Preparation of Catalytically Active, Covalent Î±-Polylysineâ Enzyme

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Biomacromolecules 2011, 12, 134–144

Preparation of Catalytically Active, Covalent r-Polylysine-Enzyme Conjugates via UV/Vis-Quantifiable Bis-aryl Hydrazone Bond Formation Andrea Grotzky, Yuichi Manaka, Taisuke Kojima, and Peter Walde* Department of Materials, ETH Zu¨rich, Wolfgang-Pauli-Strasse 10, CH-8093 Zu¨rich, Switzerland Received September 10, 2010; Revised Manuscript Received November 24, 2010

Covalent UV/vis-quantifiable bis-aryl hydrazone bond formation was investigated for the preparation of conjugates between R-poly-D-lysine (PDL) and either R-chymotrypsin (R-CT) or horseradish peroxidase (HRP). PDL and the enzymes were first modified via free amino groups with the linking reagents succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic, at pH 7.6) and succinimidyl 4-formylbenzoate (S-4FB, at pH 7.2), respectively. The modified PDL and enzymes were then conjugated at pH 4.7, whereby polymer chains carrying several enzymes were obtained. Kinetics of the bis-aryl hydrazone bond formation was investigated spectrophotometrically at 354 nm. Retention of the enzymatic activity after conjugate formation was confirmed by using the substrates N-succinimidyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (for R-CT) and 2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (ABTS, for HRP). Thus, not only a mild and efficient preparation and convenient quantification of a conjugate between the polycationic R-polylysine and enzymes could be shown, but also the complete preservation of the enzymatic activity.

Introduction Coupling peptides/proteins to synthetic polymers is a wellknown and intensively studied topic.1-7 Special cases are polymer-enzyme conjugates that need to be prepared in a way such that the catalytic activity of the enzyme is preserved. Polymer-enzyme conjugates are investigated for applications in various areas including biocatalysis in nonaqueous media, for the preparation of biosensors and in the case of biocompatible polymers in the field of drug delivery.5,7 Enzymes bound to polymers may have an enhanced thermal stability.8-10 In contrast to PEGylated enzymes, which carry a number of polymer chains on one and the same enzyme (Scheme 1A),11 or in contrast to enzymes that carry a single polymer chain (Scheme 1B),5,12 we focus our research on attaching several enzymes to one and the same polymer chain (Scheme 1C). This can be achieved with the “grafting-to” approach3,4,13 by covalently linking the enzymes to the polymer. For covalently linking enzymes to polymers on both the enzyme and the polymer, available reactive groups have to be present. The most reactive (nucleophilic) functional groups in proteins (enzymes) are free thiols and amino groups of cysteines and lysines, respectively. Because often most of the cysteines will form a disulfide bridge within the enzyme, free thiols may not be available for modification unless the protein is denatured by reducing the disulfide bridge. Denaturing enzymes, however, will result in the loss of the tertiary structure and with that in loss of catalytic activity. This may also occur during harsh reaction conditions like the use of organic solvents or elevated temperatures. To preserve the activity of the enzyme, very mild reaction conditions have to be applied. To achieve that, all steps involved in making the conjugate have to take place in aqueous solutions and preferentially at ambient temperature. A number of homo- and heterobifunctional reagents are known to be compatible with these requirements, like linkers containing * To whom correspondence [email protected].

should

be

addressed.

E-mail:

aldehydes, N-hydroxysuccinimide (NHS)-, or pentafluorophenyl (PFP)-activated esters.14 Using glutaraldehyde will most likely result in uncontrolled cross-linking and thus in highly polymeric and insoluble material.15 Additionally, the formation of enzyme-enzyme and polymer-polymer conjugates that would lower the yield significantly have to be avoided. Heterobifunctional linkers like sulfosuccinimidyl-4-[p-maleimidophenyl]butyrate (sulfo-SMPB), which need a thiol and an amide functional group, circumvent these problems.16 However, if only amine functional groups are available on both the polymer and enzyme, neither homo- nor heterobifunctional linkers are appropriate. Recently, Schwartz invented a new linking system to form biomolecule conjugates using aromatic aldehyde and hydrazine moieties to obtain a stable covalent bis-aryl hydrazone bond.17-19 In this case, one first needs to separately modify both macromolecules with an aldehyde or hydrazine linker and then conjugate them in a second step. Because during the conjugation reaction these linkers can only react with each other but not with themselves and not with other functional groups present in the polymer or enzyme, only the desired conjugate will be formed. Parts of that linking chemistry have been used mainly to conjugate oligonucleotides together,20 to form oligonucleoScheme 1. Schematic Representation of Different Types of Polymer-Enzyme Conjugatesa

a (A) One enzyme to which several polymer chains are bound;11 (B) one end-functionalized polymer chain bound to one enzyme;5,12 (C) several enzymes bound to one and the same polymer chain. The bonds between the enzyme and the polymer may be covalent or noncovalent.

10.1021/bm101074s  2011 American Chemical Society Published on Web 12/20/2010

Preparation of R-Polylysine-Enzyme Conjugates

Biomacromolecules, Vol. 12, No. 1, 2011

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Scheme 2. Reaction Scheme of the Modification and Conjugation Reactionsa 19

a Modification reactions (a) of the polymer (PDL) with S-HyNic and (b) of the enzyme (R-CT and HRP) with S-4FB and conjugation reaction (c) using bis-aryl hydrazone bond formation.

tide-antibody conjugates,21 to label proteins with 99mTc,22 and for forming protein-protein complexes.23 Herein we report the preparation, quantification, and characterization of a water-soluble, covalent polymer-enzyme conjugate (Scheme 1C) using the UV/vis-quantifiable bis-aryl hydrazone bond formation to conjugate R-poly-D-lysine (PDL) as the polymer part to R-chymotrypsin (R-CT) or horseradish peroxidase (HRP) as enzymes for possible analytical application e.g. in the field of ex vivo biosensors. With biosensor we mean in our case “a device that uses specific biochemical reactions mediated by isolated enzymes [..] to detect chemical compounds by [..] optical signals”,24 whereby the chemical compounds will not necessarily be in a biological environment. To modify some of the amino groups of the PDL and enzyme the linking reagents succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic) and succinimidyl 4-formylbenzoate (S4FB) were used. The reactions involved are shown in Scheme 2. Two different lengths of PDL were used: a low molar mass PDL (15000-30000 g/mol for the HBr salt) and a high molar mass PDL (150000-300000 g/mol for the HBr salt). R-Polylysine bioconjugates reported in literature include conjugates with nucleic acids,25 as well as with enzymes,26-28 for achieving enhanced cellular uptake. Starting with R-chymotrypsin as a well-known, stable, and easy quantifiable proteinase we wanted to investigate whether PDL-enzyme conjugates can be prepared via bis-aryl hydrazone bond formation and whether both the modification and the conjugation occur under retention of the enzymatic activity. Because the preparation of a PDL-R-CT conjugate was indeed successful and no loss in enzymatic activity could be observed, we decided to further use HRP, which was expected to be more demanding with respect to retention of the catalytic activity. In addition, we wanted to have a closer look at the actual modification and conjugation reactions by means of kinetic studies. HRP as a hydrogen peroxide oxido-reductase is a versatile enzyme and is used for a wide range of applications,29 including the synthesis of polyaromatic polymers under environmentally friendly conditions.30 Native HRP exists in different isoenzymes of which isoenzyme C1A is the most predominant. Compared to R-CT, which has 14 modifiable lysine residues, HRP has only six lysines and a blocked N-terminus.31 Only three of the six

