A Stable Three-Enzyme Creatinine Biosensor. 1. Impact of Structure, Function and Environment on Pegylated and Immobilized Sarcosine Oxidase

ActaBIOMATERIALIA

Acta Biomaterialia 1 (2005) 173–181 www.actamat-journals.com

A stable three-enzyme creatinine biosensor. 1. Impact of structure, function and environment on PEGylated and immobilized sarcosine oxidase

Jason A. Berberich c,1, Lee Wei Yang a, Jeﬀ Madura b, Ivet Bahar a, Alan J. Russell c,*

a Center for Computational Biology & Bioinformatics and Department of Molecular Genetics & Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA b Department of Chemistry, Duquesne University, Pittsburgh, PA, USA c Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA

Received 11 October 2004; received in revised form 26 November 2004; accepted 28 November 2004

Abstract

The determination of creatinine levels in biological fluids is an increasingly important clinical requirement. Amperometric biosensors have been developed based on a three-enzyme system which converts creatinine to amperometrically measurable hydrogen peroxide. The development of the amperometric creatinine biosensor has been slow due the complexity of the three-enzyme system. This paper, the first of three, discusses the chemical modification of sarcosine oxidase and the immobilization and stabilization of this enzyme using polyurethane prepolymers. Sarcosine oxidase was completely inactivated after modification using poly(ethylene glycol) activated with isocyanate. The addition of a competitive inhibitor during enzyme modification was effective in protecting the enzyme from inactivation. Computational analysis of the structure of sarcosine oxidase suggests that there is a lysine in the active site that may be hyper-reactive. The enzyme was irreversibly immobilized using polyurethane prepolymers and retained significant activity. The enzymeÕs half-life at 37 °C increased from seven days to more than 50 days after immobilization. Ó 2004 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction life conditions, such as room temperature or at 37 °C while in contact with ﬂuid. Enzyme immobilization is The utility of enzymes in biosensors is limited by their especially critical for continuous use biosensors where stability. Clinical blood analyzers require enzymes to be enzyme leaching can be a concern. Enzyme immobiliza- used repeatedly while in contact with whole blood [1,2]. tion into a ‘‘biopolymer’’ by multipoint covalent attach- Many blood analyzers operate at 37 °C which further ment aﬀords a straightforward and convenient method limits enzyme stability [3]. A variety of immobilization for preparation of immobilized enzymes for biosensors. procedures are described in the literature for use with Not only does multipoint covalent immobilization pre- biosensors. Although most of the procedures described vent enzyme leaching, but it also increases enzyme sta- suggest utility for biosensors, they are often not tested bility to heat, pH, organic solvents, peroxides and under conditions that would be applicable under real- proteolytic and microbial degradation [4,5]. We have previously described the immobilization of enzymes into polyurethane materials [6–8]. In this * Corresponding author. Tel.: +1 412 235 5200; fax: +1 412 235 5290. series of papers we extend this work, describing the E-mail address: [email protected] (A.J. Russell). use of polyurethane prepolymers for the preparation 1 Present address: Agentase, LLC, Pittsburgh, PA, USA. of an enzyme-containing membrane for multiple use

1742-7061/$ - see front matter Ó 2004 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2004.11.006 174 J.A. Berberich et al. / Acta Biomaterialia 1 (2005) 173–181

CH3 O

H2C N H2N C N CH2 C OH

C NH +H2O NH CH3 O CNH Creatinine amidohydrolase (Creatininase) EC 3.5.2.10 Creatine Creatinine

O H2N C NH2

H2N C N CH2 C OH Urea +H2O O NH CH3 Creatine amidinohydrolase Creatine (Creatinase) EC 3.5.3.3 H3CCHNH 2 C OH Sarcosine

O O H2NCHCOH +HCHO+H2O2 H3CCHNH 2 C OH +O2 +H2O H Sarcosine Sarcosine Oxidase EC 1.5.3.1 Glycine

