data is then re-plotted in Figure 4c in the Michaelis-Menten form of 1/V vs 1/[S], to determine kcat and KM. To calculate kcat, we take the actual enzyme concentration to be 2.0 × 10-5 M,


Figure 4. Kinetic analysis of cross-linked alkaline phosphatase: (a) a typical plot used to extract the rate v, showing the increase in fluorescence intensity over time as the enzymatic reaction progresses, where the ELF substrate concentration was 6 × 10-4M; (b) plot of reaction rate (v) against substrate concentration, [S], showing the expected hyperbolic dependence from the Michaelis-Menten equation; (c) Michaelis-Menten plot of 1/v against 1/[S], which yielded kcat/KM on the order of 10-5 M-1 s-1 which is in good agreement with literature values, indicating enzymatic activity was retained following the two-photon cross-linking process.

based on the increase in fluorescence of labeled BSA at the same cross-link density. The first column of Table 1 summarizes the resulting values for KM, kcat, and the specificity constant (kcat/KM) for AP. The observed value of approximately 105 M-1 s-1 for kcat/KM is consistent with that reported from other systems,32-36 indicating that the enzyme indeed stays active following the multiphoton excited crosslinking process, whereas a dramatic decrease in kcat/KM would have implied that the AP was significantly denatured. The active site in AP consists of three metal ions and three amino acids and is fairly compact and protected. Furthermore, the amino acids (Asp 101, Ser 102, and Ala 103) are not aromatic and unlikely to become cross-linked via the singlet oxygen mechanism of Rose Bengal photoactivation discussed earlier. Thus, the site remains available for its biological phosphatase function. Additionally, due to the lack of aromaticity, these residues are not expected to be excited by three photon absorption at the potentially damaging 800 nm wavelength (266 nm via three-photon absorption) used

578


Basu and Campagnola

Table 1. Kinetics Data for Alkaline Phosphatase under Different Conditions

KM kcat kcat/KM (M-1 s-1)

AP-AP

AP-BSA

AP-acrylamide (25%)

AP-acrylamide (15%)

0.10 ( 0.03 mM 13.5 ( 2.4 s-1 1.35 ( 0.47 (x 105)

0.21 ( 0.05 mM 21.9 ( 4.5 s-1 1.04 ( 0.33 (x 105)

0.11 ( 0.01 mM 393.1 ( 36.8 s-1 3.57 ( 0.46 (x 106)

0.015 ( 0.001 mM 102.3 ( 4.7 s-1 6.82 ( 0.55 (x 106)

Figure 5. Michaelis-Menten plots showing retained enzymatic activity when alkaline phosphatase is in different hosts: (a) crosslinked with BSA; (b) entrapped inside a 25% polyacrylamide gel; (c) entrapped inside a 15% polyacrylamide gel. The kinetic parameters have been calculated in each case and are listed in Table 1. The increase for the polyacrylamide over the proteins in terms of kcat/KM is likely due to the increase in mobility in the gel and availability of the active site over the cross-linked protein matrix.

here, because the σ f σ* transitions will lie further to the blue. Thus, it is chemically reasonable for this enzyme to retain its activity following the MPE cross-linking process. (b) AP ActiVity in an AP/BSA Matrix. We performed the analogous kinetic experiments on MPE cross-linked matrixes consisting of BSA and AP. Figure 5a shows the resulting Michaelis-Menten plot for the AP activity, and the resulting