lysines are available for the modification with NHS-activated esters, namely, Lys-232, Lys-241, and Lys-174.32 Lys-232 is known to react completely with NHS-esters, while Lys-241 and Lys-174 react only partially.32 Thus, when modifying HRP with one aldehyde moiety per enzyme (Scheme 2), it is likely that Lys-232 is modified predominantly.

Materials and Methods Materials. Horseradish peroxidase isoenzyme C (HRP, EC 1.11.1.7, M ≈ 44 kDa) was purchased from Sigma-Aldrich, Switzerland (type XII, RZ g 3), or from Toyobo Enzymes, Japan (grade I, RZ g 3). R-Chymotrypsin from bovine pancreas (R-CT, EC 3.4.21.1, M ≈ 25 kDa, type II) and R-poly-D-lysine · hydrobromide M ) 15000-30000 g/mol and M ) 150000-300000 g/mol (PDL, M(monomer, Lys · HBr, C6H13N2OBr) ) 209.09 g/mol) were also from Sigma. The linking reagents succinimidyl 6-hydrazinonicotinamid acetone hydrazone (SHyNic) and succinimidyl 4-formylbenzoate (S-4FB) were obtained from the SoluLink Company, U.S.A.,19 and the quantification reagents 4-nitrobenzaldehyde and 2-hydrazinopyridine hydrochloride from Fluka, Switzerland. The enzyme substrates N-succinimidyl-L-Ala-L-Ala-L-ProL-Phe-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) and 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were obtained from Fluka and Sigma. The standard marker for Isoelectric Focusing IEF Mix pI 3.6-9.3 was from Sigma. All other chemicals, including buffer salts in SigmaUltra, BioUltra or BioChemika quality, BCA assay kit, and Sephadex G25 fine were purchased from Sigma or Fluka. For all experiments, ultrapure water (filtered with a Synergy water preparation apparatus from Millipore) was used; all solutions were stored at 4 °C after preparation unless otherwise specified. Because PDL adheres to glass surfaces, all reactions, purification, and quantification steps were carried out in polypropylene reaction vessels. The following buffer solutions were prepared. The abbreviations given will be used in the following experimental procedures. PB1: Phosphate buffer saline 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2. PB2: Phosphate buffer 0.1 M sodium phosphate, 1 M NaCl, pH 7.2. PB3: Phosphate buffer 0.1 M sodium phosphate, pH 6.0. MesB1: MES buffer 0.1 M MES hydrate, 0.15 M NaCl, pH 4.7. MesB2: MES buffer 0.1 M MES hydrate, pH 5.0. TrisB: 0.1 M Tris-HCl, 10 mM CaCl2, pH 8.0. Equipment. The UV/vis-spectroscopic measurements were carried out on a PerkinElmer Lambda 20, Varian Cary 1E, or Nano-Drop spectrophotometer ND 1000. The circular dichroism measurements were done on a Jasco J-715 spectropolarimeter. For the UV/vis and CD measurements, the following quartz cuvettes (Hellma, Switzerland) were

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used: Macro Cell QX 0.35 mL l ) 0.1 cm, Macro Cell QS 1.75 mL l ) 0.5 cm, Semi-Micro Cell QS 1.4 mL l ) 1 cm, Ultra-Micro Cell QS 0.1 mL l ) 1 cm, TrayCell l ) 0.1 and 0.02 cm. Ultrafiltration was carried out with the following Millipore filter units: Mcutoff ) 10 kDa, 4 and 15 mL, and Mcutoff ) 100 kDa, 0.5 mL. Isoelectric focusing was carried out on a Biorad Criterion system with a pH 3-10 gel (from Biorad). Analytical reverse phase HPLC was carried out on a Waters Alliance system with a Phenomenex Gemini-NX 3 µm, C18, 110 Å, 4.6 × 100 mm column. For the mass spectrometric analysis, a Bruker Daltonics maXis (ESI/NanoSpray-Qq-TOF) was used. Experimental Section. HyNic Modified PDL. A PDL stock solution of 2 mg/mL was prepared in PB1 for low molar mass PDL and in PB2 for high molar mass PDL. Due to solubility problems upon addition of DMF, the concentration of the PDL stock solution could not exceed 2 mg/mL. For a typical modification of low molar mass PDL, 500 µL (44 nmol PDL respectively 4.7 µmol Lys, 1 equiv Lys) of PDL stock solution in PB1 and 48 µL (238 nmol, 5 equiv/100 Lys) of 5 mM S-HyNic solution in anhydrous DMF were mixed. For stability reasons the S-HyNic solution in anhydrous DMF was used within one month after preparation. To prevent cleavage of the hydrazine protecting group in S-HyNic during the modification reaction, the pH of PB1 was increased to 7.6 by adding 15 µL of saturated aqueous disodium hydrogenphosphate solution, as determined beforehand with buffer solutions not containing PDL. The pH could not be measured directly because PDL adheres to the pH glass electrode. After a 4 h reaction time at room temperature, the mixture was directly loaded onto a desalting column Sephadex G25 swollen in Milli-Q water (bed volume 30 mL). The fractions were identified either by thin layer chromatography (stationary phase, aluminum oxide on glass thin layers; mobile phase, BuOH/AcOH/H2O ) 6:2:1; coloring reagent, ninhydrin stain) or by UV/vis spectroscopy, collected, and their volume reduced on Millipore ultrafiltration units (Mcutoff ) 10 kDa) by centrifuging 15 min at 3000 rpm. A total of 1 mL of MesB1 was added to the retentate on the membrane and the filter unit was again centrifuged. That process was repeated three times. To ensure complete buffer exchange and that no excess reagent was present each time the conductivity of the filtrate and the UV/vis-spectrum were measured. During this workup, the hydrazine protecting group was cleaved off, yielding an aromatic hydrazine modified PDL (HyNic-PDL). Finally, the retentate was removed and diluted to half the starting volume with MesB1. The modification of high molar mass PDL was carried out similarly. A stock solution of 2 mg/mL in PB2 was prepared. A total of 1 mL (9 nmol PDL, respectively, 9.5 µmol Lys, 1 equiv Lys) of the latter solution were mixed with 48 µL (476 nmol, 5 equiv/100 Lys) of 10 mM S-HyNic solution in anhydrous DMF. After addition of 30 µL saturated aqueous disodium hydrogenphosphate solution the mixture was allowed to react for 4 h. The reaction workup was done as described above. Additionally, to follow the reaction kinetics an aliquot of the above mixture was directly prepared in a 350 µL quartz cuvette (l ) 0.1 cm) and time-dependent UV/vis-spectra were recorded for 4 h. After the incorporation of the hydrazine linker the molar substitution ratio MSR(HyNic) was determined via bis-aryl hydrazone formation with 4-nitrobenzaldehyde. The MSR(HyNic) is the ratio of HyNic to PDL and defined as

