An Optical Method for the Detection of Oxidative Stress Using Protein

Anal. Chem. 2001, 73, 957-962

An Optical Method for the Detection of Oxidative Stress Using Protein-RNA Interaction F. Lisdat,*,† D. Utepbergenov,‡ R. F. Haseloff,‡ I. E. Blasig,‡ W. Sto 1 cklein,† F. W. Scheller,† and § R. Brigelius-Flohe´

University of Potsdam, Institute of Biochemistry and Biology, 14415 Potsdam, PF 601553, Germany, Research Institute of Molecular Pharmacology, Alfred Kowalke Str. 4, 10315 Berlin, Germany, and German Institute of Human Nutrition, Arthur Scheunert Allee 114-116, 14558 Bergholz-Rehbru¨cke, Germany

The cytosolic 4Fe-4S protein aconitase can be converted under the influence of reactive oxygen species into an ironregulatory protein (IRP1). Therefore, the IRP1 level is considered as an indirect marker of oxidative stress. An experimental approach is presented here to detect the concentration of this marker protein by surface plasmon resonance. The optical method exploits the natural binding affinity of IRP1 to an iron-responsive element (IRE) which was in vitro transcribed with a linker sequence and subsequently immobilized on a BIACORE sensor chip. The detection was found to be reproducible and sensitive in the range 20-200 nM IRP. Conditions of the binding process, such as pH and thiol concentration, were characterized. Feasibility of the method to detect and quantify IRP1 in physiological media was demonstrated. Reactive species involved in oxidative stress, such as hydroperoxides, superoxide, nitric oxide, or peroxynitrite, can act as signal molecules within the cell, as well as in the intercellular communication. However, if they are locally produced at higher concentrations or exogenously delivered, the resulting oxidative stress leads to toxicity and pathophysiological reactions.1,2 Examples are reperfusion syndrome, rejection of transplants, inflammation, etc.3-6 Organisms have developed a sophisticated system that provides a balance between the production and degradation of reactive species. The system includes, on one hand, generating enzymes such as NADPH oxidase, NO synthase, or lipoxygenases. On the other hand, the defense system comprises direct enzymatic and nonenzymatic scavengers, such as superoxide dismutase, catalase, glutathione peroxidases, ascorbic acid, vitamin E, etc.7-9 †

University of Potsdam. Research Institute of Molecular Pharmacology. § German Institute of Human Nutrition. (1) Poli, G., Albano, E., Dianzani, M. U., Eds. Free Radicals: From Basic Science to Medicine; Birkha¨user: Basel, Boston, Berlin, 1993. (2) Scandalios, J. G., Ed. Oxidative Stress and the Molecular Biology of Antioxidant Defenses; Cold Spring Harbor Laboratory Press: Plainview, NY; 1997. (3) Ambrosio, G.; Tritto, I. Am. Heart J. 1999, 138 (2), 69-75. (4) Hogg, N. Semin. Reprod. Endocrinol. 1998, 16 (4), 241-248. (5) Scheller, W.; Jin, W.; Ehrentreich-Fo¨rster, E.; Ge, B.; Lisdat, F.; Bu ¨ ttemeyer, R.; Wollenberger, U.; Scheller, F. W. Electroanalysis 1999, 11 (10-11), 703706. (6) Blasig, I. E.; Shuter, S.; Garlick, P.; Slater, T. V. Free Radical Biol. Med. 1994, 16, 35-41. (7) Sies, H. Exp. Physiol. 1997, 82 (2), 291-295. (8) Stahl, W.; Sies, H. Diabetes 1997, 46 (2), 14-18. ‡

