Oxidative Protection of Hemoglobin and Hemerythrin by Cross

The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-cre...
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Oxidative Protection of Hemoglobin and Hemerythrin by CrossLinking with a Nonheme Iron Peroxidase: Potentially Improved Oxygen Carriers for Use in Blood Substitutes Denisa Hathazi,† Augustin C. Mot,† Anetta Vaida,† Florina Scurtu,† Iulia Lupan,‡ Eva Fischer-Fodor,§ Grigore Damian,∥ Donald M. Kurtz, Jr.,⊥ and Radu Silaghi-Dumitrescu*,† †

Faculty of Chemistry and Chemical Engineering, “Babes-Bolyai” University, 11 Arany Janos St., Cluj-Napoca, Romania Institute of Interdisciplinary Research in Bio-Nanosciences, Molecular Biology Center, 42 Treboniu Laurean St., Cluj-Napoca, 400271-Romania ∥ Faculty of Physics “Babes-Bolyai” University, 1 Kogalniceanu St., Cluj-Napoca, Romania § Ion Chiricuta Cancer Institute - Comprehensive Cancer Center, 34-36 Republicii St., Cluj Napoca, Romania ⊥ Department of Chemistry, University of Texas at San Antonio, One UTSA Circle, San Antonio, Texas 78249-0698, United States ‡

S Supporting Information *

ABSTRACT: The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.

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INTRODUCTION The use of hemoglobin (Hb)-based artificial oxygen carriers in blood substitutes has been described and examined extensively.1,2 However, their applicability so far has been limited due to toxicity issues, at least some of which can be linked to oxidative stress. Strategies for limiting this toxicity have included the addition of antioxidant small molecules or enzymes to chemically derivatized or encapsulated particles of Hb.1,3,4 The nonheme iron oxygen carrying protein, hemerythrin (Hr) has recently been proposed as an alternative to Hb in blood substitutes.5 Hr is an oxygen transport and storage protein found in marine invertebrates that employs a nonheme diiron site Fe(II)−Fe(II) to reversibly bind dioxygen. Hr has some potential advantages over Hb for a blood substitute, including a higher molecular weight (108 kDa vs 64 kDa Hb), which can lead to lower extravasation or elimination through the kidney (hypertension and vasoconstriction are inversely correlated with the size of the blood substitute protein),4 lower reactivity toward hydrogen peroxide, nitric oxide, and nitrite and a remarkably lower tendency to generate toxic free radicals in such reactions.5−7 © 2014 American Chemical Society

In the absence of the enzymatic system in red blood cells, Hb undergoes a series of reactions generating potentially toxic reactive oxygen species such as hydrogen peroxide or superoxide. As a strategy to scavenge the reactive oxygen species generated by Hb, PolyHb-superoxide dismutase-catalase was designed so that the blood substitute contains copolymerized antioxidant enzymes.3 We propose to combine alternative antioxidant enzymes with either Hb or Hr. One of these alternative antioxidant enzymes, rubrerythrin (Rbr) is a nonheme iron protein with peroxidase (hydrogen peroxide reductase) function.8 Unlike heme-based peroxidases or catalases, the reduced (all-ferrous) Rbr reduces hydrogen peroxide to water at a diiron site, which does not involve high-valent iron (FeIV or FeV) or free radicals (porphyrin- or protein-based). Moreover, the Km for hydrogen peroxide is 2 orders of magnitude lower for Rbr compared to those of peroxidases and catalases.9−12 The oxidized (all-ferric) Rbr also shows a low level of aromatic substrate peroxidase Received: March 19, 2014 Revised: April 9, 2014 Published: April 9, 2014 1920

