A novel method for determination of peroxynitrite based on hemoglobin catalyzed reaction

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Analytica Chimica Acta 530 (2005) 317324A novel method for determination ofzedRu-X, Wuhaber 200er 2004AbstractA novel sp roposereaction wit fluorecoupled dim new mof the reacti endedwith a detec n grap5.60 107 terfereseen in biolo are s 2004 ElseKeywords: Peroxynitrite; Enzymatic method; Hemoglobin; l-Tyrosine; Kinetics1. IntroduReactivean importantosis and nperoxidatiochain destrexperimenttrite, the reoxide and strite is conries of diseareperfusion[5,6]. Evalperoxynitrilow concenproaches has fluorescfluorescein CorresponE-mail ad0003-2670/$doi:10.1016/jctionoxygen species (ROS) have been implicated ast causative factor in cell damage, including apop-ecrosis. Their proposed actions comprise lipidn, DNA damage, the mitochondrial respiratoryuction and protein modifications [13]. Recents have fastened on the importance of peroxyni-action product of the two reactive species nitricuperoxide [4]. Cells injury aroused by peroxyni-sidered to contribute to the pathogenesis of a se-ses, including inflammatory processes, ischemia-, septic shock and neurodegenerative processesuation of the potentially injuring mechanism ofte has proved difficult due to its short life span,tration in vivo [7,8]. In recent years, various ap-ave been tried to detect peroxynitrite [913], suchent probes method (usually use dihydrodichloro-(DCFH) and dihydrorhodamine-123 (DHR-123)ding author. Tel.: +86 27 62710603; fax: +86 27 87647617.dress: zhhliu.whu@163.com (Z.-H. Liu).as the probes), which is considered to be ideal and has beenwidely employed to monitor peroxynitrite in various biolog-ical systems [14,15]. Generally, the above methods are basedon the oxidation of reduced, non-fluorescent forms of fluo-rescent dyes such as fluorescein and rhodamine by perox-ynitrite to produce the parent dye molecule, resulting in adramatic increase in fluorescence response. However, thoughthese assays are commonly used, there are some controversyin the literatures concerning the mechanism of peroxynitrite-mediated oxidation of fluorescent indicators [1618], and thesynthesis of these probe molecules is rather difficult and in-convenient [7]. Additionally, the use of organic dyes is verylikely to result in environmental pollutants and usually shouldbe avoided.Since peroxynitrite always originates and functions invivo, it is significant to seek for some methods for the deter-mination of peroxynitrite that fit to living biological system.On basis of our experiences in researching ROS utilizing en-zymatic reactions [1922], we consider this kind of methodmight also be an interesting and qualified approach for per-oxynitrite that meets the above demand. In the present ten-tative work, a peroxidase catalyzed redox reaction between see front matter 2004 Elsevier B.V. All rights reserved..aca.2004.09.025on hemoglobin catalyJu Liang, Zhi-Hong Liu,Department of Chemistry, Wuhan UniversityReceived 25 May 2004; received in revised form 13 SeptemAvailable online 7 Decembectrofluorimetric method for the determination of peroxynitrite is ph hemoglobin as the catalyst and l-tyrosine as the substrate. A newmer of tyrosine, which, instead of nitryl-tyrosine, is likely to be aon is studied and the possible reaction mechanism is also recommtion limit of 5.00 108 mol L1 of peroxynitrite. A liner calibratioto 2.10 105 mol L1, with a correlation coefficient of 0.9983. Ingical samples, and also some anions structurally similar to ONOOvier B.V. All rights reserved.peroxynitrite basedreactioniu Cain 430072, China4; accepted 13 September 2004d. The method is based on a mimetic enzyme catalyzedscent substance is produced that might probably be thearking substance of ONOO injury in vivo. Kinetics. The proposed method is simple and highly sensitiveh is obtained over the peroxynitrite concentration rangences from some amino acids and metal ions normallytudied.318 J. Liang et al. / Analytica Chimica Acta 530 (2005) 317324peroxynitrite and another reducer is studied and applied indetermination. Hemoglobin (Hb) is used as the mimetic en-zyme for substituting horseradish peroxidase (HRP). Thereare two mavious workconditionsing