Scheme 1. Three-enzyme creatinine biosensor. amperometric creatinine biosensors. A critical step in linked to the enzyme by a cysteine residue [13]. Herein the development of biosensors is effective enzyme immo- we describe the structure–function–environment rela- bilization. Incorporation into a polyurethane polymer tionships that are required to maintain sarcosine oxidase network through multipoint attachment is a rapid and in a polymeric sensor environment. The reaction cata- effective general strategy for enhancing the stability of lyzed by sarcosine oxidase is shown below. enzymes, while retaining activity [9]. This strategy involves the production of bioplastics in a single step, employing oligomers capable of chemical reaction with 2. Materials and methods specific functionalities on the enzyme surface. The covalent binding provides protein retention in the 2.1. Materials matrix so that the advantages of immobilization can be maximized. Sarcosine oxidase (from Arthrobacter sp., SAO-341) Determination of the amount of creatinine in biolog- was purchased from Toyobo Co., Ltd. Horseradish per- ical fluids is an increasingly important clinical measure- oxidase was purchase from Sigma–Aldrich (St. Louis, ment used in the evaluation of renal function and muscle MO). All enzymes were used without further purifica- damage. There has been considerable interest in the de- tion. Poly(ethylene glycol) activated with isocyanate sign of biosensors specific for creatinine. Amperometric (mPEG–NCO) (Mw 5000) and succinimidyl propionic biosensors have been developed based on a three-en- acid (mPEG–SPA) (Mw 5000) were obtained from zyme system which converts creatinine to amperometri- Shearwater Polymers Inc. (Huntsville, AL). Hypol cally measurable hydrogen peroxide (H2O2) as described 2060G prepolymer, a toluene diisocyanate based pre- in Scheme 1 [10]. Due to the complexity of the three-en- polymer [14], was purchased from Hampshire Chemical zyme system, development of these biosensors has been (Lexington, MA). All other reagents were purchased slow [11]. from Sigma–Aldrich Chemicals (St. Louis, MO) and In this report, we discuss the chemical modification were of the highest purity available. and immobilization of sarcosine oxidase into polyurethane polymers. Sarcosine oxidase (EC 1.5.3.1) catalyzes 2.2. PEGylation of sarcosine oxidase the oxidative demethylation of sarcosine (N-methylgly- cine) and forms equimolar amounts of formaldehyde, Sarcosine oxidase was dissolved in an aqueous buffer glycine, and hydrogen peroxide. Sarcosine oxidase from (50 mM phosphate buffer, pH 7.5 or 50 mM borate buf- the Arthrobacter sp. is a monomer with a molecular fer, pH 8.5) at a concentration of 1 mg/mL. PEG–NCO weight of 43 kDa [12]. The monomeric sarcosine oxi- or PEG–SPA was added in excess to the enzyme at a dases (MSOX) are flavine proteins that contain 1 mol molar ratio of 1:100 to ensure complete enzyme modifi- of flavine adenine dinucleotide (FAD) that is covalently cation. In some experiments sarcosine oxidase inhibitors J.A. Berberich et al. / Acta Biomaterialia 1 (2005) 173–181 175