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activity.13 On this basis, Rbr could conceivably be a distinctly safer enzyme than heme-based peroxidases or catalases for scavenging hydrogen peroxide in vivo. In order to furnish Rbr with reducing equivalents the flavoprotein, NADH/rubredoxin oxidoreductase (NROR) can be used.14 In this case, the small nonheme iron protein, rubredoxin (Rd) is needed to shuttle reducing equivalents between the NROR and Rbr. Prereduced Rd by itself can also furnish reducing equivalents to polymerized Rbr to reductively scavenge hydrogen peroxide (Scheme 1).11

solution was then added, and the reaction mixture was incubated for 15 min at 4 °C under mechanical agitation in order to reduce the Schiff bases formed and the excess glutaraldehyde. Rbr and NROR (33 μM) was also cross-linked in the absence of the other two proteins using 5 mM glutaraldehyde as described above. The protein components were then separated from the other reagents using a PD-10 desalting column. The polymerization yield was assessed by SDS-PAGE and analytical gel filtration chromatography. When needed, the polymerized or copolymerized forms were separated from the unreacted proteins using a HiTrap SP HP anion exchange column using a linear gradient from 100% buffer A (20 mM TrisHCl, NaCl 150 mM, pH 7.4) to 100% buffer B (20 mM Tris HCl, NaCl 1 M, pH 7.4) and HiPrep 16/60 Sephacryl S300 size exclusion column (GE Healthcare) using buffer A, controlled by a FPLC system. Analytical size exclusion chromatography was carried out on a Superdex 200 5/150 GL column (GE healthcare). Molecular weights were determined based on a calibration curve employing a molecular weight standard kit (Sigma-Aldrich) containing carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), amylase (200 kDa), apoferritin (443 kDa), thyroglobulin (669 kDa), and blue dextran (void volume marker). Copolymers containing His-tagged Rbr and Hr were purified by the use of both Niagarose (Qiagen) affinity chromatography and size exclusion chromatography. For affinity chromatography, the protein sample (containing 5 mM imidazole) was loaded onto a 1 mL Ni-agarose affinity column equilibrated in washing buffer (5 mM imidazole, 100 mM NaCl, 20 mM MOPS, pH 7.4) and further washed with this buffer followed by the elution of the protein using 250 mM imidazole, 100 mM NaCl, and 20 mM MOPS, pH 7.4. The collected protein fractions were analyzed by SDS-PAGE and size exclusion chromatography. Ferryl Heme Formation and Decay. In a quartz cuvette metHb (oxyHb was treated with ferricyanide and the excess was removed using a PD-10 desalting column) was added to attain a final concentration of 10 μM protein in PBS followed by addition of H2O2 to a final concentration of 80 μM. The reaction was monitored by absorbance at 425 nm for several minutes.18 For the EPR experiment metHb and metcopolyHbRbr were mixed with hydrogen peroxide to attain a concentration of 400 μM of Hb and 800 μM of H2O2. The mixture was quenched by freezing in liquid nitrogen after 25 s. For selected experiments Rbr was reduced with 30 μM of reduced Rd to ensure electrons for several catalytic cycles. Formation of high valent species was followed anaerobically. Rubredoxin Peroxidase Activity. The activity of the cross-linked Rbr/Hb or Rbr/Hr was measured using the following protocol. To an anaerobic quartz cuvette was added 70 μM as isolated (oxidized) Rd (determined using ε492nm = 8700 M−1 cm−1)11 in PBS buffer, to which a slight molar excess of sodium dithionite was added in order to reduce the Rd, followed by controlled air oxidation to ensure no extra dithionite remained. The solution was purged for 5 min with argon in order to ensure an anaerobic atmosphere. To this, a small volume of anaerobic 70 mM hydrogen peroxide solution was added with a Hamilton gastight microsyringe to attain 130 μM H2O2 in the assay mixture, followed by the addition of the cross-linked proteins to achieve 3.7 μM Hb or Hr concentration in the assay mixture. The rate of absorbance increase at 492 nm due to oxidation of the reduced Rd was monitored. NADH Peroxidase Activity. For assessing the NADH peroxidase activity of the multimer Hb/Rbr/NROR the following protocol was employed: in an anaerobic quartz cuvette was added 50 μM NADH (determined using ε340nm = 6.220 M−1 cm−1) in PBS and 1.5 μM oxidized Rd. To this mixture a volume of H2O2 was added to attain a final concentration of 100 μM. The reaction was initialized by the addition of the trimer. In the assay mixture the proteins concentrations were 5 μM Hb, 166 nM Rbr, and 166 nM NROR. The oxidation of NADH was monitored as the decrease in absorbance at 340 nm. Effects of Cross-Linked Proteins on Cell Viabilities. Cell viability tests were conducted using previously described protocols.19 The Human Umbilical Vein Endothelial Cell line (HUVEC) was a generous gift from Assoc. Prof. Marina Nechifor from the University of Bucharest, Faculty of Biology. Cells were defrosted carefully, cultivated