MSR(HyNic) )

A380nm [HyNic] ) [PDL] [PDL] · ε380nm · l

(1)

whereby ε380nm is the molar absorption coefficient of the formed hydrazone (ε380nm ) 22000 M-1 cm-1 33) and l the absorption path length of the cuvette (l ) 1 cm). For determining A380nm, a 50 µM 4-nitrobenzaldehyde reagent solution in MesB2 was prepared by diluting 10 µL of a 50 mM 4-nitrobenzaldehyde solution in anhydrous DMF with 990 µL of MesB2. A total of 100 µL of the latter solution was mixed with 5 or 10 µL of HyNic-PDL solution, depending on the concentration of HyNic

in PDL. In addition, a blank sample was prepared with 5 or 10 µL of MesB2 instead of the HyNic-PDL solution. Both solutions were prepared three times and were incubated in a water bath at 37 °C for 1 h. After cooling to room temperature, UV/vis-spectra were recorded, and the difference in absorbance of sample and blank was read at 380 nm. The concentration of PDL was determined with the Trypan Blue assay,34 which includes quantitative precipitation of a PDL-trypan blue complex and measuring the decrease in absorbance of the supernatant. A total of 10 or 20 µL of HyNic-PDL solution were diluted to 500 µL with MesB1. A total of 50 µL of the latter solution were further diluted to 500 µL with MesB1. A total of 125 µL of that dilution were mixed with 5 µL of trypan blue solution (1 mg/mL in MesB1). The samples were incubated at 37 °C for 1 h and after cooling to room temperature centrifuged at 8000 rpm for 20 min to sediment the precipitate. A UV/ vis-spectrum of the supernatant was recorded against MesB1 and the absorbance was read at 580 nm. A calibration curve was made with known amounts of low and high molar mass PDL, using average molar masses of 22500 and 225000 g/mol. 4FB Modified R-CT. A R-CT stock solution of ≈2 mg/mL (80 µM R-CT, determined by measuring the absorbance at 280 nm, ε280 nm ) 51000 M-1 cm-1,35 corresponding to 1.12 mM total Lys) was prepared in PB1. To 500 µL (40 nmol, 1 equiv R-CT) of R-CT stock solution in PB1, 40 µL (200 nmol, 5 equiv with respect to R-CT) of 5 mM S-4FB solution in anhydrous DMF was added. After a 4 h reaction time at room temperature, the mixture was directly loaded onto a desalting column Sephadex G25 swollen in MesB1 (bed volume 30 mL). The fractions were identified with UV/vis-spectroscopy at 280 nm (aromatic amino acids). Enzyme containing fractions were collected and their volume reduced on Millipore ultrafiltration units (Mcutoff ) 10 kDa) by centrifuging for 15 min at 3000 rpm. To the retentate on the membrane, 1 mL of MesB1 were added and the filter unit was again centrifuged. That process was repeated three times. Each time the conductivity of the filtrate was measured and the UV/vis-spectrum was recorded to ensure complete buffer exchange and that excess reagent had been removed. Finally, the retentate was recovered and diluted to half the starting volume with MesB1. The quantification of 4FB incorporation was determined via bisaryl hydrazone formation with 2-hydrazinopyridine. The molar substitution ratio MSR(4FB) is the ratio of 4FB to R-CT and defined as

MSR(4FB) )

A350nm [4FB] ) [R-CT] [R-CT] · ε350nm · l

(2)

whereby ε350nm is the molar absorption coefficient of the formed hydrazone (ε350nm ) 18000 M-1 cm-1 33) and l is the absorption path length of the cuvette (l ) 1 cm). For the measurement of A350nm, a 50 µM 2-hydrazinopyridine reagent solution in MesB2 was prepared by diluting 10 µL of a 50 mM 2-hydrazinopyridine solution in anhydrous DMF with 990 µL of MesB2. A total of 100 µL of the latter solution were mixed with 5 or 10 µL of 4FB-R-CT solution, depending on the 4FB concentration. In addition, a reagent blank sample was prepared with 5 or 10 µL of MesB2 instead of 4FB-R-CT. All samples were prepared three times and were incubated in a water bath at 37 °C for 1 h. UV/vis-spectra were recorded after cooling to room temperature. Then, the difference in absorbance of the sample and the reagent blank was read at 350 nm. The concentration of R-CT was determined with the BCA assay.36 A total of 6 µL of 4FB-R-CT solution were diluted to 60 µL with PB1. The latter solution was mixed with 1.2 mL BCA working reagent solution (BCA solution/Cu2+ solution ) 50:1 (v/v)). The blank contained PB1 instead of 4FB-R-CT. All samples were incubated at room temperature for 12 h. The absorbance was read at 562 nm and the concentration determined from a calibration curve with known amounts of R-CT. The hydrolytic activity of R-CT was measured with the substrate Suc-Ala-Ala-Pro-Phe-pNA via formation of p-nitroaniline as described