(50 mM methylthioacetic acid or 50 mM pyrrole-2-car- reaction was initiated by adding an enzyme solution boxylic acid) were added to help prevent inactivation (1 lL) or an enzyme-containing polymer (10–100 mg of the enzyme. The reaction mixture was mixed for cut into small pieces) to 5.0 mL of substrate (50 mM sar- 30 min followed by dialysis (12,000 Mw cutoff) against cosine in 50 mM phosphate buffer, pH 7.5). Before mea- 50 mM phosphate buffer [15]. surement, the assay solution was allowed to equilibrate to 37 °C with air. Oxygen consumption was measured for 5–10 min. 2.3. Synthesis of sarcosine oxidase-containing polyurethane 2.7. Thermostability of sarcosine oxidase Hypol prepolymer 2060G (0.4 g) was added to a buffered solution (3.6 g of 50 mM phosphate buffer, 50 mM Sarcosine oxidase was added to a buffered medium inhibitor, pH 7.5) containing sarcosine oxidase (0–200 (50 mM sodium phosphate, 2 mM EDTA, pH 7.5). units enzyme per gram of prepolymer). The aqueous The native enzyme concentration used was 0.06 mg/ polymer solution was vigorously mixed for 30 s in a mL. The activity of sarcosine oxidase was followed over weigh boat until the onset of gelation. Polymerization time at room temperature (22 °C), and at 4 °C and 37 °C was very rapid and gelation usually took place within using the end point assay described above. one minute. Thermoinactivation of PEG–sarcosine oxidase was monitored at 37 °C in buffer (50 mM sodium phosphate, 2 mM EDTA, pH 7.5) as described for the native 2.4. Characterization of enzyme modification enzyme. The enzyme concentration in all samples was adjusted to 0.05 mg/mL. MALDI-MS analyses were performed using a Per- Enzyme polymer samples were cut into small pieces spective Biosystems Voyager Elite MALDI-TOF. The and added to buffer (50 mM phosphate, pH 7.5) and acceleration voltage was set to 20 kV in a linear mode. incubated at 37 °C. Samples were removed over time One microliter of PEGylated enzyme solution (0.1 mg/ and assayed for enzymatic activity using the oxygen mL) was mixed with 1 lL of matrix solution (0.5 mL electrode. water, 0.5 mL of acetonitrile, 1 lL of trifluoroacetic acid, and 10 mg of sinapinic acid) and then spotted on 2.8. Inhibition of sarcosine oxidase by silver ions the target plate. Spectra were recorded after evaporation of the solvent mixture and were calibrated externally To determine the effect of silver ions on sarcosine oxi- with equine cytochrome C (12,361.96 Da (ave.)), rabbit dase, 0.07 mg/mL of sarcosine oxidase were incubated in muscle aldolase (39,212.28 Da (ave.)) and bovine serum 20 mM Tris–HCl (pH 7.5) with silver nitrate (0–1 mM) albumin (66,430.09 Da (ave.)). at room temperature. Samples were removed periodi- cally and assayed using the end point assay for sarcosine 2.5. Measurement of sarcosine oxidase activity using an oxidase activity. end point assay

The production of hydrogen peroxide was measured 2.9. Computational analysis of sarcosine oxidase by using the 4-aminoantipyrene–peroxidase system structure to predict lysine reactivity [16]. Typically, an enzyme solution (0.05 mL) was incubated with a mixture (1.0 mL total) of 95 mM sarcosine, 2.9.1. Homology modeling 0.47 mM 4-aminoantipyrene, 2.0 mM phenol, 0.045% An energy-minimized structural model for the mono- Triton X-100, 50 mM sodium phosphate (pH 8.0) and meric sarcosine oxidase (MSOX) of the strain TE1826 5 units/mL of horseradish peroxidase at 37 °C for from Arthrobacter sp. [12] was obtained from a compar- 10 min. The reaction was terminated by addition of ative modeling approach [17]. The template was chosen 2.0 mL of 0.25% sodium dodecyl sulfate (SDS) solution from the closest homologous enzyme of known struc- and the absorbance at 500 nm was measured. One unit ture, MSOX from Bacillus sp. (Protein data bank was deﬁned as the amount of enzyme that catalyzed (PDB) code:1EL5), which has a sequence identity of the oxidation of 1 lmol of substrate per minute. 83% [18]. The Molecular Operating Environment package (MOE) was used for this modeling work. It utilizes a 2.6. Measurement of sarcosine oxidase activity using an segment-matching procedure [19] together with model- oxygen monitor assay ing of insertions and deletions [20]. The insertions were modeled from available structural motifs for generating The initial rate of oxygen consumption was also mea- the possible conformations of groups of residues fol- sured at 37 °C with a Clark oxygen electrode from Yel- lowed by a standard energy minimization approach. low Springs Instruments (Yellow Springs, OH). The Amber 94 force ﬁeld was used in this process [21,22]. 176 J.A. Berberich et al. / Acta Biomaterialia 1 (2005) 173–181