Scheme 1. Diagram Showing the Flow of Reducing Equivalents from Reduced Rd (Rdred) to H2O2 Catalyzed by Rbr in the Rubredoxin Peroxidase Activity

Reported here are protocols for chemical cross-linking of Hb and Hr with relatively small amounts of Rbr leading to products with increased capability for reductively scavenging hydrogen peroxide, offering promise as less toxic artificial oxygen carriers/ blood substitutes. For selected experiments, a copolymerized trimer (Hb-Rbr-NROR) was used to examine the extra protection that this substitute can offer against oxidative stress.

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EXPERIMENTAL SECTION

Materials and Apparatus. Recombinant P. gouldii Hr,5 Desulfovibrio (D.) vulgaris Rbr,9,15 D. vulgaris Rd,11 Clostridium acetobutylicum NROR,16 and native bovine Hb17 were purified as previously described. An N-terminal His-tagged Rbr was obtained by subcloning the Rbr gene into the NdeI and BamHI restriction sites of pET28a (Novagen) to generate pET28a-Rbr. The pET28a-Rbr was transformed into E. coli strain BL21(DE3) and Rbr was expressed and purified by the previously described protocol.9−15 Unless otherwise mentioned, the Rbr experiments used the non-His-tagged protein. Hb concentrations are given per heme. Hr, Rbr, and NROR concentrations are given per protein monomer (i.e., per active site). The UV−vis absorption spectra were recorded on Agilent 8453 (Agilent, Inc.) and Cary 50 (Varian, Inc.) instruments. EPR spectra were recorded on a Bruker EMX Micro spectrometer with a liquid nitrogen cooling system. Instrument conditions were microwave frequency, 9.43 GHz; microwave power, 15.89 mW; modulation frequency, 100 kHz; modulation amplitude, 3 G; sweep rate, 22.6 G/s; time constant, 81.92 ms; average of three sweeps for each spectrum; temperature, 100 K. Glutaraldehyde Cross-Linking. All cross-linking reactions were carried out at 4 °C. All column chromatographies (size exclusion, anion exchange, and Ni−Agarose affinity) were carried out at room temperature (∼23 °C). Hr and Hb were polymerized with glutaraldehyde following previously described protocols5,6 with some modifications. Either 150 μM met-Hr or 1 mM oxy-Hb in 137 mM NaCl, 2.7 mM KCl, 12 mM NaH2PO4, pH 7.4 (PBS) was mixed with 5 mM glutaraldehyde and allowed to react for 2 h at 4 °C under mechanical agitation. For copolymerization, Rbr was added to the reaction mixture at 30:1 mol Rbr/mol Hb or 1:30 mol Rbr/mol Hr prior to glutaraldehyde addition. To attain the polymerization mixture for the trimer a ratio of 1:1:15 mol Rbr/mol NROR/mol Hb was employed. A 4-fold molar excess (with respect to glutaraldehyde concentration) of sodium borohydride from a freshly prepared stock 1921