previously.35,37 The following stock solutions were prepared: 50 µM R-CT in PB1 as a reference for modified and conjugated R-CT and a 450 µM substrate solution in TrisB. The concentrations of these solutions were determined with UV/vis-spectroscopy using molar adsorption coefficients ε315nm ) 14000 M-1 m-1 35 for the substrate and ε280nm ) 51000 M-1 m-1 35 for R-CT. In a cuvette (l ) 1 cm), the enzyme solution was diluted with TrisB such that after the addition of substrate the R-CT concentration was 100 nM. The reaction then was started upon addition of the substrate solution and the formation of p-nitroaniline was followed spectrophotometrically at 410 nm (ε410nm ) 8800 M-1 cm-1 37) at 25 °C. The substrate concentration in the cuvette was 30 µM. The relative activity was determined as initial velocity. To compare the activity of the PDL-R-CT conjugate and 4FBR-CT to native R-CT the initial velocity of native R-CT was set to 100%. 4FB Modified HRP. A HRP stock solution of ≈4 mg/mL (75 µM HRP, determined by measuring the absorbance at 403 nm, ε403 nm ) 102000 M-1 cm-1,38 corresponding to 225 µM accessible Lys) in PB1 was prepared. To avoid cross-linking during the conjugation reaction the enzyme must carry on average one aldehyde only. To achieve that, 7.5 equiv S-4FB per enzyme (2.5 equiv per accessible Lys) were used. Thus, 900 µL (73 nmol, 1 equiv HRP) of HRP stock solution were mixed with 61.5 µL (615 nmol, 7.5 equiv with respect to HRP) of 10 mM S-4FB solution in anhydrous DMF. The S-4FB solution was used within one month after preparation. After a 4 h reaction time at room temperature the mixture was directly loaded onto a desalting column Sephadex G25 swollen in MesB1 (bed volume 30 mL). The fractions were identified with UV/vis-spectroscopy at 403 nm (Soret band of the heme group). Enzyme containing fractions were collected and their volume reduced on Millipore ultrafiltration units (Mcutoff ) 10 kDa) by centrifuging for 15 min at 3000 rpm. To the retentate on the membrane, 1 mL of MesB1 was added and the filter unit was again centrifuged. That process was repeated three times. Each time the conductivity of the filtrate was measured and the UV/vis-spectrum was recorded to ensure complete buffer exchange and removal of excess reagent. Finally, the retentate was recovered and diluted to half the starting volume with MesB1. After the modification, the incorporated aldehyde moiety is called 4FB (short for 4-formylbenzoate) and, thus, yielding 4FB-HRP. In addition, a kinetic measurement of the modification reaction of HRP with S-4FB was carried out by recording time-dependent spectra of an aliquot of the above-mentioned mixture diluted to half its concentration in a 350 µL quartz cuvette (l ) 0.1 cm). The quantification of 4FB incorporation was determined via bisaryl hydrazone formation with 2-hydrazinopyridine. The molar substitution ratio MSR(4FB) is the number of 4FB per HRP and defined as

MSR(4FB) )

A350nm [4FB] ) [HRP] [HRP] · ε350nm · l

(3)

whereby ε350nm is the molar absorption coefficient of the formed hydrazone (ε350nm ) 18000 M-1 cm-1 33) and l is the absorption path length of the cuvette (l ) 1 cm). To determine A350nm, a 50 µM 2-hydrazinopyridine reagent solution in MesB2 was prepared by diluting 10 µL of a 50 mM 2-hydrazinopyridine solution in anhydrous DMF with 990 µL of MesB2. A total of 100 µL of the latter solution was mixed with 5 or 10 µL of 4FB-HRP solution, depending on the concentration. In addition, a reagent blank sample was prepared with 5 or 10 µL MesB2 instead of 4FB-HRP. All samples were prepared three times and were incubated in a water bath at 37 °C for 1 h. UV/vis spectra were recorded after cooling to room temperature. Due to the Soret band, HRP has a significant absorption at 350 nm. Therefore, another blank sample was necessary. Thus, a mixture of reagent solution and 4FB-HRP was measured immediately after mixing (not incubated). Then, the difference in absorbance of sample and said HRP blank was read at 350 nm. The HRP concentration was determined directly via the absorbance at 403 nm (ε403nm ) 102000 M-1 cm-1 38).

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Covalent binding of 4FB to HRP was also analyzed by electrospray ionization mass spectrometry (ESI-MS) by using samples that first were thoroughly desalted by ultrafiltration (Mcutoff ) 10 kDa). The activity of HRP was determined with the substrate 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) by following the formation of the ABTS radical cation at 414 nm.39 This method has some advantages over others because of the high stability of the formed ABTS radical cation.40 The following starting concentrations in the sample cell were used: [HRP] ) 1.13 nM, [ABTS]0 ) 0.25 mM, and [H2O2]0 ) 0.05 mM in PB3. For the enzyme assay, 880 µL of PB3, 50 µL of 5 mM ABTS solution in PB3, and 20 µL of 56.8 nM HRP solution in PB3 were mixed in a quartz cuvette (l ) 1 cm). The reference cell contained PB3 only. The reaction was started upon addition of 50 µL of 1 mM hydrogen peroxide solution in PB3. After shaking quickly but thoroughly, the increase of the absorbance at 414 nm (ε414 nm (ABTS•+) ) 36000 M-1 cm-1 39) was measured at room temperature against time. The relative activity was determined as initial velocity. To compare the activity of PDL-HRP conjugate and 4FB-HRP to native HRP the initial velocity of freshly prepared native HRP was set to 100%. PDL-R-CT Conjugate. The reaction was carried out at room temperature in MesB1. To conjugate 4FB-R-CT to HyNic-PDL, both linking moieties were mixed stoichiometrically. It turned out, however, that a 20 mol % excess of 4FB-R-CT increased the conversion. Thus, the concentrations of HyNic in HyNic-PDL and 4FB in 4FB-R-CT were calculated via the MSRs and mixed in a molar ratio of 1.2:1 ([4FB]/ [HyNic]). Because the formation of the bis-aryl hydrazone bond gives rise to a new absorbance band, it was possible to follow the kinetics of the conjugation reaction with UV/vis spectroscopy. The formed bisaryl hydrazone has an absorption maximum at 354 nm (ε354 nm ) 29000 M-1 cm-1 19). Additionally, the conversion of PDL-R-CT conjugate formation after a 24 h reaction time at 25 °C was determined by comparing the measured absorbance at 354 nm with the absorbance calculated for 100% conversion. Furthermore, the activity of the conjugation reaction mixture was measured at different reaction times to detect possible activity loss during the reaction. Hence, every 30 min a small amount of reaction mixture was removed and the activity was measured with Suc-AlaAla-Pro-Phe-pNA, as described above. PDL-HRP Conjugate. The concentrations of HyNic in HyNic-PDL and 4FB in 4FB-HRP were calculated via the MSRs and 4FB-HRP and HyNic-PDL, both in MesB1, were mixed in a molar ratio of 1.2:1 ([4FB]/[HyNic]). A kinetic measurement was carried out by following the bis-aryl hydrazone bond formation with UV/vis-spectroscopy at 354 nm (ε354nm ) 29000 M-1 cm-1 19). In addition, the conversion of PDL-HRP conjugate formation was determined by comparing the measured absorbance at 354 nm after 24 h reaction time at 25 °C with the absorbance at 354 nm calculated for 100% conversion. To detect possible activity loss during the reaction, the enzymatic activity of the conjugation reaction mixture was measured at different reaction times. Therefore, every 30 min an aliquote of reaction mixture was taken and the activity was measured with ABTS as described above. For comparison the activities of native HRP and 4FB-HRP were also measured. After 24 h reaction time the conjugation reaction mixture was directly loaded onto a 0.5 mL Millipore ultrafiltration unit (Mcutoff ) 100 kDa) which was washed one time with 500 µL 0.1 M NaOH to remove the membrane preservatives followed by two times washing with PB1 beforehand. The filter unit was centrifuged for 5 min at 8000 rpm. To the retentate 500 µL of PB1 were added and the unit was centrifuged again. This process was repeated several times. Each time a UV/visspectrum of the retentate (with the TrayCell, l ) 0.02 cm) and of the filtrate (with the Ultra-Micro Cell QS 0.1 mL, l ) 1 cm) were measured to follow the filtration process. From the spectra of the retentates, the ratio of the absorbances at 403 and 354 nm was calculated. This ratio allowed estimation of the conjugate purity. The ultrafiltration process was repeated until a ratio of 1.6 was reached. This ratio was obtained