2.9.2. Prediction of solvent accessibility 120 The solvent accessibility of individual residues was calculated in Swiss-PdbViewer [23]. The relative value 100 is shown in percentage scale. One hundred percent sol- 80 vent accessibility for a single residue X is achieved when the residue X has the same exposed surface area as that 60 when it is presented in a pentapeptide form, ﬂanked by two glycines on each side (GGXGG) in the solution. 40 When residue X is totally surrounded by its neighboring Activity Retention (%) residues, its relative solvent accessibility is 0%. 20

2.9.3. Prediction of lysine pK values 0 a 0 20 40 60 80 100 Some amino acids in a protein are titratable residues Native Protein (%) which exchange protons with their environment. These residues (His, Lys, Arg, Asp, Glu, Tyr and Ser) possess Fig. 1. Activity retention of PEGylated MSOX modified using PEG– NCO at pH 7.5 (closed circles) and pH 8.5 (closed squares). pKas that can be influenced by their environments. The charged states of these residues have a direct impact on protein function. UHBD (University of Houston philicity of the e-amines of non-critical lysines with in- Brownian Dynamics), is a software package capable of creased pH. solving the linearized and non-linear Poisson–Boltz- Since a single modification by isocyanate appeared to mann equation using a finite-difference method [24,25]. deactivate sarcosine oxidase, a method will be required The program calculates the electrostatic free energy dif- to protect the enzyme during immobilization within ferences for ionization of a given site in the model amino polyurethane polymers. If modification is occurring at acid and in the protein for a given residue. For N ioniz- or near an active site residue, an inhibitor may be added able residues, there will be 2N possible ionization states. to solution to help increase activity retention by block- The average pKa is obtained over all states with proba- ing the active site cleft. Although this method of protec- bilities based on Boltzmann weighting factor [26]. tion is often suggested, the literature contains precious few examples of where inhibitor protection strategies work effectively [27,28]. A number of inhibitors have 3. Results and discussion been identified for sarcosine oxidase [29]. Pyrrole-2-carboxylic acid (Ki = 1.37 mM) and (methylthio)acetic acid 3.1. Effect of PEGylation on enzyme activity and stability (Ki = 2.60 mM) were selected since they had low Ki values and do not contain functional groups that are highly Since the immobilization of enzymes into polyure- reactive with isocyanate. The native substrate, sarcosine thane polymers involves isocyanate reacting with nucle- (KM = 0.6), was not used as a protecting agent since the ophilic residues on the enzyme surface it is convenient to catalytic oxidation product, hydrogen peroxide, can model the process of chemical modification using solu- inactivate the enzyme and may also oxidize the PEG ble polymers. Modification using this technique allows backbone. Modification of sarcosine oxidase at pH 7.5 us to mimic the effect of covalent immobilization on en- showed considerably improved activity retention com- zyme activity while still working with a soluble enzyme. pared to modification without inhibitors (Fig. 2). MAL- Once the enzyme is incorporated into a polymer, mass DI-TOF analysis confirmed that the majority of the transfer effects can complicate the analysis. enzyme was modified with PEG and that the presence Monomeric sarcosine oxidase was modified using of the inhibitor had minimal effect on the modification PEG–NCO (5000 Mw) at pH 7.5 (50 mM phosphate process itself. buffer) and pH 8.5 (50 mM borate buffer) using different The stabilities of native and PEG-modified sarcosine isocyanate to enzyme ratios. The amount of native en- oxidases were measured at 37 °C in 50 mM phosphate zyme remaining was quantified using MALDI-TOF buffer (Fig. 3). Enzyme with an average of one PEG analysis and plotted versus the activity retention of sar- chain attached per molecule of enzyme had a stability cosine oxidase following modification (Fig. 1). When the similar to native enzyme (half-life of 7 days). Enzyme enzyme was modified at pH 7.5, the percentage of activ- with an average of three PEG chains attached per mol- ity retained was proportional to the percentage of ecule of enzyme had an improved half-life (17 days). unmodified enzyme. This suggests that a critical residue from the perspective of the enzyme mechanism (or 3.2. Understanding the nature of chemical modification group of residues) dominates the reaction with the isocyanate. However, modification at pH 8.5 led to increased Isocyanates are capable of reacting with amino, sulf- activity retention probably due to the increased nucleo- hydryl, carboxyl, phenolic hydroxyl, imidazole, and J.A. Berberich et al. / Acta Biomaterialia 1 (2005) 173–181 177

