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J. Phys. Chem. B 2001, 105, 6278-6280
Horseradish Peroxidase Adsorption on Silica Surfaces as an Oscillatory Dynamical Behavior Ewa S. Kirkor† and Alexander Scheeline*,† Department of Chemistry, UniVersity of Illinois at Urbana-Champaign, Urbana, Illinois 61801 ReceiVed: March 27, 2001; In Final Form: May 23, 2001
Oxidation of Horseradish Peroxidase (HRP) to Compound I (CpI) induces release of the protein from adsorbing silica surfaces. Adsorption and desorption can be modulated by changes in concentration of hydrogen peroxide in solution in contact with the adsorber. Coupling of the HRP adsorption-desorption cycles to the chemistry of the peroxidase in bulk solution by oscillations in concentration of hydrogen peroxide is postulated as a plausible explanation for a qualitative change in the dynamics of the peroxidase oscillator at low concentrations of the enzyme. We hypothesize that the relative change in the HRP concentration in solution will be most pronounced at low concentrations of the enzyme and high reactor surface-to-volume ratio.
Introduction Our interest in retention of horseradish peroxidase (HRP, native state ferriperoxidase, Per3+) on silica (quartz) surfaces stems from a potential effect of adsorption on the dynamics of an oscillatory oxidation of NADH catalyzed by peroxidases (the PO reaction1). A prototype for biological oscillatory reaction, the PO reaction is a much-studied model of nonlinear systems.2-4 In general, proteins are known to bind to various oxides, silica among them. Adsorption of proteins to these surfaces is nonspecific and practically irreversible.5 As a rule, an ability to manipulate adsorption of a protein requires information on its properties, or at least its class.6 HRP adsorption properties are important for its multitude of analytical applications. Often, HRP is used adsorbed on silica particles, glass, or metal oxides. Compound I (CpI), formed in a reaction of native enzyme with hydrogen peroxide, initiates oxidations catalyzed by HRP. The dissolved protein activity depends on its pKa, pI, and the acidity of the solvent.7 Complexation or protonation of the native enzyme, which decreases its reactivity toward H2O2, lowers the observed rate constant for CpI formation. At pH values at or below the pKa for protonation of the imidazole group of the distal histidine of the enzyme cavity (pKa ) 4.1), the rate constant for CpI formation significantly decreases. In the presence of acetate, the rate constant of CpI formation (2 × 107 M-1 s-1) remains stable in the pH 5-8 range.7 A decline of the activity of adsorbed enzyme is interpreted in terms of conformational relaxation induced by adsorption.8 The magnitude of the decline is disputed, and the enzyme regains its full activity upon reentering solution. A slow permanent loss of activity attributed to structural distortion progresses over tens of hours.9 Adsorption on hydrophilic silicas (silanol termination) is presumably different from hydrophobic ones (siloxane termination). The extent of adsorption of the native enzyme may reach ten-monolayer coverage on some silica-containing surfaces.10 Current work has been conducted under mildly acidic conditions (0.1 M sodium acetate buffer, pH 5.1, our oscillatory reaction medium) on HRP nominally containing mainly isoform C1, and on hydrophilic silicas. Concentration of the enzyme in solution is an important experimental parameter controlling reaction dynamics. Existing †
E-mail:
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[email protected].