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for a conjugate with an average of one bis-aryl hydrazone per heme group and was calculated with the following formula:

Table 1. Dependence of the MSR(HyNic) in PDL on the Relative Amount of S-HyNic Useda PDL

A403nm ε403nm(heme in HRP) ) 1.6 ) A354nm ε354nm(heme in HRP) + ε354nm(hydrazone)

low molar massb

(4) high molar massc

In addition, analytical isoelectric focusing on a pH 3-10 polyacrylamide gel was carried out to detect unconjugated HRP. Solutions of (i) native HRP, (ii) a freshly prepared mixture of 4FB-HRP and HyNicPDL, (iii) conjugation reaction mixture before ultrafiltration, and (iv) purified conjugate were applied onto the gel. In each case the sample was diluted with the IEF sample buffer containing 50 v/v % glycerol (from Biorad) to 5, 10, and 15 µg. As standard markers, the IEF Mix 3.6-9.3 from Sigma was used. After loading the samples onto the gel, a current was applied (1 h, 100 V; 1 h, 250 V; 0.5 h, 500 V). Afterward, the gel was put into a fixation solution containing 4 w/v % sulfosalicylic acid, 12.5 v/v % trifluoroacetic acid, and 30 v/v % methanol in ultrapure water for 2 h. Afterward, the gel was dyed overnight with a solution containing 0.04 w/v % copper(II) sulfate, 0.04 w/v % Coomassie blue, 10 v/v % acetic acid, and 27 v/v % isopropanol in ultrapure water. Afterward, the gel was washed with ultrapure water to remove excesses of the dye. The PDL-HRP conjugate was further analyzed by reverse phase (RP)-HPLC with photodiode array (PDA) detection. The following samples were applied onto a C18 column: aqueous solutions of (i) 4FBHRP, (ii) conjugation reaction mixture before ultrafiltration, and (iii) purified conjugate in PB1. The chromatographic separation was carried out with a gradient of 15:85 (v/v) to 60:40 (v/v) mobile phase A (0.05 v/v % trifluoroacetic acid in acetonitrile) to mobile phase B (0.1 v/v % trifluoroacetic acid in ultrapure water) for 40 min. Circular dichroism (CD) measurements were carried out with solutions of (i) native HRP, (ii) 4FB-HRP, and (iii) purified conjugate at a concentration of 6 µM HRP in PB1. For the spectra measurements between 200 and 300 nm, a 350 µL quartz cell (l ) 1 mm) was used, and in the Soret band region between 300 and 500 nm, a 1.5 mL quartz cell (l ) 0.5 cm) was used. Each measurement was carried out at 25 °C, and four measured spectra were accumulated and averaged. Moreover, temperature scans from 25 to 90 °C and back to 25 °C were measured at 222 nm in a 350 µL quartz cell (l ) 1 mm).

Results and Discussion HyNic Modification of PDL. The modification reaction was carried out at pH 7.6 (see Experimental Section). Some of the amino groups of PDL were modified with S-HyNic (Scheme 2) to yield a PDL with a defined average number of hydrazine linkers per polymer chain. The modification reaction was carried out with varying amounts of S-HyNic. The modified PDL is abbreviated as HyNic-PDL. To compare the degree of modification of both low and high molar mass PDL, the equivalents of S-HyNic were calculated on the basis of the lysine concentration in PDL and are given in equiv/100 lysine residues, see Table 1. After the incorporation of HyNic, the MSR(HyNic) was determined spectrophotometrically via bis-aryl hydrazone formation with 4-nitrobenzaldehyde (see Experimental Section). Both the modification and quantification reactions of low and high molar mass PDL were easy to carry out and highly reproducible. Preliminary experiments showed that increasing the pH from 7.2 to 7.6 significantly lowered the amount of S-HyNic reagent needed (data not shown). Therefore, the modification reaction was carried out at pH 7.6. The kinetics of the modification reaction of high molar mass PDL was followed with UV/vis spectroscopy (see Figure 1a,b). Figure 1a depicts the time-dependent absorption spectra. The

equiv S-HyNic/ 100Lys

MSR(HyNic)/ 100Lys

HyNic/ PDL chain

2.5 5.0 7.5 5.0 7.5 10.0

0.8 2.1 3.7 2.1 3.5 4.3

0.9 2.2 4.0 22.5 37.5 46.1

a PDL solution 2 mg/mL in PB1. b Low molar mass PDL M ) 15000-30000 g/mol: MSR(HyNic) calculated assuming an average molar j ) 22500 g/mol (107 Lys). c High molar mass PDL M ) mass of M 150000-300000 g/mol: MSR(HyNic) calculated assuming an average j ) 225000 g/mol (1072 Lys). molar mass of M