100 tion is reversible upon dilution, due to the lability of the carbamylated sulfhydryl group. Chymotrypsin was 80 also shown to be inactivated by cyanate [35]. Modifica- tion of the active site serine is the cause of the inactivation and similar effects have been reported for trypsin 60 and subtilisin [35]. In order to determine if sarcosine oxidase modified 40 with isocyanate was modified at a residue giving an unstable adduct (a modified sulfhydryl or tyrosyl residue

Activity Retention (%) 20 for example), the enzyme was ‘‘re-activated’’ in a number of ways. Neither dilution nor dialysis overnight at 0 pH 7.5 was successful in reactivating the enzyme. Treat- No inhibitor Pyrrole-2-carboxylic (Methylthio)acetic ment with hydroxylamine is a method frequently used to acid acid remove weakly bound conjugates from enzymes [38]. Fig. 2. Activity retention of MSOX after PEGylation in the presence Treatment of isocyanate-modified sarcosine oxidase of inhibitors (10:1 (solid) and 20:1 (white) isocyanate to amine ratio). with hydroxylamine (500 mM, pH 7) for up to 24 h The inhibitor concentration was 50 mM. was ineffective in reviving the enzyme. Thus, we can rea- sonably presume that the PEG–isocyanate has reacted with an amine on the surface of the protein and thereby 120 inactivated the enzyme. 100 In order to determine if inactivation was specific for isocyanate, or whether other amine-modifying PEGs 80 would have a similar effect, sarcosine oxidase was modified using PEG–SPA. PEG–SPA is an NHS-modified 60 PEG that forms stable amide linkages with amines 40 [32]. Interestingly, low degrees of modification (average