10.1021/ac000786j CCC: $20.00 Published on Web 01/30/2001

© 2001 American Chemical Society

Furthermore, indirect cascades triggered by oxidative stress are involved. For example, several iron-sulfur proteins that are targets of reactive species can be modified by these interactions and converted into nucleic acid-binding proteins that subsequently regulate the expression of proteins of the defense system.10 Cytosolic aconitase (EC 4.2.1.3.), which is a 4Fe-4S protein, is considered to be such a typical bioprobe.11-13 Three Fe of the cluster are bound to the protein chain, whereas the fourth Fe has only the inorganic S as a ligand and holds a free coordination site. This site is involved in the substrate’s binding to the active center and the catalytic conversion of cis-aconitate to aconitate. The enzymatic activity is lost under the influence of oxidative stress. Upon release of the iron-sulfur cluster, aconitase is converted into a nucleic acid-binding protein, the so-called “ironregulatory protein 1” (IRP1).14-18 As such, it regulates the expression of ferritin and the transferrin receptor by binding to a specific secondary RNA structure called “iron responsive element” (IRE). Multiple IREs are located in the 3′ nontranslated region of the transferrin receptor mRNA.19 By the binding of IRP1 to IREs, the expression of this transport protein is facilitated, due to mRNA stabilization. In contrast, the binding of IRP1 to the IRE of the 5′ nontranslated region of ferritin mRNA inhibits the translation of the iron-storage protein ferritin.20 The IRP1 concentration can be considered as an indicator of the level of oxidative stress in a biological system. Further examples of such sensory proteins are the iron-sulfur protein SoxR10 and the fumarate nitrate reduction protein (FNR) in Escherichia coli.21 (9) Brigelius-Flohe´, R. Free Radical Biol. Med. 1999, 27 (9-10), 951-965. (10) Hidalgo, E.; Ding, H.; Demple, B. TIBS 1997, 22, 207-210. (11) Flint, D. H.; Allen, R. M. Chem. Rev. 1996, 96, 2315-2334. (12) Cowan, J. A.; Lui, S. M. Adv. Inorg. Chem. 1998, 45, 313-350. (13) Grune, T.; Blasig, I. E.; Sitte, N.; Roloff, B.; Haseloff, R. G.; Davies, K. J. A. J. Biol. Chem. 1998, 273, 10857-10862. (14) Hausladen, A.; Fridovich, I. J. Biol. Chem. 1994, 269, 29405-29408. (15) Bouton, C.; Hirling, H.; Drapier, J.-C. J. Biol. Chem. 1997, 272 (32), 1996919975. (16) Castro, L.; Rodriguez, M.; Radi, R. J. Biol. Chem. 1994, 269 (47), 2940929415. (17) Mumby, S.; Koizumi, M.; Taniguchi, N.; Gutteridge, J. M. C. Biochim. Biophys. Acta 1998, 1380, 102-108. (18) Klausner, R. D.; Roault, T. A.; Harford, J. B. Cell 1993, 72, 19-28. (19) Casey, J. L.; Hentze, M. W.; Koeller, D. M.; Caughman, S. W.; Roault, T. A.; Klausner, R. D.; Harford, J. B. Science 1988, 240, 924-928. (20) Hentze, M. W.; Caughman, S. W.; Roault, T. A.; Barriocanal, J. G.; Dancis, A.; Harford, J. B.; Klausner, R. D. Science 1987, 238, 1570-1573. (21) Rouault, T. A.; Klausner, R. D. TIBS 1996, 21, 174-177.