reagent itself (S-HyNic) has an absorption maximum at 327 nm (dashed line in Figure 1a). This peak decreased over time, while a new peak centered around 270 nm appeared, which is due to the absorption of the incorporated HyNic moiety and the absorption of the leaving group N-hydroxysuccinimide (NHS), which itself has an absorption maximum at 260 nm.41 The changes in the difference spectra, that is, the absorption spectrum measured at time t minus the absorption spectrum measured at t ) 0, are shown in the Supporting Information, Figure S1a. Figure 1b is a plot of the absorbance difference measured at 327 and 270 nm as a function of time. It shows that the reaction was completed after approximately 4 h under the conditions used. Table 1 shows the results of the modification reaction. As expected, the higher the molar ratio of S-HyNic to lysine residues is, the higher is the MSR(HyNic). A total of 5 equiv of S-HyNic per 100 lysines led to approximately 2 HyNic modified lysines per 100 lysine residues in PDL. This means that on average the low molar mass PDL had about 2 HyNic modifications per chain and the high molar mass PDL around 20. In Figure 1c, the UV/vis spectrum of purified HyNic-PDL is shown. The absorption above 240 nm is due to the HyNic linker only which has an absorption maximum at 262 nm with a shoulder at 300 nm. PDL itself showed no absorption in this region of the spectrum. Stability measurements carried out at different storage conditions showed that some of the HyNic moieties in PDL lose their reactivity over time (Figure 1d) which might be due to hydrazine oxidation.42 Therefore, for further reactions the modified PDL was used as soon as possible, namely, within 1 day after the modification. 4FB Modified r-CT and HRP. Some of the lysine residues of R-CT and HRP were modified with S-4FB (Scheme 2). The modification reactions were carried out at pH 7.2. The modified enzymes are abbreviated as 4FB-R-CT and 4FB-HRP. The quantification of 4FB incorporation was determined via bisaryl hydrazone formation with 2-hydrazinopyridine. The MSR(4FB) values are listed in Table 2. To avoid cross-coupling during the conjugation reaction, one enzyme molecule must not carry more than one aldehyde linker. By systematically varying the amount of S-4FB reagent, we found out that, for R-CT, 5 equiv of S-4FB reagent, and for HRP, 7.5 equiv of S-4FB reagent per enzyme (2.5 equiv per accessible Lys) lead to the desired MSR(4FB) of about 1 or less (Table 2). In the case of HRP, the modified enzyme was also analyzed by nanoelectrospray ionization mass spectrometry (ESI-MS). A comparison between native and modified HRP was made (Figure 2). The presence of HRP molecules containing one 4FB in the modified HRP sample is obvious (Figure 2b(ii)). The



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Figure 1. Modification of high molar mass PDL with S-HyNic: (a,b) kinetics of the modification reaction of PDL with 5 equiv S-HyNic/100 Lys (25 °C, l ) 0.1 cm): (a) time-resolved spectra from t ) 0 min (---), 10, 30, 50, 90, 130, 170, to 250 min ( · · · ); (b) difference in absorbance at 270 nm (O) and 327 nm (9) vs time (the values recorded at t ) 0 min were subtracted from all other data); (c) UV/vis-spectra of unmodified PDL (---) and HyNic-PDL (s; MSR(HyNic) ) 2.1, l ) 0.1 cm), (d) stability of incorporated HyNic expressed as determined MSR(HyNic) vs time at different storing conditions: at room temperature (9), 4 °C (O), and -20 °C (0). Table 2. Dependence of the MSR(4FB) in the Enzyme on the Relative Amount of S-4FB Used enzyme R-CT HRP

equiv S-4FB

MSR(4FB)

5.0 6.9 6.0 7.5

1.1 1.6 0.5 0.8

relative activitya (%) 98 100

100% activity refers to initial velocity of the native enzyme at 25 °C; conditions R-CT: [R-CT] ) 100 nM, [Suc-Ala-Ala-Pro-Phe-pNA] ) 10 µM, TrisB; conditions HRP: [HRP] ) 1.13 nM, [ABTS]0 ) 0.25 mM, [H2O2]0 ) 0.05 mM, PB3. a

presence of unmodified HRP could also be detected (Figure 2a(ii)). The measured mass difference was in good agreement with the calculated value for the 4FB linker (134 Da). The presence of HRP molecules containing two 4FB moieties cannot be excluded completely, although this doubly modified HRP was certainly not the predominant species. In addition, kinetic measurements of the modification reaction for HRP are shown in Figure 3a-c. S-4FB itself has an absorption maximum at 274 nm. Because the incorporated 4FB moiety also absorbs at this wavelength, there was no dramatic change in absorbance at 274 nm during the modification reaction. The changes become obvious, however, if the difference spectra are considered (Figure 3b). Now the increase at 274 and 260 nm (NHS leaving group) can be seen clearly. In Figure 3c, the absorbance at 274 nm is plotted against time. This shows that the modification reaction was completed after approximately 4 h under the experimental conditions used.

The UV/vis-spectra of the purified 4FB-enzymes and of the native enzymes are shown in Figure S2a (for R-CT) and Figure 3d (for HRP). The absorption at 274 nm arises from the 4FB moiety. Unlike in the case of HyNic-PDL, the aromatic amino acids of the enzymes have a contribution to the absorbance at 274 nm, which did not allow direct quantification. The quantification of 4FB with 2-hydrazinopyridine was however possible. In the case of R-CT the quantification was straightforward with a standard deviation of less than 3%. When calculating the MSR(4FB) of HRP with this method, however, the standard deviation of 10% was considerably higher due to the significant contribution of the heme group to the absorption at 350 nm. The spectrum obtained after the reaction of the modified HRP with 2-hydrazinopyridine demonstrates the difficulties in the precise quantification of incorporated 4FB moieties in HRP (Figure 3e) compared to R-CT (Figure S2b). Due to the high molar absorption coefficient of the Soret band of HRP compared to the much smaller molar absorption coefficient of the formed bis-aryl hydrazone, the difference in absorbance was relatively small compared to the total absorbance. Nevertheless, the quantification was possible. The modification reaction itself could be carried out under mild conditions and the enzymatic activity could be fully preserved (see Table 2). As indicated in Figure 3f the stability of the incorporated 4FB moiety was quite high. In contrast to the HyNic moiety incorporated into PDL (Figure 1d) the 4FB linker did not drastically lose its reactivity and was quite stable during the time measured, even if the solution was kept at room temperature.

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Figure 2. Nanoelectrospray MS of (a) native HRP and (b) 4FB modified HRP (MSR(4FB) ) 0.8). Shown are (i) the spectra of multiply charged species in m/z and (ii) the deconvoluted spectra in Da. The mass difference of 134 Da (or 131 Da, respectively) in (b(ii)) between native HRP 40808 Da (or 41631 Da, respectively) and 4FB modified HRP 40942 Da (or 41762 Da, respectively) indicated the presence of the 4FB linker (calculated difference 134 Da). The peak at 40808 in a(ii) and b(ii) can be assigned as HRP, which misses the C-terminal serine, while the additional peak in a(ii) at 40895, which is not visible in b(ii), is an HRP molecule with said serine (mass difference 87). Moreover, because HRP is a glycosylated protein, the mass difference of 823 between 40808 and 41631 Da in a(ii) is due to a different glycoform of HRP. For a more detailed discussion of the native HRP mass spectrum, see Wuhrer et al.43