Relative Activity (%) of 1 to 3 PEGs per enzyme) of sarcosine oxidase with 20 PEG–SPA caused a loss of less than 5% activity. At 0 higher degrees of modification (>5 PEG chains at- 0102030 40 tached) more significant loss of enzyme activity was Time (days) apparent which could be prevented using the inhibitors pyrrole-2-carboxylic and (methylthio)acetic acid (data Fig. 3. Stability of sarcosine oxidase as a function of number of PEGs attached at 37 °C. Native enzyme (open squares); one PEG attached not shown). This suggests that the reactivity of PEG– (closed squares); three PEGs attached (closed circles). SPA with residues on the surface of sarcosine oxidase is different than the reactivity of PEG–NCO with sarcosine oxidase. phosphate groups in proteins [30,31]. Reactions with Since modification of sarcosine oxidase at pH 7.5 sulfhydryl, imidazole, tyrosyl, and carboxyl groups give caused a greater inactivation than at pH 8.5, it seems relatively unstable adducts that may decompose upon likely that the modification of the terminal amine may dilution or change in pH [32]. Due to the high nucleo- be responsible for the loss of activity (the terminal amine philicity of amino groups, these are the most easily mod- has a lower pKa than the e-amine of lysine). In fact, ified groups that form a stable adduct. Since hydroxyl modification of sarcosine oxidase by PEG–NCO caused and phenolic groups generally have nucleophilicities no noticable shift in the visible absorption spectrum of very similar to that of water, they are not typically easily sarcosine oxidase (data not shown) suggesting that mod- modified. ification did not occur in the core of the protein. The Inactivation by cyanate and isocyanate has been re- absorption spectrum of the enzyme-bound FAD of ported for a number of enzymes including pepsin [33], Corynebacterium sarcosine oxidase has been reported papain [34], trypsin and chymotrypsin [35,36], and to change (red shift of absorption peak from 455 to glutathione reductase [37]. The proteolytic activity of 462 nm) upon modification of a histidine residue with pepsin was inhibited when tyrosine residues were carb- diethylpyrocarbonate (DEP), probably in the vicinity amylated by potassium cyanate; however treatment with of the flavin moiety [39]. DEP-modified sarcosine oxi- hydroxylamine was effective in reversing the inactivation dase is completely inactive; however, activity can be by decarbamylating the residues [33]. Papain is also recovered by treatment with hydroxylamine. Although inactivated by cyanate; in fact, the active site thiol of the absorption spectrum of Arthrobacter sarcosine oxi- papain is about 3000 times more reactive with cyanate dase did not shift upon modification with PEG–NCO, than the thiol group of free cysteine [34]. The inactiva- this is not absolute evidence that modification did not 178 J.A. Berberich et al. / Acta Biomaterialia 1 (2005) 173–181 occur in the active site. Since the terminal amine is lo- Table 1 cated 44 A˚ from the nearest catalytic residue (Gly Solvent accessibility and pKa predicted for the terminal amine and the 344), it seems unlikely that the modification of this res- lysine residues in sarcosine oxidase idue is responsible for the complete inactivation of the Residue number Solvent accessibility pKa enzyme. MET 1 83.40 7.39 LYS 4 43.22 10.66 3.3. Prediction of chemical modification site LYS 5 29.67 11.30 LYS 27 30.40 12.22 LYS 31 31.87 11.51 In order to better understand the effect of chemical LYS 80 58.97 11.38 modification on sarcosine oxidase activity, computa- LYS 85 30.04 11.79 tional studies were performed to predict the most impor- LYS 89 34.07 10.72 tant residues for enzyme activity/stability and the most LYS 98 37.36 11.34 LYS 112 54.95 11.22 reactive residues for modification. LYS 128 56.04 10.80 Glocker et al. have measured the reactivity of lysine LYS 145 36.63 11.07 residues in RNAse A as a function of the solvent acces- LYS 169 31.14 11.36 LYS 187 20.88 10.69 sibility (SA) of a residue and the pKa value of lysines [40]. Similar data have been collected for deoxy-hemo- LYS 199 19.05 11.88 LYS 210 29.30 13.04 globin [41] and horseradish peroxidase [42]. Specifically, LYS 214 35.53 12.09 reactivity increases with increasing SA. However, even LYS 236 57.51 10.61 with low SA, the reactivity increases dramatically when LYS 237 26.74 11.86 LYS 268 2.20 9.68 the pKa of a given lysine residue is especially low (signi- fying a very good nucleophile). Hence, lysine residues LYS 277 48.72 11.38 LYS 299 46.52 11.23 located on the surface of the enzyme will have a reactiv- LYS 313 38.10 12.12 ity that is proportional to SA. However, residues located LYS 322 24.18 13.67 in an environment which may have a significant effect on LYS 351 4.76 9.18 LYS 368 45.42 10.90 pKa, like the active site lysine of bovine ribonuclease A, will have a significantly different pK and altered LYS 384 45.42 10.86 a LYS 386 43.96 11.34 reactivity. ˚ The predicted pK s for all lysine residues in sarcosine Rows in bold show the residues located in or within 6 A of the active a site of the enzyme. oxidase (FAD free-MSOX) are shown in Table 1. The SA was calculated from SWISS PDBviewer and esti- 120 mated pKa was obtained from the UHBD algorithm. Based on these results, we believe it is likely that lysine 100 351, an active site residue, may be the most reactive lysine since the pKa of this residue is 9.18. Lysine 268 and 80 322 are within 6 A˚ of the active site. Lysine 268 has the 60 second lowest pKa (9.68) and is probably the second most reactive residue. Lysine 322 has a significantly in- 40 creased pK (13.67) and is not likely to be easily a Relative Activity (%) modified. 20 0 3.4. Immobilization of sarcosine oxidase in 0 50 100 150 200 polyurethane polymers Enzyme Concentration (Units/g polymer)