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The analysis of the level of oxidative stress is of principal importance in understanding biological signal processes, as well as in the evaluation of antioxidative therapies which should intervene efficiently into a disturbed system where the balance between production and degradation of reactive species is disturbed. Sensorial approaches for the detection of individual radicals have been mainly focused on superoxide22-24 and nitric oxide25-27 and have been reviewed recently.28 An analysis based on a biological effect can serve as a valuable alternative to these specific elements, giving a summarized information on the state of a biological system. The technique of surface plasmon resonance (SPR) can be efficiently used for the direct analysis of binding events of biomolecules and has been applied for screening experiments, as well as for quantitative analysis with the determination of binding constants.29-31 In the field of nucleic acid-protein interaction, biomolecular recognition analysis has mainly been used for mechanistic investigations. This includes the identification of binding sites on proteins and on DNA/RNA,32-35 as well as the determination of kinetic constants for the binding process.36-41 Until now, there has been only limited work on the sensorial use of these interactions, in particular with additional fluorescence labeling of a DNA probe.42 Here we report on the use of a specific nucleic acid-protein binding process to evaluate the concentration of a marker protein of oxidative stress. The binding of IRP1 to the biological binding partner will be adopted with an IRE-modified gold chip, which will be read out by SPR. (22) Cooper, J. M.; Greenough, K. R.; McNeil, C. J. J. Electroanal. Chem. 1993, 347, 267-275. (23) Lvovich, V.; Scheeline, A. Anal. Chem. 1997, 69, 454-462. (24) Lisdat, F.; Ge, B.; Reszka, R.; Kozniewska, E. Fresenius’ J. Anal. Chem. 1999, 365, 494-498. (25) Malinski, T.; Taha, Z. Nature 1992, 358, 676-677. (26) Barker, S. L. R.; Clark, H. A.; Swallen, S. F.; Kopelman, R.; Tsang, A. W.; Swanson, J. A. Anal. Chem. 1999, 71, 1 (9), 1767-1772. (27) Privat, C.; Trevin, S.; Bedioui, F.; Devynck, J. J. Electroanal. Chem. 1997, 436, 261-265. (28) Lisdat, F.; Scheller, F. W. Anal. Lett. 2000, 33 (1) 1-16. (29) Karlsson, R.; Michaelsson, A.; Mattson, L. In Structure of Antigens; Van Regenmortal, M. H. V., Ed.; CRC Press: Boca Raton, FL, 1991; Vol. 1, pp 127-148. (30) Malmqvist, M. Curr. Opin. Immun. 1993, 5, 282-286. (31) Biacore Application, Note 305 1994, Kinetic Characterization of Thrombin Aptamer Interactions, Pharmacia Biosensors AB, Uppsala, Sweden. (32) Fisher, R. J.; Rein, A.; Fivash, M.; Urbaneja, M. A.; CasasFinet, J. R.; Medaglia, M. J. Virol. 1998, 72 (3), 1902-1909. (33) Van-Ryk, D. I.; Venkatesan, S. J. Biol. Chem. 1999, 274 (25), 17452-17463. (34) Dekker, J.; Kanellopoulos, P. N.; van Oosterhout, J. A.; Stier, G.; Tucker, P. A.; van der Vliet, P. C. J. Mol. Biol. 1998, 277 (4), 825-838. (35) Dutreix, M. J. Mol. Biol. 1997, 273 (1), 105-113. (36) Esser, D.; Rudolph, R.; Jaenicke, R.; Bohm, G. J. Mol. Biol. 1999, 291 (5), 1135-1146. (37) Cornille, F.; Emery, P.; Schuler, W.; Lenoir, C.; Mach, B.; Roques, B. P.; Reith, W. Nucleic Acids Res. 1998, 26 (9), 2143-2149. (38) Hartmann, R.; Norby, P. L.; Martensen, P. M.; Jorgensen, P.; James, M. C.; Jacobsen, C.; Moestrup, S. K.; Clemens, M. J.; Justesen, J. J. Biol. Chem. 1998, 273 (6), 3236-3246. (39) Galio, L.; Briquet, S.; Cot, S.; Buillet, J. G.; Vaquero, C. Anal. Biochem. 1997, 253 (1), 70-77. (40) West, M. L.; Ramsdale, T. E. Rev. Biomed. Pept. Protein Nucleic Acids 199697, 2 (3), 85-88. (41) Chinami, M.; Inoue, N.; Masunaga, K.; Fukuma, T.; Shingu, M.; Toyoda, T. J. Virol. Methods 1996, 59 (1-2), 173-176. (42) Potyrailo, R. A.; Conrad, R. C.; Ellington, A. D.; Hieftje, G. M. Anal. Chem. 1998, 70, 0 (16), 3419-3425.

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Figure 1. Schematic structure of the iron-responsive element (IRE). (In the mutant form of the IRE used within this study, the boxed nucleotides were omitted.)