Conjugation of 4FB-Enzyme to HyNic-PDL. The PDLenzyme conjugates were prepared by mixing a 4FB-enzyme solution with a HyNic-PDL solution in a molar ratio of 1.2 to 1 (4FB to HyNic). The experimental conditions and the results are summarized in Table 3. The following conclusions can be drawn. The conjugation of 4FB-R-CT to HyNic-PDL resulted in higher conversions as compared to the conjugation of 4FBHRP to HyNic-PDL. This might be due to the smaller size of R-CT. Nevertheless, high conversions could be obtained for both cases if highly concentrated solutions of 4FB-enzyme (0.1 mM enzyme) were used. Furthermore, a 20% excess of 4FB-enzyme increased the conversions notably. The time-resolved difference spectra measured during the conjugation reaction were very similar for both enzymes (Figure 4a-c for PDL-HRP and S3a-c for PDL-R-CT). The formation of the bis-aryl hydrazone bond led to an increase in absorption at 354 nm (Figure 4b for HRP and Figure S3b for R-CT). Under the experimental conditions used, the conjugation reaction of 4FB-HRP to HyNic-PDL was almost complete after 12 h (Figure 4c). For R-CT (Figure S3), the conjugation reaction to HyNic-PDL was completed after 24 h (Figure S3c). Because we were interested in preserving the enzymatic activity of the conjugated enzymes, the relative activity was measured during the actual conjugation reaction to detect a possible loss in activity. Figure 4d (for HRP) and Figure S3d (for R-CT) show that there were no losses in enzymatic activity during the conjugation. Given that the presence of 4FB modified HRP containing two 4FB moieties per HRP could not be excluded completely (see above), there was the possibility of the formation of intermolecular cross-linked structures in which HRP would act as a cross-linker between two different polymer chains. However, such intermolecular cross-linking is unlikely to occur for steric reasons due to the proximity of the accessible lysine residues (Lys-174, Lys-232, and Lys-241, which are located in the same area of the enzyme surface).28 Furthermore, no precipitation was observed which one would expect for intermolecular cross-linking. It is likely that HRP molecules with two 4FB moieties would undergo intramolecular conjugation with HyNic linkers on the same polymer chain which would be of no further consequence.

The foremost advantage of using the UV/vis-quantifiable bisaryl hydrazone bond formation is that the concentration of formed conjugate can be determined which then allows direct calculation of the average number of enzymes per polymer chain. In the case of high molar mass PDL, approximately 20 enzymes could be bound to one and the same polymer chain. PDL-HRP Conjugate. After the conjugation reaction reached equilibrium, the reaction mixture did not only contain the desired conjugate but also unreacted 4FB-enzyme and unmodified enzyme. Purification of the PDL-HRP conjugate was carried out by repetitive ultrafiltration (see Experimental Section). The increase in the purity of the PDL-HRP conjugate during the ultrafiltration process was followed by determining the ratio of the absorbances at 403 nm (heme group) and 354 nm (bis-aryl hydrazone bond) (see also eq 4 in Experimental Section). Additionally, analytical isoelectric focusing (IEF) was carried out on a pH 3-10 gel to detect the presence of unconjugated enzyme. Figure 5a shows that the purified PDL-HRP conjugate did not contain significant amounts of free HRP (lanes 9-11), in contrast to the unpurified conjugate (lanes 6-8) and a freshly prepared mixture of 4FB-HRP and HyNic-PDL (lanes 3-5). Native HRP is shown in lanes 1 and 2. In addition to the IEF analysis, reverse phase HPLC with photodiode array (PDA) detection was used to analyze the purity of the PDL-HRP conjugate. The elution diagram displayed at 254 nm is shown in Figure 5b(i). The very hydrophilic PDL had a rather short retention time (1.5 min, data not shown), followed by the protein part which eluted much later (30-35 min). Because the column was run under denaturing conditions, the heme group was washed out and gave an additional peak (35.7 min). Most importantly, the conjugate peak was not only much broader than the HRP peak, but also contained different peak maxima arising from different kinds of conjugates which are probably due to the polydispersity of the PDL and the different number of HRP molecules per PDL chain. Small amounts of unreacted 4FB-HRP in the reaction mixture could also be seen (34.4 min). However, the amount of unreacted 4FBHRP was e5% as calculated from the magnitude of this peak compared to the entire peak. Thus, a purity of the conjugate of g95% could be achieved. Before ultrafiltration, the purity was around 70%. In addition, the retention times of both the PDL-



141

Figure 3. Modification of HRP with S-4FB: (a-c) kinetics of the modification reaction of HRP with 6 equiv of S-4FB/HRP (25 °C, l ) 0.1 cm): (a) (i) time-resolved spectra from t ) 0 min (---), 20, 40, 60, 80, 120, to 200 min ( · · · ), (ii) magnification of the spectra between 245 and 300 nm; (b) difference spectra (the spectrum at t ) 0 min was subtracted from all other spectra) from t ) 0 min (---), 20, 40, 60, 80, 120, to 200 min ( · · · ); (c) difference in absorbance at 274 nm (O) vs time; (d) UV/vis-spectra of unmodified HRP (---) and 4FB-HRP (MSR(4FB) ) 0.6; (s); l ) 0.1 cm), (e) determination of MSR(4FB): quantification spectra of 4FB-HRP (MSR(4FB) ) 0.6) reacted with 2-hydrazinopyridine after 1 h reaction time (s); the spectrum of freshly mixed 4FB-HRP and 2-hydrazinopyridine (---) and the spectrum of 2-hydrazinopyridine in MesB2 ( · · · ) (for specific reaction and measuring conditions, see Experimental Section); (f) stability of incorporated 4FB expressed as determined MSR(4FB) vs time at different storing conditions: at room temperature (9), 4 °C (O), and -20 °C (0).

HRP conjugate and unreacted 4FB-HRP were close together, which indicates that the interaction of the conjugate with the column was dominated by the enzyme part and not by the polymer part. Figure 5b(ii) shows the UV/vis-spectra of the highest peak of the conjugate (at 33.1 min in curve 1 in Figure 5a) and of 4FB-HRP (at 34.4 min in curve 3 in Figure 5a). The spectrum of the PDL-HRP conjugate had an additional absorption band

with a maximum at 354 nm. This clearly indicates the presence of the bis-aryl hydrazone bond. The CD spectra recorded in the UV (Figure 6a) and the Soret band region (Figure 6b) showed that the secondary and tertiary structures of HRP did not change upon 4FB modification and conjugation to PDL. The spectra of modified and conjugated HRP are very similar to the ones of native HRP and they are

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Table 3. Reaction Conditions for the Conjugation of 4FB-Enzyme to HyNic-PDL enzyme MSR (4FB) R-CT

HRP

HRP

0.6 0.5 0.6 0.8 0.9 0.8 0.9 0.7 0.7 0.8

PDL low molar massd low molar massd

high molar masse

MSR(HyNic) /100Lys ratio HyNic/4FB [conjugate]theor.a (M) conversionb (%) enzymes/polymerc 1.8 0.8 1.5 3.7 1.7 2.1 2.1 2.7 2.1 2.1

1:0.6 1:1.0 1:1.6 1:1.0 1:1.0 1:1.2 1:1.2 1:1.0 1:1.2 1:1.2

2.5 × 10-5 2.2 × 10-5 2.9 × 10-5 2.6 × 10-5 2.2 × 10-5 2.5 × 10-5 7.0 × 10-5 8.0 × 10-5 6.0 × 10-5 8.0 × 10-5