kcat and KM parameters are given in the second column of Table 1. It is observed that within experimental error a comparable kcat/KM of approximately 1 × 105 M-1 s-1 is obtained relative to that of the pure AP case, indicating a similar retention of enzymatic activity. Given the similar environments of the two protein matrixes, comparable diffusion and reactivity are indeed expected. (c) ActiVity of AP Inside Polyacrylamide Gels. Due to the biocompatibility in the polymerized form, we choose to use polyacrylamide as a model host in which to measure the activity of AP following MPE cross-linking. Even though monomeric acrylamide is toxic, the nontoxic polymeric form has been used as a model delivery system.37,38 Thus, polymerized structures could be fabricated in vitro and then used as implantable devices. Additionally, this is a good model system because, unlike many synthetic polymers, acrylamide is a hydrogel and the water content can be varied over a wide range, allowing for large changes in diffusion coefficients and migration rates under electric fields for making microscale gels. Three-dimensional polyacrylamide matrixes were fabricated as before, and Figure 5b shows the resulting Michaelis-Menten plot for the 25% V/V matrix, and the resulting kinetic parameters for acrylamide are summarized in Table 1 (column 3). This data shows that the kcat/KM value for AP in 25% acrylamide is approximately 3.0 × 106 M-1 s-1, or about 20-fold larger than the protein cases. More specifically, the KM value is similar to that observed in the protein matrix, but kcat is significantly higher, as would be expected for the increased diffusion and active site accessibility for the entrapped enzyme in the hydrogel relative to the covalently bound protein matrixes. The analogous Michaelis-Menten analysis was carried out for 15% V/V polyacrylamide, where the laser power, and therefore the cross-link density, was kept constant. The data are shown in Figure 5c, where a 2-fold increase in kcat/KM (∼7 × 106 M-1 s-1) over the 25% acrylamide is observed. This is perhaps due to the fact that the active site of the enzyme is more accessible in the more porous polyacrylamide gel at the lower volume fraction. It should be noted that, although kcat/KM increases for the case of acrylamide over the protein, this does not imply that the activity in the protein cases was limited, as the differences in the chemical environment will affect the diffusion within the matrixes. Conclusions We have demonstrated the fabrication of three-dimensional micron scale structures made from bioactive proteins or polymers whose physical and biological properties can be well-controlled. In particular, we have quantitatively shown that alkaline phosphatase retains its activity in cross-linked matrixes consisting of itself, BSA/AP mixtures, and within


polyacrylamide gels. We find specificity constants, kcat/KM, to be on the order 105-106 M-1 s-1 and these agree well with literature values for AP from other systems. We have shown that the relative reaction rates can be controlled by varying the cross-link density of the protein matrix and also determined the nature of binding of the enzyme in both the protein and polymer matrixes. We observed no apparent denaturing at higher energy excitation that could in principle excite aromatic residues on the protein molecules or lead to bond breaking. Our results show that in both protein and polyacrylamide environments it is possible to retain biological activity and this opens up the possibility of creating in vivo devices through multiphoton excited cross-linking. Acknowledgment. Support under NIH R01 GM60703 is gratefully appreciated. We also thank Rajesh Nagarajan, Prof. Rex Pratt, Dr. Jonathan Pitts, Dr. Steven Goodman, and Victoria Scranton for helpful discussions. References and Notes (1) Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster, J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo, C. H.; Burke, D. T. Science 1998, 282, 484-487. (2) Weigl, B. H.; Yager, P. Science 1999, 283, 346-347. (3) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Biotechnol. Prog. 1998, 14, 356-363. (4) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73-76. (5) Strickler, J. H.; Webb, W. W. Opt. Lett. 1991, 16, 1780-1782. (6) Maruo, S.; Nakamura, O.; Kawata, S. Opt. Lett. 1997, 22, 132134. (7) Cumpston, B. H.; Ananthavel, S. P.; Barlow, S.; Dyer, D. L.; Ehrlich, J. E.; Erskine, L. L.; Heikal, A. A.; Kuebler, S. M.; Lee, I.-Y. S.; McCord-Maughon, D.; Qin, J.; Marder, S. R.; Perry, J. W. Nature 1999, 398, 51-54. (8) Bhawalkar, J. D.; Swiatkiewicz, J.; Pan, S. J.; Samarabandu, J. K.; Liou, W. S.; He, G. S.; Berezney, R.; Cheng, P. C.; Prasad, P. N. Scanning 1996, 18, 562-566. (9) Witzgall, G.; Vrijen, R.; Yablonovitch, E.; Doan, V.; Schwartz, B. J. Opt. Lett. 1998, 23, 1745-1747. (10) Kawata, S.; Sun, H.-B.; Tanaka, T.; Takada, K. Nature 2001, 412, 697-698. (11) Belfield, K. D.; Liu, Y.; Negres, R. A.; Fan, M.; Pan, G.; Hagan, D. J.; Hernandez, F. E. Chem. Mater. 2002, 14, 3663-3667. (12) Olson, C. E.; Previte, M. J.; Fourkas, J. T. Nat. Mater. 2002, 1, 225228.