EXPERIMENTAL SECTION Materials. IRP-1 (human, recombinant) was supplied by MBI Fermentas (Vilnius, Lithuania) and streptavidin, by Boehringer/ Mannheim (Germany). The 5′ biotinylated DNA oligomer (5′ TGCCCTCCCCACCCA 3′) and the oligonucleotides for IRE cloning (see below) were synthesized by Biotez (Berlin, Germany). Ethanolamine, and N-hydroxysuccinimide (NHS) were from Fluka (Neu-Ulm, Germany), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was from Sigma (Deisenhofen, Germany). Sensor chips CM5 (carboxymethyldextran-modified gold chips) were provided by BIACORE AB, (Uppsala, Sweden). Phosphate buffered saline (PBS) and Dulbecco’s modified Eagle’s medium (DMEM) were from Biochrom KG Seromed (Berlin, Germany). Digitonin and cis-aconitate were supplied by Fluka (Neu-Ulm, Germany) and sucrose, by ICN (Eschwege, Germany). Tris-(hydroxymethyl)-aminomethane (TRIS), piperazine-N,N′bis[2-ethanesulfonic acid] (PIPES), N-[2 hydroxyethyl]piperazineN′-[2 ethane sulfonic acid] buffer (HEPES), hydrochloric acid, sodium hydroxide, and acetic acid were of analytical grade. All solutions were prepared with 18 MΩ water (Millipore, Eschborn, Germany). IRE and Mutant Preparation. The sequence of the IRE corresponded to a consensus structure of ferritin and transferrin receptor mRNAs.18,43 (Figure 1). It was prolonged at the 3′ end by a linker sequence (15mer) for immobilization on the sensor chip. To construct the plasmid encoding IRE and linker, two oligonucleotides, 5′-A-ATTCCTTAAGCTTCAACAGTGCTTGAACTTAAG-TGGGTGGGGAGGGCAG-3′ and 5′-GATCCTGCCCTCCCCACCCACTTAAGTTCAAGCACTGTTGAAGCTTAAGG-3′ (IRE sequence, not bold; linker sequence, underlined) were synthesized (Biotez, Berlin Germany), annealed, cloned in pGEM-3Z vector (Promega, Mannheim, Germany), and cut with EcoRI and BamHI (New England Biolabs, Schwalbach, Germany). The mutant form of IRE lacking the four boxed nucleotides in the stem region (Figure 1) was constructed using a QuickChange mutagenesis kit (Stratagene, Heidelberg, Germany) using the following primers: 5′-GAATTCCTTAAGCTCAGTGCTTGAAGTT-3′ and 5′AACTTCAAGCACTGAGCTTAAGGAATTC-3′. The in vitro tran(43) Paraskeva, E.; Atzberger, A.; Hentze, M. W. Proc. Natl. Acad. Sci., U.S.A. 1998, 95, 951-956.