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in special culture flasks (Nunclon) with RPMI-1640 (Sigma) culture medium, supplemented with fetal calf serum (FCS, Sigma), penicillin− streptomycin (Sigma), and glutamine in humidified Heto Holten Cellhouse 154 incubator at 37 °C and 5% CO2 level. Cells were adherent and several cell passages were performed using enzymatic procedures. Experiments were made when confluence of the cells achieved 80% on the flask surface. Cells were then plated on 96-well flat-bottomed microtiter plates (Nunclon) and were kept 24 h in the incubator. To obtain a suitable density of cells on each well on the day of the measurement, and considering the HUVEC cells proliferation rate, we used different cell densities pro well: 1 × 105 per well cell for the 24 h experiment, 7.5 × 103 cells for 48 h incubation, and 6 × 103 cells for the 72 h long experiment. Wells were then treated with solutions to be tested (10 μL of PBS solution of polyHb, polyHr, copolyHb-Rbr, or copolyHr-Rbr) with untreated cells left as reference; blank cell-free wells contained cell culture media: as coloration reference, cell-free wells were treated with culture media, and compound. Each solution was tested in triplicate, and three different experiments were completed. Cytotoxicity was assessed using a quantitative colorimetric MTT assay.20,21 After 24, 48, or 72 h incubation with solutions to be tested, polyHb, polyHr, copolyHb-Rbr, or copolyHr-Rbr at final concentration of 180 μM Hb or Hr, MTT solution was added to each well at a final concentration of 1 mg/mL per well, and the plates were incubated at 37 °C for another hour. Dimethylsulfoxide was added to each well to dissolve the formazan, and spectrophotometric absorbance measurements were made using a BioTek Synergy 2 multimodal fluorescence microplate plate reader. Statistical analysis employed the GraphPad Prism 5 biostatistics software. One-way analysis of variance (ANOVA) and Dunnett Multiple Comparison test (p < 0.05, r2 = 0.94−0.86) were completed for all individual inhibitory effects. Dioxygen Affinity and Autoxidation. OxyHb was obtained by treatment of the met form of the derivatized globins with dithionite, followed by aerobic passage over a PD-10 desalting column. OxyHr was prepared as previously described.22 For dioxygen affinity measurements an anaerobic UV−vis cuvette, containing PBS and 10 μM of the oxy protein, was purged for 10 min with argon gas until the protein was transformed completely into the deoxy form. Titration with dioxygen was then achieved by addition of small aliquots of airequilibrated buffer (240 μM O2)23 and collecting UV−vis absorption spectra subsequent to each addition. Occupation fractions Y were calculated by dividing the concentration of the oxy form by the total protein concentration in the cuvette. The former was assessed by monitoring the absorbance ratio at 415 nm (oxy Hb)/430 nm (deoxyHb) (r = AU415/AU430) and at 300 nm/500 nm for oxyHr (r = AU300/AU500), at which wavelengths the latter ratio has a minimum and a maximum value for the deoxy and the oxy forms, respectively. The normalization was performed by defining the fractional saturation with dioxygen of the protein, Y = (rdeoxy − r)/(rdeoxy − roxy), as obtained from the measurements. Data were plotted in the form of Y versus dioxygen concentration and were fitted to a Hill equation using a least-squares protocol within the Microsoft Excel software package using the Solver module. The P50 were obtained by transforming the oxygen concentration (obtained by adding small quantities of buffer to the sample) into mmHg (KHoxygen × [O2] × 10−6 atm and 1 atm = 760 mmHg). The autoxidation half-times for 10 μM of oxyHbs were measured in aerobic PBS buffer solution at 37 °C for 30 h monitoring changes at 415 and 405 nm. Autoxidation for 20 μM oxyHr were carried out in aerobic PBS buffer at room temperature (∼23 °C), for 18 h, following the changes in absorbance at 330 and 500 nm. Data were plotted in the form of AU415/405 = f (time), for Hb and for Hr AU500/380 = f (time), and fitted to a first order decay.