albumesimilarity ois capablecatalytic oxand enzymour laboratmore weighbody it is morganism.TyrosineAs we allwith peroxas the detecwith spectrmarking suwork, withperoxynitrinitro-tyrosiperoxynitrianism of thThe othsince all porganism ais reasonabanother painjured byular structunew markithe work mon peroxyn2. Experim2.1. Reagel-TyrosChina), a sby dissolvter (contai(Lizhu Relution (1.00.3400 gtase (SOD0.10 mol Lpared; 0.20pH valuesthroughout(4 C).For fluorometric measurements, a Perkin-Elmer LS55spectrofluorometer equipped with 1 cm quartz cell is used.The excitation and emission wavelength slits are respectively15.0 aotomerimentzer (Aure, brches,For precisioPrepae staneportere, 25nalyticled wandure iscontaimovedmol L2 andd witthe coimatelite areminePrepabout 5epare2.00).paredixed aonor ientratiwithMethoa set o 108.50)L oder, anthe bueraturn is rec0.0 nmin reasons for doing so. Firstly, just like in our pre-, it is for the purpose of the mildness of reactionand the minimization of cost. Hb is the respir-n in blood cells of the amniotes, due to the highf its porphyrin (Fe) structure to that of HRP, Hbof being used as a peroxidase substitute in manyidation reactions. Its peroxidatic characteristicsatic kinetics have been studied systematically inory during last few years [2225]. Secondly andtily, since Hb is a kind of nature protein in humanuch likely to coexist with peroxynitrite in cell oris chosen as the reduced substrate in this work.have known, based on the nitration of tyrosineynitrite, 3-nitro-tyrosine has normally been actedting target for the determination of peroxynitriteophotometry [26,27]. It is also considered as thebstance of ONOO injury in vivo. Whereas in thisHb catalyzing, l-tyrosine is directly oxidized byte to produce a fluorescent product other than 3-ne. Thus a new fluorometric method is built up forte determination. The kinetics and possible mech-e enzymatic reaction is also discussed.er notable and important point of the work is thatartners of the reaction system are seen in livingnd such reaction is very likely to occur in vivo, itly presumed that the enzymatic reaction may bethway in which the biologic macromolecules areONOO and the fluorescent product, the molec-re of which is still in further research, may be ang substance of ONOO injury. And we believeight give a fillip to peroxidase-based researchesitrite in future.entalnts and apparatusine (Tianyuan Biotechnological Co., Wuhan,tock solution (1.00 103 mol L1) is prepareding 0.0090 g of l-tyrosine in 50.00 mL wa-ning 1.00 mL 0.10 mol L1 HCl); hemoglobinagent Co., Shanghai, China), a stock so-0 104 mol L1) is prepared by dissolvingHb in 50.00 mL water; Superoxide dismu-, 3000 U mL1) is purchased from Sigma; A1 dimethyl sulfoxide (DMSO) solution is pre-mol L1 TrisHCl buffer solutions with varyingare prepared. Distilled, de-ionized water is used. All the solutions are stored in a refrigeratorset attrophexpeanalyperatreseabath.3C p2.2.Thods rceduof adistiloxidemixttionis re0.30MnOmineandproxynitrdeter2.3.Ais prpH =is preare mNO dconcrately2.4.In(1.00(pH =20.00in orwithtemplutioof 32nd 5.0 nm. An UVIKON-941 (Kontron Inc.) spec-ter is used for absorbance measurements. Kinetics are performed on an SX18MV-R stopped-flowpplied Photophysics, UK). The experimental tem-oth for the quantitative determination and kineticis controlled with a Shimadzu TB-85 thermostatH measurement, a TOA Electronics Model PHS-n pH meter (Shanghai, China) is used.ration of peroxynitrite standard solutiondard peroxynitrite is prepared according to meth-d previously [28], to simply describe the pro-.00 mL solution containing 1.00 g sodium nitriteal reagent purity is added with 25.00 mL ofater containing 1.50 mL of 35% hydrogen per-0.40 mL of 96% sulphuric acid. The resultingpromptly poured into 50 mL of aqueous solu-ning 2.50 g sodium hydroxide. The excess H2O2by 0.40 g MnO2. The product is diluted by1 NaOH and filtrated to eliminate the excessplaced in a thermostat. The product is deter-h UVvis spectrophotometry (max = 302.0 nm)ncentration of peroxynitrite is found to be ap-y 4.007.00 104 mol L1. Aliquots of perox-monitored spectrophotometrically to accuratelythe concentration before each experiment.ration of NO donor standard solution0.00 mL of