Biomacromolecules, Vol. 5, No. 2, 2004 579 (13) Pitts, J. D.; Campagnola, P. J.; Epling, G. A.; Goodman, S. L. Macromolecules 2000, 33, 1514-1523. (14) Campagnola, P. J.; R., H. A.; Delguidas, D.; Epling, G. A.; Pitts, J. D.; Goodman, S. L. Macromolecules 2000, 33, 1511-1513. (15) Pitts, J. D.; Howell, A. R.; Taboada, R.; Banerjee, I.; Wang, J.; Goodman, S. L.; Campagnola, P. J. Photochem. Photobiol. 2002, 76, 135-144. (16) Sridhar, M.; Basu, S.; Scranton, V. L.; Campagnola, P. J. ReV. Sci. Instrum. 2003, 74, 3474-3477. (17) Xu, C.; Williams, R. M.; Zipfel, W. R.; Webb, W. W. Bioimaging 1996, 4, 198-207. (18) Foote, C. S. In Free Radicals in Biology; Pryor, W. A., Ed.; Academic Press: New York, 1976; Vol. 2, pp 85-134. (19) Webster, A.; Britton, D.; Apap-Bologna, A.; Kemp, G. Anal. Biochem. 1989, 179, 154-157. (20) Balasubramanian, D.; Du, X.; Zigler, J. S. J. Photochem. Photobiol. 1990, 52, 761-768. (21) Verweij, H.; Dubbelman, T. M.; Van Steveninck, J. Biochim. Biophys. Acta 1981, 647, 87-94. (22) Shen, H. R.; Spikes, J. D.; Kopecekova, P.; Kopecek, J. J. Photochem. Photobiol. B 1996, 34, 203-210. (23) Shen, H. R.; Spikes, J. D.; Kopeckova, P.; Kopecek, J. J. Photochem. Photobiol. B 1996, 35, 213-219. (24) Li, M. Y.; Cline, C. S.; Koker, E. B.; Carmichael, H. H.; Chignell, C. F.; Bilski, P. Photochem. Photobiol. 2001, 74, 760-764. (25) Neckers, D. C. J. Photochem. Photobiol. A 1989, 47, 1-29. (26) Gilbert, D. L.; Kim, S. W. J. Biomed. Mater. Res. 1990, 24, 12211239. (27) Konig, K.; So, P. T. C.; Mantulin, W. W.; Gratton, E. Opt. Lett. 1997, 22, 135-136. (28) Hopt, A.; Neher, E. Biophys. J. 2001, 80, 2029-2036. (29) Patterson, G. H.; Piston, D. W. Biophys. J. 2000, 78, 2159-2162. (30) Brown, E. B.; Wu, E. S.; Zipfel, W.; Webb, W. W. Biophys. J. 1999, 77, 2837-2849. (31) Zubay, G. Biochemistry Vol. 1. Energy, Cells and Catalysis; W. C. Brown Publishers: Dubuque, IA, 1993. (32) Kim, E. E.; Wyckoff, H. W. J. Mol. Biol. 1991, 218, 449-464. (33) Portmann, P.; Joerg, A.; Furrer, K.; Walker, H. S.; Leuthard, P.; Sudan, J. F.; Perriard, F.; Comment, J. F.; Leva, G.; Nell, J. P. HelV. Chim. Acta 1982, 330, 2668-2681. (34) Shan, Y.; McKelvie, I. D.; Hart, B. T. Anal. Chem. 1993, 65, 30533060. (35) Zhang, Y. X.; Zhu, Y.; Zhou, H. M. Int. J. Biochem. Cell. Biol. 2000, 32, 887-894. (36) Cox, W. G.; Singer, V. L. J. Histochem. Cytochem. 1999, 47, 14431456. (37) Clark, H. A.; Hoyer, M.; Philbert, M. A.; R., K. Anal. Chem. 1999, 71, 4831-4836. (38) Clark, H. A.; Kopelman, R.; Tjalkens, R.; Philbert, M. A. Anal. Chem. 1999, 71, 4837-484843.

BM0344194

Enzymatic Activity of Alkaline Phosphatase Inside Protein and Polymer

Recommend Documents