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Figure 1. Size exclusion chromatograms of Hb and Hr cross-linked with Rbr using glutaraldehyde. The void time (t0) is that for blue dextran elution, and molecular mass calibration axis was estimated using the molecular masses were established by a calibration curve indicated above the chromatograms.

expected to function as a catalyst. High-molecular weight aggregates were formed similar to those previously characterized for glutaraldehyde polymerization of Hb and Hr in the absence of Rb,5,6 as also confirmed by the SDS-PAGE analysis (Supporting Information). The polymers/copolymers eluted at ≤750 kD for the Hr mixtures and at ≤600 kD for the Hb mixtures. Lower molecular weight products (≥400 kD) in the glutaraldehyde-treated proteins eluted coincidentally with proteins not treated with glutaraldehyde. To confirm that the above-described higher molecular weight fractions in the Figure 1 chromatograms correspond to copolymers rather than a mixture of individual protein polymers, a His-tagged Rbr was alternatively employed. The resulting polymerization mixture contained species that bound to a Ni-agarose column (Figure 2); the UV−vis absorption spectra (Figure 3, left panel) of the high molecular weight subfraction of the Ni-agarose eluted copolymerized Hr-Histagged Rbr showed UV−vis spectral signatures characteristic of both Hr and Rbr (Figure 3, left panel). The unpolymerized region seen in the size exclusion chromatogram (Figure 3, left panel) of the protein fraction after Ni-agarose affinity column is a minority (fractions with MW lower than 480 kD) that could contain dimers of Hr-Rbr His-tagged, poly His-tagged Rbr, or native His-tagged Rbr. Native Hr and polyHr do not bind to the Ni affinity column (Figure 2). Unfortunately, the imidazole purification step on the Ni-agarose affinity column impinged on

RESULTS AND DISCUSSION

Glutaraldehyde Cross-Linking. Figure 1 shows sizeexclusion chromatograms of glutaraldehyde-treated mixtures of Hr + Rbr and Hb + Rbr at mol ratios of 30:1 Hb/Hr:Rbr. The relatively lower amount of Rbr was chosen because it is 1922

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spin ferric Hb, the EPR spectra of the higher molecular weight copolyHb-Rbr and copolyHr-Rbr (>600 kDa for Hb-Rbr, and >750 kDa for Hr-Rbr) are dominated by the g = 4.3 feature characteristic of the Fe(SCys)4 site in ferric Rbr.12 The relatively intense absorptions at ∼490 and 560 nm in the UV−vis absorption spectra of the Hr-Rbr polymer are also characteristic of the Rbr Fe(SCys)4 site.12 The copolyHr-Rbr also retains the intense UV−vis absorption features at ∼320 and 360 nm characteristic of the metHr diiron site. The UV− vis absorption spectrum of the Hb-Rbr polymer is dominated by the heme, so that the Rbr features cannot be detected. Comparison of Hb absorption features for copolyHb-Rbr versus polyHb in Figure 4 indicates a slightly lower tendency of the Hb to undergo autoxidation during cross-linking in the presence of Rbr, as evidenced by the larger absorbance in the α and β region at ∼575 nm and slightly lower absorbance at 630 nm in the Hb−Rbr copolymer. The differences were not so obvious in the Soret region, although based on initial and final absorptions in the α and β region (((AUi(575−630) − AUf(575−630))/AUi(575−630)) × 100) we determined that, compared to native Hb, the mixture containing Rbr after derivatization had 23% more met species, whereas simple polyHb had 32%. Based on the intensity of the g = 4.3 EPR signals compared to that of an oxidized Rd standard,11,25 it can be estimated that 20−30% of the Rbr present in the initial reaction mixtures has been incorporated in the Hb-Rbr and HrRbr copolymers when using a Rbr/Hr or Hb 1:30 mol ratio in the cross-linking reaction, after size exclusion chromatography to separate out the unpolymerized Rbr (MW ≤ 60 kDa). O2 Affinity and Autoxidation Rates. Table 1 shows that the dioxygen affinity of the copolyHb-Rbr is higher than that of the native Hb, although still low enough to be able to deliver the O2 to myoglobin for discharge the oxygen to muscles and tissues. For the derivatized copolyHr-Rbr, the affinity for dioxygen is lower than for native Hr and closer to that of native Hb, which is expected to be an advantage for a blood substitute. The autoxidation rates of the poly- and copolyHb-Rbr are approximately five times faster than that the native Hb and roughly two times faster for poly- and copolyHr-Rbr than that of the native Hr (Table 1). Ferryl Formation and Decay. One of the main problems of Hb-based blood substitutes is their autoxidation, which leads to the ferric form of the heme, which in turn reacts with peroxides in vivo generating ferryl heme and toxic free radicals (Scheme 3).1 The reaction of the met forms of these