reduced GSH (2.00 102 mol L1)d in citric acid buffer (1.00 103 mol L1,About 50.00 mL of KNO2 (2.00 102 mol L1)in NaCl solution (9.0%). The above two solutionsnd the pH is adjusted to 2.00. A yellow solution ofs obtained and stored in a refrigerator (4 C). Theon of this NO donor solution is determined accu-UVvis spectrophotometry (max = 338.0 nm).ds for determination of peroxynitritef 10 mL-colorimetric tubes, 0.20 mL of l-tyrosine3 mol L1), 5.00 mL of TrisHCl buffer solution, a series of different amount of ONOO andf hemoglobin (1.00 104 mol L1) are addedd then the reaction solution is diluted to the markffer solution. The reaction solution is kept at roome for 10 min. The fluorescence intensity of the so-orded at 410.0 nm with the excitation wavelength.J. Liang et al. / Analytica Chimica Acta 530 (2005) 317324 3192.5. Detection of peroxynitrite in adriamycin-treatedyeast cellsWhen yare harvestsubsequent(pH = 8.5).same volumgroup withgroup are pcubated forcentrifugat(pH = 8.5),nized comp10 min at 1for analysisOH and Sare introduble interferperimentaland controladditions avarious con2.6. ProceStoppedquire kineamount ofstream cona 20L csion wavelis set to 1tation/emiswithin therate (repreof l-tyrosinFurther, anplotted so tare obtaine3. Results3.1. Spectrstructure oThe subthe maximu225.0 andONOO wdecreasednew fluoression waveletra of bothIt is seen t(a) (1) The excitation spectra of the product, (2) the emission spectraproduct; (b) (1) the excitation spectrum of l-tyrosine, (2) the emissionum of l-tyrosine, (1) the excitation spectrum of l-tyrosine after adding, (2) the emission spectrum of l-tyrosine after adding ONOO.he spectral signal of the product is fully free from in-ence from that of the substrate. The fluorescence of thect has two excitation peaks, which locates at 250.0 andnm, respectively. Consider that strong violet light maybly result in the decomposing of the compounds in theion system, the relatively long wavelength, 320.0 nm isn as the excitation wavelength in the following experi-s.those works concerning ONOO injury, it is well estab-d that the tyrosine residue is nitrified to generate 3-nitro-ine that has no intrinsic fluorescence but a maximum ab-nce around 428.0 nm [29,30], which is detected spec-otometrically. Whereas in our experiments, the prod-s a strong fluorescent substance with no absorbanced 428.0 nm.Thus it is safely excluded the productionnitro-tyrosine in this reaction. As has been proved in lit-res, peroxidase-catalyzed oxidation of substrates havingar molecular structure to that of tyrosine, i.e., p-cresol,east culture enters the stationary phase, cellsed by centrifugation for 3 min at 5000 rpm andly washed twice with 0.20 mol L1 TrisHClThe same amounts of cells are suspended in thee of buffer in closed screw-cap cuvettes. A takenadriamycin (100g mL1) added and a controlrepared synchronously. Both groups are then in-20 h with shaking at 28 C. Cells are harvested byion and washed twice with 0.20 mol L1 TrisHCltransferred to a glass homogenizer and homoge-letely on ice. The homogenate is centrifuged for2000 rpm under 4 C. The suspension is taken. DMSO (0.10 mol L1), a specific scavenger forOD (3 U mL1), a specific scavenger for O2,ced to the cell extraction to eliminate the possi-ence of OH and O2. Under the optimal ex-conditions, the samples including the taken groupgroup are detected fluorimetrically. For standardnd recovery experiments, yeast cells treated withcentration of adriamycin are taken as samples.dures for dynamic experiments-flow fluorescence analysis is performed to ac-tic parameters. One stream containing variousl-tyrosine, buffer and Hb is mixed with the othertaining ONOO and buffer in the flow cell withubage. The slit width of excitation and emis-ength are both set at 2.5 nm and the dead timems. The fluorescence at 320.0/410.0 nm (exci-sion) is recorded under a constant temperatureinitial 10 s after sampling, and the initial reactionsented as dFI/dt) under different concentrationse are calculated from such kinetic curves (FIt).initial rate versus tyrosine concentration curve ishat the MichaelisMenten constant Km and Vmaxd.and discussiona characteristics of the system and possiblef the productstrate, l-tyrosine has its inherent fluorescence withm excitation and emission wavelength located at305.0 nm, respectively. After being oxidized byith Hb catalyzing, the fluorescence of l-tyrosinegreatly or disappeared completely, meanwhile acent substance appeared with the maximum emis-ngth located at 410.0 nm. The fluorescence spec-the substrate and the product are shown in Fig. 1.he oxidization of the substrate is quite completeFig. 