Article pubs.acs.org/Langmuir
Structure and Enzymatic Properties of Molecular Dendronized Polymer−Enzyme Conjugates and Their Entrapment inside Giant Vesicles Andrea Grotzky,† Emiliano Altamura,†,‡ Jozef Adamcik,§ Paolo Carrara,∥ Pasquale Stano,∥ Fabio Mavelli,‡ Thomas Nauser,⊥ Raffaele Mezzenga,§ A. Dieter Schlüter,† and Peter Walde*,† †
Laboratory of Polymer Chemistry, Department of Materials, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland Chemistry Department, University “Aldo Moro”, via Orabona 4, 70125 Bari, Italy § Laboratory of Food & Soft Materials, Institute of Food, Nutrition and Health, Department of Health Sciences and Technology, ETH Zürich, Schmelzbergstrasse 9, 8092 Zürich, Switzerland ∥ Sciences Department, University of Roma Tre, Viale Guglielmo Marconi 446, 00146 Rome, Italy ⊥ Laboratory of Inorganic Chemistry, Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland ‡
S Supporting Information *
ABSTRACT: Macromolecular hybrid structures were prepared in which two types of enzymes, horseradish peroxidase (HRP) and bovine erythrocytes Cu,Zn-superoxide dismutase (SOD), were linked to a fluorescently labeled, polycationic, dendronized polymer (denpol). Two homologous denpols of first and second generation were used and compared, and the activities of HRP and SOD of the conjugates were measured in aqueous solution separately and in combination. In the latter case the efficiency of the two enzymes in catalyzing a two-step cascade reaction was evaluated. Both enzymes in the two types of conjugates were highly active and comparable to free enzymes, although the efficiency of the enzymes bound to the second-generation denpol was significantly lower (up to a factor of 2) than the efficiency of HRP and SOD linked to the first-generation denpol. Both conjugates were analyzed by atomic force microscopy (AFM), confirming the expected increase in object size compared to free denpols and demonstrating the presence of enzyme molecules localized along the denpol chains. Finally, giant phospholipid vesicles with diameters of up to about 20 μm containing in their aqueous interior pool a first-generation denpol−HRP conjugate were prepared. The HRP of the entrapped conjugate was shown to remain active toward externally added, membrane-permeable substrates, an important prerequisite for the development of vesicular multienzyme reaction systems.
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INTRODUCTION Conjugates between synthetic polymer molecules and enzymes are molecular hybrid structures in which properties of enzymes and polymer molecules are combined. Such hybrids (or conjugates) are often synthesized with the aim of modifying the physicochemical properties of an enzyme. For example, (i) attaching polymer chains to trypsin resulted in an enzyme− polymer conjugate that was more stable and even apparently more active than the native enzyme;1,2 (ii) attaching a photoresponsive polymer chain to endoglucanase 12A led to an enzyme−polymer conjugate with photoswitchable enzyme activity (“on” after irradiation with visible light, “off” after irradiation with UV light);3 and (iii) attaching a hydrophobic polymer to α-chymotrypsin resulted in a polymer−enzyme conjugate that was active at an oil−water interface.4 In all these and other5−16 examples, one or several polymer chains were bound to one and the same enzyme molecule. The opposite case is a hybrid structure in which several enzymes are bound to one and the same polymer molecule. In such type of polymer− © XXXX American Chemical Society
enzyme conjugates, the polymer is a kind of macromolecular linker, allowing several enzyme molecules to be brought in close proximity,17,18 the number of bound enzymes being dependent on the length of the polymer chain, on the number of reactive groups along the polymer chain, and on the number of enzyme molecules that one is able or one wishes to bind to the polymer molecule. Recently, we succeeded in preparing a polymer−enzyme hybrid structure in which multiple copies of two different types of enzymes were bound to one and the same polymer molecule.19 The two enzymes were horseradish peroxidase (HRP) and bovine erythrocytes Cu,Zn-superoxide dismutase (SOD) and the polymer was de-PG1, a water-soluble, polycationic, first-generation dendronized polymer (denpol) with amine-terminated dendrons and an average degree of Received: May 17, 2013 Revised: July 4, 2013
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dx.doi.org/10.1021/la401867c | Langmuir XXXX, XXX, XXX−XXX
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Figure 1. Schematic representation of the dendronized polymer (denpol)−enzyme conjugates used in the work, abbreviated as de-PG12000−BAH− (FLx,HRPy,SODz) and de-PG22000−BAH−(FLx,HRPy,SODz). The denpols had on average 2000 ru and were of first or second generation, dePG12000 and de-PG22000, obtained through deprotection of PG12000 and PG22000 (see the text and Supporting Information for details). Fluorescein (FL), HRP, and SOD were bound to the denpol through a stable bis-aryl hydrazone (BAH) bond. The subscripts x, y, and z, refer to the average number of FL, HRP, and SOD molecules bound to one denpol chain of 2000 ru length.
glass tubes,28 and inside microfluidic channels.29 Enzymatic cascade reactions could be carried out by using sequentially immobilized β-galactosidase, glucose oxidase, and HRP.28,29 In a more recent work, short de-PG1176 was found to be a promising polymer for the stabilization of a proline-specific endopeptidase in the gastrointestinal tract of rats through covalently linking the denpol chains to the enzyme.30 In the present work we explored the novel type of denpol− enzyme conjugate shown in Figure 1 in more detail. We particularly investigated the denpol generation dependence of the activity of the conjugated enzymes HRP and SOD. Furthermore, we visualized the conjugates by AFM, and we examined whether it is possible to encapsulate this type of denpol−enzyme conjugate into the aqueous interior of giant phospholipid vesicles (GVs), a prerequisite for the development of vesicular multienzyme reaction compartments. If bound to the dendronized polymer at a defined ratio, the two enzymes can be entrapped inside the vesicles at precisely this ratio through entrapment of the conjugate. This is particularly important if small vesicles are used, where for statistical reasons the defined coentrapment of different enzymes is difficult to achieve.