Figure 2. Ni-agarose affinity chromatography elution profiles of copolyHr-His-tagged Rbr (1), unpolymerized His-tagged Rbr (2), and polyHr (3).

the stability of the His-tagged Rbr so that further tests were performed using only the nontagged protein. The chromatographic profiles (anion exchange, size exclusion) of polyHb + polyRbr mixtures are different from those of copolyHb-Rbr. The same differences in the chromatographic profiles were also observed for polyHr + polyRbr versus Hr-Rbr (Supporting Information). This suggests that polymerization of binary protein mixtures (Hb/Rbr and Hr/Rbr) does lead to copolymers. These interprotein cross-linked species are hereafter referred to as “copolymer”. Such lower molecular weight components are not expected to be an issue for a blood substitute since they should be readily removable by size exclusion chromatography. To prevent tetramer dissociation, methods for covalent cross-linking of monomer proteins have been developed (this method also suppressed renal filtration).24 Figure 4 shows UV−vis absorption and EPR spectra of glutaraldehyde-cross-linked oxyHb or metHr5,6 immediately after the glutaraldehyde polymerization step, as well as the corresponding spectra obtained for the higher molecular weight fractions obtained from the size exclusion chromatography shown in Figure 1 of the same two proteins that were glutaraldehyde-cross-linked in the presence of substoichiometric ferric Rbr. Besides the g = 5.7 feature characteristic of high-

Figure 3. Left panel: Size exclusion chromatography of the copolyHr-His-tagged-Rbr fraction that was bound to and eluted from the Ni-agarose affinity column in Figure 2. Right panel: UV−vis absorption spectra of the higher molecular weight fractions from the left panel chromatogram. 1923

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Figure 4. UV−vis absorption spectra of void volume fractions from size-exclusion chromatography as shown in Figure 1 of Hr and Hb cross-linked with glutaraldehyde in either the absence or presence of Rbr for 1:30 mixture, PBS buffer, pH 7.4. Insets show corresponding EPR spectra of the high molecular weight fractions of the copolymers; the g values are indicated by arrows.

Table 1. P50 Values (Expressed in mm Hg) and Autoxidation Half-Time (t1/2; Expressed in Hours) of Hb, polyHb, copolyHb-Rbr; Hr, polyHr, and copolyHr-Rbr Mb Hb polyHb copolyHbRbr Hr polyHr copolyHrRbr

P50(mmHg)

T1/2(hours)

3.5 18.3 10.3 8.9 7.28 14.1 12.8

3.2 24.9 4.7 4.9 9.4 5.8 6.3

courses in Figure 6 show that, when supplied with reduced Rd, copolyHb-Rbr accumulates significantly less ferryl, consistent with scavenging of H2O2 by reduced Rbr. Rubredoxin Peroxidase Activity. The combined anaerobic system of reduced Rbr, copolyHbRbr, or copolyHrRbr and hydrogen peroxide, does indeed retain Rbr-peroxidase activity. Figure 7 shows the time course of anaerobic oxidation of reduced Rd by Rbr/H2O2, as monitored at 492 nm. Another method for the reduction of Rbr in the copolymers was also employed. The electron transfer pathway from NADH to H2O2 can be reconstituted using the protein NROR, that has previously been shown to efficiently catalyze reduction of Rd,26 as shown in Scheme 2. The time courses in Figure 8 show that the resulting high molecular fractions of this multimer (HbRbr-NROR) was found to indeed also have the ability to scavenge very efficiently H2O2 seen by a decrease at 340 nm due to NADH consumption. As seen in Figure 9 one may reduce metHb-Rbr-NROR to its oxy form using NADH. This pathway could be useful in maintaining a functional state for the globin, by rereducing the met Hb resulted from the intrinsic autoxidation reaction. Effects of Poly- and CopolyHb and -Hr on Cell Viabilities. The effect of Hb-based potential blood substitutes on human cell cultures, including the HUVEC line, has previously been used to identify possible toxic effects of cellfree Hb or modified Hb.1,27−30 Figure 10 shows these effects for high molecular weight fractions of copolyHb-Rbr and coplyHr-Rbr as obtained after the derivatization step. As previously shown, polyHb shows slight toxicity toward these cells,19 while polyHr reveals a small protective effect relative to the control.31 The copolyHb-Rbr and copolyHr-Rbr appear to provide a slightly better protective effect compared to those of native Hb or Hr at 24 h. At longer times (48 and 72 h, respectively), this advantage of the protein copolymers is no longer evident