1.of thespectrONOOand tterferprodu320.0probareactchosementInlishetyrossorbatrophuct iarounof 3-eratusimil320 J. Liang et al. / Analytica Chimica Acta 530 (2005) 317324Fig. 2. Relatder different2.40 105 mpH 8.50, 22 would alwaearly reseaby H2O2 walyzing to pmation of tdonorsinhdard solutiperoxynitricates that thproduct.3.2. KinetiWith dycatalytic refluorescencconcentratitial reactioconcentratiis discovercentrationlevel, whicreactions; wreaction tufinally, whhigher, i.e.initial ratereaction hathat Hb, thsine, the ssponding tenzyme-batyrosine bytypical enzof MichaelisMenten constant Km and Vmax are acquired as2.27 mmol L1 and 3.34 FI s1, respectively.udies have shown that the oxidation of various substratesroxynt pathrates;e specrate oe preseine byfic scaeforem is fonot colarly, ad to thlso shD, pre fluortion obut noe resuerningand tylows.metheroxynte radn, tyrobin iuct is f.Optims is wionship between the reaction rate and reaction time un-concentrations of l-tyrosine. Concentration: ONOO,ol L1; Hb, 2.00 106 mol L1; TrisHCl buffer solution,C.ys yield polymers of the substrate [31,32]. And anrch reported that l-tyrosine was directly oxidizedith manganese-tetrasulfonatophthalocyanine cat-roduce coupled dimer [33]. In this work, the for-he dimer of tyrosine is further verified with NOibitors of dimer [34]. With introducing the stan-on of NO donor to the reaction mixture beforete, the fluorescence signal disappears, which indi-e NO donor restrains the formation of the dimericcs and possible mechanism of the reactionnamic experimental approach, the kinetics of thisaction is studied. By recording the FIt (relativeStby peferensubstactivsubstIn thtyrosspeciture bsystedoesSimiaddetem aof SOof thoxidaself,abovconctriteas folformof pebonaizatiomoglprodicals3.3.Ae intensity versus time) curves under differentons of tyrosine, the relationship between the ini-n rate (represented as dFI/dt) and the substrateon is thus obtained, which is shown in Fig. 2. Ited that the initial rate is proportional to the con-of tyrosine at comparatively lower concentrationh accords with the kinetic property of first-orderith the concentration of tyrosine increasing, therns to be the so-called mixed order reaction. Anden the concentration of tyrosine becomes even, up to 4.90 104 mol L1 in this system, thedoes not increase any longer, in other words, thes turned into a zero-order reaction due to the facte enzyme, has already been saturated by tyro-ubstrate. All the experimental results are corre-o the MichaelisMentens law, which describessed kinetic behaviors, indicating the oxidation ofperoxynitrite in the presence of Hb is a quiteymatic reaction. According to Fig. 2, the valuesof peroxynwhich is raterminationwith buffersolutions wNa2HPO4the highesttem is inveTrisHCl bseen the depH value uwithin the pwith pH 8.reported thsay, in a sliof peroxynindicatingto yield a hconsistentitrite (ONOO/ONOOH) can take place via dif-ways [7]: (i) peroxynitrite can directly oxidize the(ii) peroxynitrite may decompose into highly re-ies (OH, NO2), which subsequently oxidizes ther hydroxylates and nitrates aromatic compounds.nt work, the possible pathway of the oxidation ofperoxynitrite is studied. DMSO (0.10 mol L1), avenger for OH, is introduced to the reaction mix-the addition of peroxynitrite, and the signal of theund to be almost unchanged, indicating that OHntribute to the fluorescence increase of the system.specific scavenger for O2, SOD (3 U mL1), ise reaction system, and the fluorescence of the sys-ows no changes compared with that in the absenceoving that O2 does not mediate the generationescent product. The above results suggest that thef tyrosine must have raised from peroxynitrite it-t its decomposed reactive species. Combined thelts of kinetic studies with other reported opinionsthe mechanism of the reaction between peroxyni-rosine [3537], the