models of the PO oscillator have difficulty in consistently simulating reaction dynamics for the whole range of experimentally accessible concentration of HRP, although significant progress in explaining low concentration behavior has recently been made.4 Bistability and bursting that develop at low enzyme concentrations (below 1 µM) are difficult to simulate. Obviously, adsorption of HRP on the walls of the reactor can diminish the enzyme concentration in solution and so affect the reaction dynamics. A selection of the reactor material (polypropylene vs quartz) influences the frequency, amplitude, and duration of oscillations in the PO reaction.11,12 Neither coupling of HRP reactivity on the walls of the reactor to the chemistry of the bulk of solution nor heterogeneity in PO reactions has been established. The presently described observation of reactive desorption of HRP from silica surfaces provides qualitative information about a mechanism through which chemistry of HRP on the walls of a quartz reactor could influence the dynamics of the chemistry in solution. Experimental Section To minimize HRP losses from solution, polypropylene containers were used. Adsorption of HRP on hydrophilic silica surfaces (NP2 chips and quartz) was monitored in SELDI-TOFMS (surface enhanced laser desorption ionization-time-of-flightmass spectrometry) experiments carried out with a Protein Chip Reader Series PBS II (Ciphergen Biosystems, Inc., Fremont, CA). The m/z calibration was based on the manufacturer’s protocol and verified using carbonic anhydrase (Sigma, Mw 29 024). HRP (Boehringer-Mannheim) 1-10 µM solutions were prepared in 0.1 M sodium acetate at pH 5.1. To assess HRP losses due to adsorption on polypropylene or quartz surfaces, a comparative solution depletion method was used. Quartz was rendered hydrophilic by a minimum 10 min soak in solution of Nochromix (Aldrich) in concentrated sulfuric acid, followed by extensive rinsing with water. The same treatment was given to polypropylene containers. SELDI samples were prepared by deposition of 0.5-5 µL aliquots of HRP on the chip surface for 2-10 min. The spots were subsequently washed twice with 5 µL of water. (HPLC quality solvents were used throughout this work.) The treated area was covered with 0.5 µL of a saturated solution of matrix forming 3-(4-hydroxy-3,5-dimethoxy-
10.1021/jp011148n CCC: $20.00 © 2001 American Chemical Society Published on Web 06/15/2001
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Figure 2. Change over time in HRP adsorption ratio from Per3+ and CpI solutions. Linked black squares indicate averages of 8 independent experiments each. Error bars show largest departures from averages.
Figure 1. Representative pair of SELDI spectra of HRP retained on silica (NP2 Chip) after H2O (top) or H2O2 (bottom) wash. See text for procedural details.
phenyl)-2-propenoic acid (sinapinic acid, Mw ) 224.212) with 0.5% trifluoroacetic acid. The matrix components were dissolved in a 50% solution of acetonitrile in water. The matrix was allowed to crystallize prior to analysis that was carried out according to the manufacturer’s protocol. HRP was converted to CpI by reaction with H2O2 in acetate buffer, where the concentration of added H2O2 exceeded the concentration of HRP by 20%. The CpI solution was used within 3 min of preparation. Confirmatory experiments demonstrating adsorption of Per3+ were carried out by spectrophotometrically monitoring absorption of native HRP (HP8453A diode array spectrometer, Hewlett-Packard Inc.). One milliliter of HRP between 0.34 and 2.2 µM, in pH 5.1 acetate buffer was placed in a polypropylene cuvette at ambient temperature (21 °C). Quartz plates, 0.5 cm × 4 cm, were cleaned with enzyme detergent, then rinsed copiously with Milli-Q water (Millipore Corp.), and air-dried. The quartz plates were dipped into the HRP solution for 30 s to a depth such that total exposed quartz surface area was approximately 6 cm2. The quartz was then cleaned, the spectrometer re-referenced, and the measurement repeated 10 times. Absorbance changes indicated the fraction of enzyme adsorptively removed. Results HRP is a glycoprotein. Isozyme C1 contains 308 amino acids in a single polypeptide, heme prosthetic group, two calcium ions, and eight carbohydrate chains. The molecular mass of the polypeptide is 33 890 Da. An addition of heme and calcium ions increases the mass to 34 590 Da. Glycosylation varies from 17 to 21% carbohydrate. The consensus average carbohydrate structure leads to a molecular mass of 43 955 Da. Oxidized forms of HRP were detected as native peroxidase in SELDI experiments, as sinapinic acid is an effective reducing substrate for the enzyme. SELDI spectra of our HRP samples (Figure 1a) indicate a higher molecular weight than MALDI (42 870), reported for a preparation from Sigma13 in the same matrix. The difference is consistent with the known variable extent of glycosylation. The HRP SELDI mass spectra are very