scription of RNA was accomplished using a T7 RiboMAX large scale RNA production system (Promega, Mannheim, Germany) with plasmids linearized with BamHI. Sensor Chip Modification. Prior to buffer preparation, the solutions were treated with 0.1% diethyl pyrocarbonate overnight and sterilized. Buffer 1 was a sodium acetate buffer of pH 5 (10 mM); buffer 2 was TRIS buffer, pH 7.5 (50 mM), with 100 mM NaCl and 1.5 mM MgCl2. The modification of the chip followed standard BIACORE procedures. A solution containing EDC (200 mM) and NHS (50 mM) was freshly prepared in water, and 50 µL of the solution was allowed to pass over the chip at a flow rate of 4 µL/min. Streptavidin was dissolved in buffer 1, desalted, and then diluted in the same buffer to a concentration of ∼400 µg/mL, and 100 µL passed over the preactivated chip at 4 µL/min. Ethanolamine (1 M, pH 8.5) was used to saturate all remaining activated groups (50 µL, 4 µL/min). Then the biotinylated DNA oligomer was immobilized (300 µg/mL buffer 2, 4 µL/min, 100 µL). For the binding of the linker-modified IRE loop and the mutant form, 150 µL of a 10 µg/mL solution in buffer 2 was pumped over the chip at a flow rate of 2 µL/min. IRP Measurements. A BIACORE 2000 instrument (BIACORE AB, Uppsala, Sweden) was used in order to control the immobilization steps and to characterize the binding of IRP1 to the IRE-modified surface. The binding buffer was buffer 2 with 10 mM cysteine. IRP1 concentrations were varied in the binding buffer between 2 and 200 nM. 20 µL of the protein solution was injected at a flow rate of 2 µL/min. Sodium carbonate (0.1 M) was used to regenerate the chip with a subsequent immobilization of linker-modified IRE. To vary the pH conditions for the binding process, PIPES buffer (50 mM) with 100 mM NaCl and 1.5 mM MgCl2 with 10 mM cysteine in the pH range 6-9 was used as the binding buffer. The influence of thiol concentration was investigated by the use of TRIS buffer (50 mM, pH 7.5, 100 mM NaCl, 1.5 mM MgCl2) with different glutathione concentrations (0-20 mM). Because glutathione was provided as free acid, the pH of the binding solutions was carefully controlled. For fitting of the binding kinetics, BIACORE evaluation software 3.0 was applied. The 1:1 Langmuir binding model was chosen. Association and dissociation phases of the sensorgrams were used for kon and koff determinations within the concentration range of IRP1 between 20 and 200 nM. For the association phase, the selected time window for curve-fitting started 40 s after sample injection and lasted 270 s. Dissociation was analyzed for a period of 250 s, starting 260 s after buffer flow. Integrated rate equations were used for the separate fitting of kon and koff; in addition, numerical integration was used for simultaneous global fitting of both kinetic constants for several sensorgrams,44 taking into account the drift of the baseline. Using the concept of limit coefficient requirement45 and experimental parameters, such as molecular weight of IRP1 (98 kDa), the amount of surface-bound IRP1 (in resonance units), and the kon value determined from the sensorgram, it was clarified that the binding observed was not mass-transport-limited. (44) Morton, T. A.; Myszka, D. G.; Chaiken, I. M. Anal. Biochem. 1995, 227, 176-185. (45) BIAtechnology Handbook; Pharmacia Biosensor AB: Uppsala, Sweden; June 1994.

Aconitase was reconstituted from IRP146 by incubating the protein (500 nM) with FeSO4 (125 µM), Na2S (125 µM), and dithiothreitol (10 mM) in argon-purged TRIS buffer (50 mM, pH 7.5) for 2 h. Aconitase activity was measured in TRIS buffer, pH 8 (+20 mM KCl), with a Beckman spectrophotometer DU 640. Substrate concentration (cis-aconitate) was 200 µM. The decrease of substrate concentration was followed at a wavelength of 240 nm. Cytosol samples were prepared from the macrophage cell line RAW 264.7 which were cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. For preparing cell extracts, the macrophage cells, which were growing on 75 cm2 cell culture flasks, were harvested, washed with phosphate-buffered saline, and resuspended in 100 mM HEPES buffer (pH 7.2, 0.25M sucrose). The cells were lysed using 0.01% digitonin. The resulting lysate was centrifuged at 15000g for 20 min to obtain mitochondria-free cytosol. This was portioned and used for the binding studies. RESULTS AND DISCUSSION Assay Principle and Sensor Chip Construction. The cellular level of the IRP1 can serve as an indicator of oxidative stress. Therefore, the sensorial detection of IRP1 by means of surface plasmon resonance is proposed to be a valuable indirect method for the analysis of reactive oxygen species. Because cytosolic aconitase does not bind to the specific ribonucleic acid loop, the detection of the binding affinity of the iron-sulfur-clusterfree protein IRP1 to this IRE can be exploited. The principle is schematically shown in Figure 2. The recognition element used here is an RNA loop representing a consensus IRE.18,43 The structure is given in Figure 1. For biosensor application, the sequence was prolonged at the 3′ end by a 15-mer oligonucleotide for immobilization on the sensor chip (for details, see Experimental Section). Figure 3 illustrates the modification steps of the chip for IRP1 detection. After EDC/NHS activation of the dextran surface (Figure 3A), streptavidin was covalently immobilized (Figure 3B). Rather high surface loadings were used (>10 000 RU) in order to provide high binding site density for the biotinylated DNA oligomer. After ethanolamine treatment, the biotinylated 15-mer was immobilized via affinity binding (Figure 3D). The IRE with the linker sequence was hybridized to the DNA oligomer (Figure 3E), which resulted in a further increase of the resonance signal (∼2000 RU). This sensor surface was used for IRP1 detection. Biomolecular Recognition Analysis of IRP1. When IRP1 passed over the sensor chip, a significant bulk index contribution was obvious. However, the association of IRP1 was clearly visible. This is shown in Figure 4 for different protein concentrations. After switching to pure buffer, the dissociation of IRP1 from the immobilized IRE could be followed. Both the kinetics of IRP1 association and the total amount of bound IRP1 showed a defined concentration dependence. A typical standard curve is shown in Figure 5. The detection was characterized from 2 to 200 nM IRP1. The lower detection limit was determined to be 20 nM IRP1. By considering the activity of cytosolic aconitase found in the cytosol of macrophages under normal physiological conditions, nanomolar (46) Phillips, J. D.; Guo, B.; Yu, Y.; Brown, F. M.; Leibold, E. A. Biochemistry 1996, 35 (49), 15704-15714.