Scheme 2. Model for H2O2 Scavenging Using NADH as an Electron Source for NADH Peroxidase Activity via NROR

Scheme 3. Diagram Showing the Electron Flow for the Autooxidation Process in Globins and the Formation of the High Valent Species or Ferryl

polymerized Hbs with hydrogen peroxide was monitored with UV−vis absorption and EPR spectroscopy (Figure 5). The UV−vis absorbance at 425 nm indicates the copolyHb-Rbr accumulates significantly less ferryl. The EPR spectra also show that the presence of ferric copolyHb-Rbr accumulates less free radical (g = 2.005) than polyHb, which may result in reduced toxicity under oxidative stress in vivo. The formation and decay of the high-valent ferryl species was also followed when Rbr was supplied with a source of reducing equivalents in the form of reduced Rd in an amount sufficient for several catalytic cycles. Indeed, Rd is known to be an efficient (and physiologic) redox partner for Rbr.8 The time

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CONCLUSIONS We demonstrate a potentially new and efficient system to provide protection against oxidative stress to oxygen-carrying proteins in blood substitutes. This protection consists of copolymerization of the oxygen-carrying protein and a nonheme iron peroxidase, Rbr. Hb-Rbr or Hr -Rbr copolymers are able to reversibly bind molecular oxygen and at the same time display increased peroxidase reactivity with decreased 1924

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Figure 5. Left: Formation and decay of ferryl heme generated from reaction of metHb, polymetHb or copolymetHb-Rbr with hydrogen peroxide (PBS buffer pH 7.4, [metHb] = 10 μM, [H2O2] = 80 μM). Right: EPR spectra obtained during during ferryl heme generation. Inset showing a detailed view of the formed radical obtained at [metHb] = 400 μM, [H2O2] = 800 uM, and a reaction time 25 s at room temperature followed by freezing in liquid N2.

Figure 6. Formation and decay of ferryl heme generated from reaction of copolymetHb-Rbr or polymetHb with hydrogen peroxide (PBS buffer pH 7.4, [metHb] = 10 μM, [H2O2] = 80 μM) in the presence or absence of 30 μM reduced Rd. Reaction was started by the addition of H2O2.

Figure 8. NADH peroxidase activity of Hb, polyHb, copolyHb-Rbr, and copolyHb-Rbr-NROR, as obtained after the derivatization step. Activities were monitored anaerobically as the decrease in absorbance at 340 nm due to NADH consumption. In a total volume of 1 mL, the concentration of components in the assay was 5 μM Hb, 166 nM Rbr, 166 nM NROR, 50 μM NADH, 1.5 μM Rd, and 100 μM H2O2, PBS pH = 7.4 PBS buffer solution. Initial rates: Hb, 2.7 μM/min; polyHb, 2.4 μM/min; copolyHb-Rbr, 2.45 μM/min; copolyHb-Rbr-NROR, 5.63 μM/min.

tendency to produce free radicals as side-products under these conditions. Addition of third protein, NROR, generated a multimer showing NADH peroxidase activity via electron transfer to Rbr via Rd. The copolymerized oxygen-carrying