broad, with two singly protonated, partly overlapping peaks present at 43 560 ( 30 and 44 540 ( 30 Da (Figure 1a). After two water washes the proportion of these peaks was stable. Their difference corresponds to 4-fold the molecular weight of the matrix; consequently, an association with the sinapinic acid is an unlikely explanation for the two peaks. Weighted by relative abundance, our average molecular weight is 43 960 ( 50 Da (n ) 24), very close to the average molecular weight reported by K. Welinder.14 In light of known variability of glycosylation,7 the two peaks most likely arise from differences in the protein’s carbohydrate content. The shape of the peaks shows no significant changes in all of our tests. Subsequent analyses were based on the amplitude of the more intense 43 560 mass peak. The amplitude of the SELDI signal was proportional to the sample loading on NP2 chips with the relative standard deviation (15% (n ) 8 in each of 3 independent experiments, surface exposure about 3 min). The results are reported as averages and ranges of departures. In differential depletion measurements, a solution of HRP was exposed to macroscopically equivalent surface areas of polypropylene or quartz reactor walls for 10 min. SELDI signals derived from equal aliquots of HRP solution exposed to polypropylene were 2-5-fold more intense than those exposed to quartz, confirming more extensive adsorption of the protein on quartz reactor walls than on polypropylene. What happens when HRP is oxidized on the silica surface? The ratio of SELDI amplitudes corresponding to a double wash of HRP adsorbed on an NP2 chip with 5 µL of water or 1 × 10-4 M H2O2 was 1 ( 0.6 and 0.07 ( 0.04 (n ) 24), respectively, clearly indicating peroxide-induced desorption of HRP (Figure 1 shows representative spectra). Do oxidized forms of HRP adsorb as readily on silica as the native Per3+? Equimolar solutions of Per3+ and CpI were applied to separate NP2 chip spots in 2.00 µL aliquots, and allowed to adsorb. The adsorption was measured at 2, 3, 5, 7.5, and 10 min. The ratio of SELDI signals changed from ca. 10:1 to 4.5: 1, while exposure time changed from 2 to 10 min. In 10 min, a large fraction of CpI in solution returns to the native ferriperoxidase, as CpI lifetime in solution is limited to ca. 20 min. An excess of H2O2 in a solution of CpI (with ferriperoxidase and CpI still equimolar) facilitated observation of larger differences in adherence to the NP2 surface for longer times, Figure 2. We conclude that, upon oxidation, HRP desorbs from silica surfaces and readsorbs upon return to the native state. Spectrometric monitoring of absorption at 402 nm indicated approximately monolayer coverage of the quartz surface. Table 1 shows that in 30 s approximately 30 pmol adsorb on 6 cm2
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TABLE 1: Changes in Ferriperoxidase Concentration after 30 s Exposure to Clean Quartz Surface initial [Per3+] (µM)
% removed by adsorption (1σ error)
apparent adsorbed moles (on 6 cm2)
apparent molecular area (nm2)
2.2 1.3 1.0 0.8 0.34
0.85 ( 0.10 3.4 ( 1.0 3.7 ( 1.0 3.8 ( 1.0 9.4 ( 2.0
1.9 × 10-11 4.4 × 10-11 3.7 × 10-11 3.0 × 10-11 3.2 × 10-11
52 23 27 33 31
of clean quartz. Given that the unit cell for a crystal of isozyme C1 of HRP has dimensions ∼15.9 × 15.9 × 11.4 nm,15 the apparent coverage is ∼5-8 molecular layers (molecular area between 160 and 225 nm2 from the X-ray structure vs ∼30 nm2 observed here). Note that the experimental procedure is immune to error from removal of bulk solution, as this would not effect solution concentration or optical absorption.
Much research has been done on surface-catalyzed oscillatory reactions such as O2 + CO on Pt18 and other reactions adjacent to surfaces.19,20 Enzymes are frequently membrane-bound and can give rise to unique dynamics due to the interaction of transport and enzyme kinetics.21,22 One of the attractions of the peroxidase-oxidase oscillator has been the simplicity of being able to treat it as a homogeneous system, allowing use of ordinary differential equation models. If the adsorption phenomena shown here are significant in oscillatory reaction experiments, there may be important limits to the precision of modeling that ignores wall effects, global coupling, and spatial heterogeneity. Acknowledgment. Ciphergen Biosystems, Inc., Fremont, CA, generously provided access to the SELDI instrumentation. Neil L. Kelleher is acknowledged for providing the sample of carbonic anhydrase with ESI-FTMS verified molecular mass. The University of Illinois and National Science Foundation grant CHE 96-15739 supported this work.