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Figure 2. General scheme of the detection principle of IRP1 as a marker protein of oxidative stress. Cytosolic aconitase can be converted from a protein with catalytic activity into an iron-responsive-element binding protein (IRP1) when the Fe-S cluster is destroyed. An optical sensor surface with an immobilized IRE (mRNA loop) can detect IRP1 binding by means of the surface plasmon resonance technique. IRE immobilization is performed by hybridization of the linker sequence by which the IRE was prolonged to a complementary biotinylated DNA.

Figure 3. Typical sensorgram during sensor chip construction. A shows the activation of the CM5 chip with EDC/NHS. B illustrates the streptavidin immobilization. In C, the chip was saturated with ethanolamine. D represents the sensor output during DNA immobilization via streptavidin-biotin interaction. In period E, the linkermodified IRE was hybridized to the DNA. Between, buffer has passed the chip.

concentrations of the enzyme can be approximated. Thus, the response range of the sensor chip should be suited to the detection of IRP1, which is the conversion product of aconitase during oxidative stress action in the cell. kon and koff values were determined from the sensorgram by analyzing the association and the dissociation kinetics of the IRP1-IRE interaction using the model of a Langmuir isotherm and accounting for baseline drift. They were calculated to be 1.9 ( 0.6 × 104 lmol-1s-1 (kon) and 1.5 ( 0.4 × 10-3 s-1 (koff), respectively. Thus, for the apparant binding constant KD, 80 nM can be calculated.47 To show the specificity of the measurement, IRP1 was allowed to pass over a sensor chip which was prepared by the same procedure except for the IRE immobilization. No binding was observed in the sensorgram with this sensor surface. Because RNA is responsible for the protein binding activity, a mutant RNA chain with a different base sequence was immobilized on the 960 Analytical Chemistry, Vol. 73, No. 5, March 1, 2001

Figure 4. Sensorgrams of IRP1 binding to the sensor chip surface. (a, 200 nM; b, 160 nM; c, 120 nM; d, 100 nM; e, 60 nM; f, 20 nM; in binding buffer TRIS pH 7.5, 50 mM, 100 mM NaCl, 1.5 mM MgCl2, 10 mM cysteine). The inset shows the sensor response with the immobilized mutant form of IRE lacking four nucleotides (IRP1, 50 nM, binding buffer).

sensor chip instead of wild-type IRE (Figure 1). No specific loop can be formed by this sequence and, thus, only 10-15% of the sensor signal with the immobilized IRE was observed in the BIACORE experiment. This is illustrated by the inset in Figure 4. The IRE mutant is not only valuable for demonstrating the specificity of the system but also for building up a reference sensor surface that accounts for potential nonspecific interactions with components in more complex physiological media. (47) The value determined here by SPR binding studies for the surfaceimmobilized IRE is much higher than the values in the picomolar range, determined in solution [e.g., Brown, N. M.; Anderson, S. A.; Steffen, D. W.; Carpenter, T. B.; Kennedy, M. C.; Walden, W. E.; Eisenstein, R. S. Proc. Nat. Acad. Sci., U.S.A. 1998, 95, 15235-15240: KD ) 60 pM]. The behaviour is obviously not governed by rebinding effects, because they should increase the affinity. Mass transfer limitation could be excluded, too. The behavior has been observed for other surface-bound recognition elements and is attributed to the changed access characteristics of the immobilized binding partner [Persson, B.; Stenhag, K.; Nilsson, P.; Larsson, A.; Uhlen, M.; Nygren, P. A. Anal. Biochem. 1997, 246 (1), 33-44].