Fig. 4. Relative activity of MSOX containing polyurethanes as a After finding the optimal conditions for modification function of enzyme content. of sarcosine oxidase to preserve enzyme activity during modification with isocyanate, we looked to immobilize the enzyme in polyurethane hydrogels. Enzyme-contain- tained no activity verifying that the effects seen with ing polymers that were prepared with enzyme concentra- the soluble modifiers translated directly to the polyure- tions of 0–200 U/g of polymer had a specific activity that thane biopolymer. Enzyme immobilized in polymers was directly proportional to enzyme concentration (Fig. prepared with inhibitor retained approximately 10% of 4). Since the reaction rate is proportional to enzyme their initial activity after rinsing to remove the inhibitor. concentration, this ensures that the rates measured The enzyme–polymers were tested for reusability by under these conditions are not diffusion limited and rep- repeatedly assaying a single gel sample for eight cycles resent only the rate from the enzyme-catalyzed reaction with no loss of apparent activity (Fig. 5). As seen with [43]. Enzyme–polymers prepared without inhibitor re- other polyurethane chemistries, the enzyme was well J.A. Berberich et al. / Acta Biomaterialia 1 (2005) 173–181 179

120 100

100

60 10

40 Relative Rate (%) Relative Activity (%) 20

0 1 12345678 0 10 20 30 40 50 60 Assay Number Time (days)

Fig. 5. Reusability of MSOX containing polyurethane hydrogels. Fig. 7. Stability of MSOX containing polyurethane hydrogels stored in buffer at 37 °C. Rates were normalized to the rate of oxygen immobilized within the gel and no leaching of enzyme consumption after the first day. Native enzyme (closed circles); from the polymer to the surrounding solvent was ob- immobilized enzyme (open circles). served [6–8]. Polymers were also prepared and stored in buffer at 4 °C under gentle agitation. Samples were re- 3.5. Effect of silver ions on enzyme activity moved from the liquid phase to determine the degree of enzyme leaching from the polymer. No activity was de- Since our goal is to use sarcosine oxidase in conjunc- tected in the liquid phase over a nearly 50-day period tion with an amperometric electrode that contains a sil- (Fig. 6). Since the enzyme is irreversibly immobilized ver/AgCl reference electrode, the effect of silver ions on in the polyurethane hydrogel material, this immobiliza- enzyme activity must be explored. Silver ions that leach tion technique will be ideal for use in clinical biosensors. from reference electrodes can bind tightly to an enzyme The thermostability of the enzyme in the polymer was and lead to inactivation [3,44]. Although no data is determined by incubating enzyme–polymer samples in available describing the concentration of silver ions gen- buffer at 37 °C and removing samples for assay period- erated by silver electrodes, the concentration of silver ically (Fig. 7). The half-life of native sarcosine oxidase ions in equilibrium with a saturated solution of silver at 37 °C is 7 days whereas immobilized sarcosine oxidase chloride is approximated 10 lM [45]. The impact of sil- retained >50% activity after incubation at 37 °Cin ver ions on sarcosine oxidase activity was determined by buffer for more than 50 days. Obviously, inhibitor- adding 1 nM to 1 mM silver nitrate to a solution of en- protected immobilization of sarcosine oxidase in poly- zyme and then assaying in the absence of silver (Fig. 8). urethane polymers is effective in stabilizing the enzyme. Obviously, silver is an effective irreversible inhibitor of High enzymatic stability, especially at elevated tempera- sarcosine oxidase. The enzyme inhibition appears to be tures, will be essential for successful application in con- slow requiring long incubation times to see an effect at tinuous use clinical blood analyzers. low silver concentrations. Although the inhibition is