Discussion The native HRP adsorbs on silica surfaces. Reactive desorption from the surface can be observed with the use of dilute hydrogen peroxide (compare Figure 1a with 1b). Oxidized forms of HRP adsorb on the silica surface much less readily than does the native peroxidase. The difference in adherence to silica might be related to the location of charges in oxidized vs native peroxidase. The structure of Compound I corresponds to a ferryl porphyrin π-cation radical. The unpaired electron is mobile, as evident from formation and cross-linking of secondary radicals.16 Manipulation of electrostatic interactions is a well-known factor controlling protein adsorption. A change in localization of a single charge can modify protein adsorption.17 We suppose, such change is responsible for the HRP desorption upon formation of the Compound I. The absorbance data indicate that, once some HRP adsorbs on quartz, additional layers also bind. The walls of a quartz reaction vessel can affect the dynamics of PO reaction by modulating concentration of the enzyme in solution in concert with oscillations of concentration of hydrogen peroxide. Increase in hydrogen peroxide concentration will drive the conversion of the native ferriperoxidase to CpI, simultaneously desorbing enzyme into solution. Such an effect could be most pronounced at relatively low concentrations of the enzyme in solution and large surface-to-volume ratio. The data in Table 1 indicate that the effect could modulate enzyme concentration at the few percent level for approximately micromolar enzyme concentrations in reaction cells of ca. 1 cm2 area. At less than micromolar concentrations, it has been noted that the enzyme’s dynamics are qualitatively different from the high concentration regime. A recently modified model explained this behavior by adding bulk reactions to those employed by previous models.3,4 The nature of this effect might also be related to the larger proportion of the enzyme occupying adsorption sites on the walls of the reactor. Our finding presents an additional explanation for the observed change in the dynamics.
References and Notes (1) Yamazaki, I.; Yokota, K. Biochim Biophys. Acta 1967, 132, 310320. (2) Scheeline, A.; Olson, D. L.; Williksen, E. P.; Horras, G. A.; Klein, M. L.; Larter, R. Chem. ReV. 1997, 97, 739-756. (3) Bronnikova, T. V.; Schaffer, W. M.; Olsen, L. F. J. Phys. Chem. B 2001, 105, 310-321. (4) Schaffer, W. M.; Bronnikova, T. V.; Olsen, L. F. J. Phys. Chem. B 2001, 105, 5331-5340. (5) Hlady, V.; Buijs, J.; Jennissen, H. P. Methods Enzymol. 1999, 309, 402-429. (6) Larive, C. K.; Lunte, S. M.; Zhong, M.; Perkins, M. D.; Wilson, G. S.; Gkulrangan, G.; Williams, T.; Afroz, F.; Schoneich, C.; Derrick, T. S.; Middaugh, C. R.; Bogdanowich-Knipp, S. Anal. Chem. 1999, 71, 389R423R. (7) Dunford, H. B. Heme Peroxidases; Wiley-VCH: New York, Chichester, Weinheim, Brisbane, Singapore, Toronto, 1999. (8) Kondo, A.; Murakami, F.; Kawagoe, M.; Higashitani, K. Appl. Microbiol. Biotechnol. 1993, 39, 726-731. (9) Lobel, K.; Hench, L. J. Biomed. Materials Res. 1998, 39, 575579. (10) Klint, D.; Erkisson, H. Protein Expression Purification 1997, 10, 247-255. (11) Lvovich, V. F. Characterization of Solution Species Relevant to the Peroxidase-NADH Biochemical Oscillator. Ph.D. Thesis, University of Illinois at Urbana-Champaign, 1998. (12) Olson, D. L. Experimental and Theoretical Studies of the Peroxidase-NADH Biochemical Oscillator: An Enzyme-Mediated Chemical Switch. Ph.D. Thesis, University of Illinois at Urbana-Champaign, 1994. (13) Gowda, D. C.; Ambekar, S. Y.; Gupta, P.; Lecchi, P.; Pannell, L. K.; Davidson, E. A. Bioconjugate Chem. 1996, 7, 265-270. (14) Welinder, K. Eur. J. Biochem. 1979, 96, 483-502. (15) Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nature Struct. Biol. 1997, 4, 1032-1038. (16) Giulivi, C.; Cadenas, E. Free Radic. Biol. Med. 1998, 24, 269279. (17) Ramsden, J. J.; Roush, D. J.; Gill, D. S.; Kurrat, R.; Willson, R. C. J. Am. Chem. Soc. 1995, 117, 8511-8516. (18) Graham, M. D.; Kevrekidis, I. G.; Asakura, K.; Lauterbach, J.; Krischer, K.; Rotermund, H. H.; Ertl, G. Science 1994, 264(5155), 80-82. (19) Salje, E. K. H. Eur. J. Mineral. 1995, 7, 791-806. (20) Zhdanov, V. P.; Kulginov, D.; Kasemo, B. Phys. ReV. E: Stat. Phys., Plasma, Fluids, Relat. Interdiscip. Top. 1996, 53, R3013-R3016. (21) Zou, X.; Siegel, R. A. J. Chem. Phys. 1999, 110, 2267-2279. (22) Larter, R. Chem. ReV. 1990, 90, 355-81.