Figure 6. pH dependence of the sensor signal (PIPES buffer, 50 mM, 100 mM NaCl, 1.5 mM MgCl2, 50 nM IRP1). Figure 5. Typical standard curve of the concentration-dependent IRP1 binding to the sensor chip with immobilized IRE (amount of bound IRP1 was determined from the sensorgram 30 s after the end of the injection.)

The selectivity of analysis for the iron-sulfur-cluster-free protein could be shown by the use of aconitase. The enzyme can be reconstituted from IRP1 in the presence of iron and sulfide. The activity was tested spectrophotometrically by following the decrease in substrate concentration. A specific activity of 6.5 U/mg could be evaluated. In the BIACORE experiment, no binding was observed for a protein concentration of 50 nM. This confirms the validity of the system. According to recent structural investigations, the binding regions at the IRP1 for the IRE and for the Fe-S cluster are distinct but overlapping.48 This can explain the nonbinding activity of the Fe-S cluster containing protein. The measuring cycle for IRP1 analysis can be completed within 12-15 min. It was not found necessary to regenerate the sensor surface after every measurement, because equilibrium was not reached. Starting from the amount of bound IRE, more than 20 binding experiments with a concentration of 100 nM IRP1 could theoretically be performed until a 90% saturation would be reached, even if the dissociation of IRP1 from the sensor surface is not taken into account.49 Furthermore, a good reproducibility of the interaction analysis was observed in the experiments. For 5 repeated injections of IRP1 (50 nM), the standard deviation was (10%. This allows quality control of the sensor chip by intermittent injections of one standard IRP1 concentration. Long-term stability was investigated by repeated IRP1 injections within a period of 15 h. A decrease of only 20% in the amount of bound protein was found when the first three and the last three injections were compared within this time scale (total number of injections, 11). The chip can be successfully regenerated by treatment with sodium carbonate solution and subsequent IRE immobilization. Influence of Solution Composition on IRP1 Binding Affinity. Because the IRP1 is to be used as a marker protein, precise concentration analysis is important. In physiological samples, however, the complex matrix may influence the binding

activity. Varying thiol and protein concentrations seems to be particularly relevant. In addition, an optimal pH range of pH 7-9 for the RNA-protein interaction has been indicated.50 To clarify the pH range for optimal binding of IRP1 to the immobilized IRE, sensorgrams were recorded at different pH levels of the buffer solution. The result is shown in Figure 6. The drastically reduced binding at pH 6 and 9 reveals that acidification and also alkaline pH have to be avoided in IRP1 binding experiments. Physiological pH was found to be optimal for the IRP1 concentration analysis; however, this needs to be carefully controlled for the analysis of unknown samples. Binding of IRP1 to IREs requires a controlled loss of the FeS cluster, which can be initiated by oxidative processes. Overoxidation, however, results in reduced RNA binding, indicating that the redox state of essential protein thiols influences the binding affinity. This has also been shown for the redox-regulated transcription factors NFκB51 or AP-1.52 These factors bind only to their responsive elements when reduced by thiols in vitro and in vivo. Thus, the presence of low-molecular-weight thiols in solution as reducing agents can ensure efficient binding, because they guarantee that the redox state of the sulfur groups at the protein is fixed to the free sulfhydryl state in order to provide the right conformation of the IRP1 for the interaction with IREs.15 In physiological solutions however, the thiol content may vary in the lower millimolar range. Therefore, the influence of glutathione as the major free cellular thiol on the binding of IRP1 to the IRE was investigated. It was found that the presence of thiols is essential for the binding, because thiol-free buffer resulted in a very weak IRP1 association to the IRE-modified sensor chip (