88 94 68 21 21 27 73 40 40 75

1.7 0.8 1.1 0.8 0.4 0.6 1.6 11.5 9.0 18.9

a Reaction conditions: MesB1, 25 °C, 24 h. b Conversion calculated by A354nm,measured/A354nm,theoretical. c Calculated on the basis of the conversion. d Low j ) 22500 g/mol (107 Lys). e High molar mass molar mass PDL M ) 15000-30000 g/mol: MSR(HyNic) calculated assuming an average molar mass of M j ) 225000 g/mol (1072 Lys). PDL M ) 150000-300000 g/mol: MSR(HyNic) calculated assuming an average molar mass of M

Figure 4. Conjugation reaction kinetics of 4FB-HRP reacted with HyNic-PDL (high molar mass; (a-c); 25 °C, l ) 0.1 cm): (a) (i) time-resolved difference spectra, (ii) magnification of the spectra between 250 and 390 nm; (b) time-resolved difference spectra; (c) difference in absorbance at 354 nm (O) vs time; and (d) relative activity during the conjugation reaction, reaction mixture (b), and additionally 4FB-HRP (O), native HRP (0), and PB3 buffer (9) as blank measurements, conditions [HRP] ) 1.13 nM, [ABTS]0 ) 0.25 mM, [H2O2]0 ) 0.05 mM, PB3.

similar to the previously published CD spectra for HRP.44 While the mean residue ellipticity at 222 nm reflects the helix content in the protein and therefore the secondary structure, the Soret band region at 407 nm arises from the tertiary structure. There is only a CD band at 407 nm if the heme group is embedded in the chiral environment of the active site of HRP. The difference in ellipticity between the spectrum of HRP and the spectrum of the PDL-HRP conjugate below 210 nm is due to PDL, which has a positive contribution (D-amino acids). Similarly, the CD heating and cooling curves (Figure S6) show that there was no measurable difference in the temperature stability between native HRP, 4FB modified HRP and HRP conjugated to PDL. The enzymatic activity of the purified PDL-HRP conjugate was measured right after purification and upon storage in

solution (Figure 7). The conjugate not only had the same enzymatic activity as native HRP but also the activity could be preserved over the time period measured (at least 14 days).

Conclusions It could be shown that the formation of a PDL-enzyme conjugate via bis-aryl hydrazone bond formation can be carried out in two easy steps, including modification of both enzyme and polymer and the final conjugation reaction. R-CT and HRP were used as model enzymes. Every step involved in making the conjugate occurred under mild conditions and could be quantified. Despite the intrinsic absorption of HRP at 403 nm, quantification of 4FB-HRP was still possible, although with a higher standard deviation than in the case of R-CT



143

Figure 5. Purity analysis of PDL-HRP conjugate (with high molar mass PDL): (a) Isoelectric focusing: lanes 1 and 2, native HRP isoenzyme C (pI ) 9;45 10 and 15 µg HRP); lanes 3-5, freshly prepared mixture of 4FB-HRP and HyNic-PDL (5, 10, and 15 µg HRP); lanes 6-8, conjugation reaction mixture before ultrafiltration containing about 30% free HRP (5, 10, and 15 µg HRP); lanes 9-11, purified PDL-HRP conjugate (5, 10, and 15 µg HRP). (b) Reverse phase HPLC: (b(i)) overlay of the elution curves at 254 nm of purified conjugate (1), unpurified conjugate (2), and 4FB-HRP (3) normalized to the same protein concentration, and (b(ii)) UV/vis spectra of purified conjugate eluting at 33.1 min (s) and 4FB-HRP eluting at 34.4 min (---).

Figure 6. CD spectra of native HRP (s), 4FB-HRP ( · · · ), PDL-HRP conjugate (with high molar mass PDL) (- · -), and PB1 (---) (a) in the UV region (l ) 0.1 cm) and (b) of the Soret region (l ) 0.5 cm). Conditions: protein concentration 6 µM, 25 °C.

which itself does not absorb light in the visible region of the spectrum. Because the quantification of HyNic-enzyme would be even more difficult, we decided to modify PDL with HyNic and not the enzyme. More importantly, the average number of enzymes per polymer chain could be easily determined spectrophotometrically as the formation of the bis-aryl hydrazone bond gives rise to a new absorption

j band with a maximum at 354 nm. Using high molar mass PDL (M ) 225000 g/mol), approximately 20 enzymes on average could be conjugated to one and the same polymer chain. The reaction conditions for both the enzyme modification and the conjugation were mild and, therefore, the enzymatic activity could be preserved, as no activity loss upon enzyme modification

144


Figure 7. Relative activity of purified PDL-HRP conjugate (with high molar mass PDL) stored at -20 °C (b) and 4 °C (O) and native HRP stored at 4 °C (0); conditions: [HRP] ) 1.13 nM, [ABTS]0 ) 0.25 mM, [H2O2]0 ) 0.05 mM, PB3, 25 °C.

or conjugation was observed. With ultrafiltration, a satisfactory PDL-HRP conjugate purity could be obtained. Characterization and stability measurements of the purified PDL-HRP conjugate further proved that the HRP structure could be preserved, which was in agreement with the retention of the enzymatic activity. Thus, the efficient preparation, purification, quantification, and characterization of a covalent, catalytically active PDL-enzyme conjugate via bis-aryl hydrazone bond formation could be shown where several enzymes were conjugated to one and the same polymer chain. Whether the same conjugation chemistry can also be applied for fully synthetic polymers from non-natural monomers is currently under investigation. Acknowledgment. The financial support by the Swiss National Science Foundation (200021-116205) is highly appreciated. We also thank Dr. Louis Bertschi and Dr. Xiangyang Zhang (LOC MS service, ETHZ) for the ESI-MS measurements, Dr. Joris Beld (ETHZ) for the help with the HPLC measurements, Reto Zbinden (Labor Spiez) for the IEF measurement, and Prof. D. Hilvert (ETHZ) and Prof. F. Diederich (ETHZ) for providing the HPLC and CD instruments, respectively. Furthermore, Dr. Y. Manaka and T. Kojima acknowledge the International Training Program (ITP) from the Japan Society for the Promotion of Science (JSPS). Supporting Information Available. Additional UV/vis spectra and data for (S1) the modification of high molar mass PDL with S-HyNic, (S2) the modification of R-CT with S-4FB, (S3) the conjugation of 4FB-R-CT to high molar mass HyNicPDL, (S4) the conjugation of 4FB-HRP to low molar mass HyNic-PDL, (S5) for RP-HPLC, and (S6) CD heating and cooling curves at 222 nm are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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Preparation of Catalytically Active, Covalent Î±-Polylysineâ Enzyme

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