polymerization, n, of 2000 (de-PG12000) (see Figure 1, left-hand side). Herein, we demonstrate that the same type of denpol− enzyme conjugate can be prepared with a homologous secondgeneration denpol, de-PG22000 (see Figure 1, right-hand side). The main differences between de-PG12000 and de-PG22000 are their main chain thickness, their amino group density at the periphery per repeating units (ru), and their chain stiffness. The chain stiffness increase with increasing denpol generation is evident from atomic force microscopy (AFM) images of adsorbed tert-butyloxycarbonyl (Boc)-protected denpols, PG1 and PG2,20 as well as from recent computer simulations.21 The denpol−enzyme conjugate synthesized and investigated so far was composed of de-PG12000 and carried on average 120 HRP and 60 SOD enzyme molecules, as well as 20 fluorescenyl residues (FL) as fluorescent marker. The ratio of HRP to SOD (2:1) was arbitrarily chosen. Furthermore, we found that 120 HRP molecules per de-PG12000 or de-PG22000 is the maximum possible loading. FL and the two enzymes were attached to the denpol through a chemically stable bis-aryl hydrazone (BAH) bond. For this molecular hybrid structure the following abbreviation is used: de-PG12000−BAH−(FL20,HRP120,SOD60). Since BAH bond formation can be quantified by analyzing the absorption at λ = 354 nm,19,22−26 determination of the average number of bound enzymes per polymer chain was possible with a conventional spectrophotometer. In addition to using the mentioned type of denpols as “macromolecular linker”,19 de-PG21000 was also applied successfully as “glue”, together with the biotin−avidin system, for the stepwise immobilization of enzymes on sputtered SiO2 surfaces, on conventional silicate glasses,27,28 inside micropipet
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MATERIALS AND METHODS
Materials and Synthesis of Denpols and Denpol−Enzyme Conjugates. The two enzymes used, horseradish peroxidase (HRP, EC 1.11.1.7, M ≈ 44 kDa) and bovine erythrocytes Cu,Zn-superoxide dismutase (SOD, EC 1.15.1.1, M = 32.6 kDa), were from the same supplier as in our previous work,19 although partially different batches were used. HRP was from Toyobo Enzymes [isoenzyme C, product B
dx.doi.org/10.1021/la401867c | Langmuir XXXX, XXX, XXX−XXX
Langmuir
Article
number PEO-131, grade I, Lot 2131616000, RZ (A403nm/A260nm) = 3.08, 278 units/mg protein] and SOD was from Sigma-Aldrich (product number S7571, Lot 080M7690 V, 3999 units/mg solid). ABTS2‑(NH4+)2, the diammonium salt of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), was from Fluka and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) from Anawa Trading SA. These two compounds were used as reducing substrates for HRP. POPC (1palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) was from Avanti Polar Lipids Inc. Mineral oil (#M5904, light oil), D-glucose, and sucrose were from Sigma-Aldrich. Details about the syntheses of the following compounds can be found in our previously published communication:19 deprotected firstgeneration dendronized polymer with a number-average degree of polymerization of n ≈ 2000, de-PG12000; the linking reagents succinimidyl 4-formylbenzoate (S-4FB) and succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic); HyNic-modified firstgeneration denpol de-PG12000−HyNic440; fluorescein-labeled HyNicmodified first-generation denpol de-PG12000−(FL20)−HyNic420; fluorescein-labeled first-generation denpol−enzyme conjugate dePG12000−BAH−(FL20,HRP120,SOD60); 4FB-modified HRP (HRP4FB); and 4FB-modified SOD (SOD-4FB). In the Supporting Information, details of the procedures for the syntheses of the following compounds are reported: the dendronization reagent DG1-NHS (2,5-dioxopyrrolidin-1-yl 3,5-bis(3-(tertbutoxycarbonylamino)propoxy) benzoate); protected second-generation dendronized polymer PG22000; deprotected second-generation dendronized polymer de-PG22000; HyNic-modified second-generation denpol de-PG22000−HyNic440; fluorescein-labeled HyNic-modified second-generation denpol de-PG22000−(FL20)−HyNic420; fluoresceinlabeled second-generation denpol−enzyme conjugate de-PG22000− BAH−(FL20,HRP120,SOD60); and fluorescein-labeled first-generation denpol−enzyme conjugate de-PG12000−BAH−(FL100,HRP100). Enzyme Activity Measurements in Bulk Solution. The enzymatic activity of the denpol−enzyme conjugates was measured by analyzing reactions catalyzed by either (i) SOD (using pulse radiolysis equipped with an optical detector device), (ii) HRP (using ABTS 2‑ as reducing substrate and H 2 O2 as oxidant and a spectrophotometer), or (iii) SOD and HRP (using ABTS2‑ and pulse radiolysis to monitor the efficiency of the enzymatic cascade reaction). The pulse radiolysis experiments, i and iii, were carried out on a Febetron 705 2.3 MeV accelerator (Titan Systems Corp.) with a pulse width of