Figure 7. Rubredoxin peroxidase activity of samples as obtained after the derivatization step. Conditions: anaerobic 70 μM reduced Rd in PBS, addition of H2O2 (130 μM final concentration), step 1, and copolymer sample addition, step 2; left panel: 1 μM Rbr in copolyHb-Rbr1 and 5 μM Rbr copolyHbRbr2; polyHb, 9 μM Rd oxidized/min, copolyHb-Rbr1 ([Hb]/[Rbr] = 150), 11 μM Rd oxidized/min, copolyHbRbr2 ([Hb]/[Rbr] = 30), 30 μM Rd oxidized/min; right panel: 1 μM Rbr in copolyHr-Rbr1 and 5 μM Rbr copolyHrRbr2; polyHr, 1 μM Rd oxidized/min, copolyHrRbr1 ([Hr]/[Rbr] =150), 7 μM Rd oxidized/min, copolyHrRbr2 ([Hr]/[Rbr] = 30), 17 μM Rd oxidized/min. 1925

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Figure 9. Reduction of metHb-Rbr-NROR using NADH. Concentration of components employed in the assay was: 10 μM of metHb-Rbr-NROR (concentration corresponding to Hb) and 80 μM of NADH. The reduction was followed for 240 min.

Figure 10. Effect of polyHb, polyHr, copolyHb-Rbr, and copolyHr-Rbr on HUVEC cultures at various exposure times. The assay is described in Experimental Section.

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proteins showed slightly increased dioxygen affinity, which has been stated to be an advantage for a blood substitute as the premature release of dioxygen can thus be prevented.32,33 The somewhat faster autoxidations of the copolymerized Hb and Hr, while undesirable, are nevertheless still on the time scale of several hours, which may be sufficient to fulfill they functions. At 24 h exposure, copolyHr-Rbr and copolyHb-Rbr exhibited a decreased toxicity toward HUVEC cultures compared to the native oxygen carrying proteins. This decreased toxicity of the copolymers disappears at longer incubation times. However, one expects that at such longer times a patient subjected to transfusion with these substitutes would have reached a condition beyond emergency, at least from the point of view of blood supply from human donors.

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AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: +40-264593833. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work shown here has been supported by the Romanian Ministry for Education and Research (Grants PNII ID565/ 2007 and ID488/2012) and by a Ph.D. scholarship to ACM (Contract POSDRU/88/1.5/S/60185, “Innovative doctoral studies in a knowledge based society”).

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ASSOCIATED CONTENT

REFERENCES

(1) Alayash, A. I. Nat. Rev. Drug Discovery 2004, 3, 152. (2) Christa, L.; Modery-Pawlowski, L. L. T.; Pan, V.; Sen Gupta, A. Biomacromolecules 2013, 14, 939. (3) D’Agnillo, F.; Chang, T. M. S. Nat. Biotechnol. 1998, 16, 667. (4) Xiong, Y.; Liu, Z. Z.; Georgieva, R.; Smuda, K.; Steffen, A.; Sendeski, M.; Voigt, A.; Patzak, A.; Bäumler, H. ACS Nano 2013, 7, 7454. (5) Mot, A. C.; Roman, A.; Lupan, I.; Kurtz, D. M., Jr.; SilaghiDumitrescu, R. Protein J. 2010, 29, 387. (6) Deac, F.; Todea, A.; Silaghi-Dumitrescu, R. In Metal Elements in Environment, Medicine and Biology Tome IX; Silaghi-Dumitrescu, R., Garban, G., Eds.; Cluj University Press: Cluj-Napoca, Romania, 2009; p 165.

S Supporting Information *

Experimental details regarding SDS-PAGE of Hr-Rbr and HbRbr cross-linked mixtures prior to gel filtration (Figure S1); size exclusion chromatograms of copolyHrRbr and polyHr + polyRbr, copolyHbRbr and polyHb + polyRbr (Figure S2); size exclusion chromatogram of Hb cross-linked with Rbr and NROR using glutaraldehyde (Figure S3); NADH peroxidase activity of Hb-Rbr-NROR at different concentrations of Rd and Michaelis−Menten curve (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org. 1926

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