120

100

40 Enzyme Leaching (%) Relative Activity (%) 20

0 0 0.001 0.01 0.1 1 10 100 1000 0 10 20 30 40 50 Time (days) AgNO3 (µM)

Fig. 6. Leaching (cumulative) of sarcosine oxidase from polyurethane Fig. 8. Silver ion induced irreversible inhibition of sarcosine oxidase hydrogels. 1 mg MSOX/g polymer (closed triangles); 2 mg MSOX/g activity. Incubation time: 5 min (closed circles); 1 h (open squares); 3 h polymer (open squares). (closed triangles); 5 h (open circles); 21 h (closed squares). 180 J.A. Berberich et al. / Acta Biomaterialia 1 (2005) 173–181 not instantaneous, since these sensors may be used over [8] Drevon GF, Danielmeier K, Federspiel W, Stolz DB, Wicks DA, a period of weeks, inhibition by silver may drastically Yu PC, et al. High-activity enzyme-polyurethane coatings. Bio- shorten sensor half-lives and we will report on strategies technol Bioeng 2002;79:785–94. [9] Russell AJ, Berberich JA, Drevon GF, Koepsel RR. Biomaterials to diminish the impact of silver ions on biocatalyst- for mediation of chemical and biological warfare agents. Annu based sensors in a separate paper. Rev Biomed Eng 2003;5:1–27. [10] Tsuchida T, Yoda K. Multi-enzyme membrane electrodes for determination of creatinine and creatine in serum. Clin Chem 1983;29:51–5. 4. Conclusions [11] Killard AJ, Smyth MR. Creatinine biosensors: principles and designs. Trends Biotechnol 2000;18:433–7. We modeled the immobilization of sarcosine oxidase [12] Nishiya Y, Imanaka T. Cloning and sequencing of the sarcosine oxidase gene from Arthrobacter sp. TE1826. J Ferment Bioeng in polyurethane polymers using PEG–NCO. Sarcosine 1993;75:139–244 (protein sequence can be found at http://www. oxidase was completely inactivated when modified by ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&val=730706). a single molecule of PEG–NCO. Using a pKa prediction [13] Trickey P, Wagner MA, Jorns MS, Mathews FS. Monomeric program, we were able to show that two residues in sarcosine oxidase: structure of a covalently flavinylated amine the active site of sarcosine oxidase are predicted to oxidizing enzyme. Structure 1999;7:331–45. K [14] Braatz JA. Biocompatible polyurethane-based hydrogel. J Bio- have decreased p a values making them hyper-reactive. mater Appl 1994;9:71–96. Two irreversible inhibitors, (methylthio)acetic acid and [15] Drevon GF, Hartleib J, Scharff E, Ru¨terjans H, Russell AJ. pyrrole-2-carboxylic acid, were effective in preventing Thermoinactivation of diisopropoylfluorophosphatase-containing complete enzyme inactivation during modification. Sar- polyurethane polymers. Biomacromolecules 2001;2:764–71. cosine oxidase was successfully and irreversibly immobi- [16] Nishiya Y, Imanaka T. Analysis of interactions between the Arthrobacter sarcosine oxidase and the coenzyme flavin adenine lized during polymerization by chemical crosslinking dinucleotide by site-direct mutagenesis. Appl Environ Microbiol into polyurethane hydrogel-forming polymers, retaining 1996;62:2405–10. approximately 10% activity. Polymers containing sarco- [17] Schulz-Gasch T, Stahl M. 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