reaction pathway is proposedFirstly, hemoglobin is oxidized by peroxynitrite tomoglobin, which will promote the isomerizationitrite. Subsequently, in the presence of the car-ical anion the product of peroxynitrite isomer-osyl radicals are generated. Meanwhile, methe-s quickly back-reduced to Hb. Finally, the dimericormed through the polymerization of tyrosyl rad-ization of determination conditionsell known, in an acid circumstance, the majorityitrite will present as its conjugate acid, ONOOH,ther unstable and easy to decompose. Thus the de-is performed under alkaline conditions controlledsolutions. Having studied several kinds of bufferith similar pH range, i.e., TrisHCl, NH3NH4Cl,KH2PO4 and so on. TrisHCl is preferred due tosensitivity. Then the pH dependence of the sys-stigated over the range from pH 7.20 to 9.10 usinguffer solution, which is shown in Fig. 3. It can betection signal (FI) of the system increases with thep to 8.40, and there exists a platform on the curveH range of 8.408.80. Hence the TrisHCl buffer50 is chosen in the subsequent experiments. It isat peroxynitrite has a pKa value of 6.80, that is toghtly alkaline environment (pH 8.50), about 85%itrite will present in the form of ONOO [27],that it is mainly ONOO who oxidized tyrosineighly fluorescent product in this system, which iswith the oxidizing nature of ONOO.J. Liang et al. / Analytica Chimica Acta 530 (2005) 317324 321Fig. 3. The influence of pH on the system. Concentration: l-tyrosine, 2.00 105 mol L1; ONOO, 1.00 105 mol L1; Hb,1.00 107 mol L1; TrisHCl buffer solution.As has been pointed out above, the Hb catalyzed reac-tion between l-tyrosine and ONOO is thoroughly differ-ent from that without Hb catalyzing. Like most works con-cerning enalyst is alwmance of tpriate amocent produrescence ining till 2.00concentraticence signmatic reacbecomes pFig. 4. The intration: l-tyroTrisHCl buffFig. 5. The iConcentrationTrisHCl buffsitivity asof hemoglotion.e cona veryn of sue incoconcee catald reacmptiotyrosiystemhe det 10llowie temzymatic reactions, the concentration of the cat-ays an important factor influencing the perfor-he method and is essential to decide its appro-unt. In our experiment, there is nearly no fluores-ct generated in the absence of Hb, and the fluo-tensity increases with Hb concentration increas- 107 mol L1, as is shown in Fig. 4. With Hbon higher than 2.00 107 mol L1 the fluores-al decrease instead, which is often seen in enzy-tions. Besides, the reproducibility of the signaloor with even higher Hb. Considering the sen-Thwaystratioof thhighto thsigneconsuof l-the sthat t2.00the foThfluence of the concentration of Hb on the system. Concen-sine, 2.00 105 mol L1; ONOO, 2.40 105 mol L1;er solution, pH 8.50.the characttal temperarate in the rFig. 6 as aature of thi3.4. AnalyThe caliabove menfrom 5.60coefficienttion limit,in which mand Sb this found todeviation (is 3.80% (nfluence of the concentration of l-tyrosine on the system.: ONOO, 2.40 105 mol L1; Hb, 1.00 107 mol L1;er solution, pH 8.50.well as the reproducibility, 2.00 107 mol L1bin is recommended in the following determina-centration of substrate in enzymatic reactions is al-important factor for the system. Too low concen-bstrate will unavoidably cause signal loss becausempleteness of reactions. Meanwhile, improperlyntration of substrate will be extremely detrimentalyst and also contribute to the blank. For the de-tion completed and the signal fully obtained, lessn of substrate is certainly better. The influencene concentration on the fluorescence intensity ofis also studied in this work. It is shown in Fig. 5ection signal (FI) reaches to the maximum at about5 mol L1 of l-tyrosine, which is then used duringng experiments.perature dependence of the reaction rate is one oferistics of enzymatic reactions and the experimen-ture is always precisely controlled. The reactionange of 450 C was obtained, which is shown intypical bell-mouthed curve. The optimal temper-s Hb catalyzed reaction is located at 22 C.tical performances of the methodbration curve for peroxynitrite obtained from thetioned determination procedure is linear ranging107 to 2.10 105 mol L1 with a correlation0.9983, which is shown in Fig. 7. The detec-calculated according to the three Sb/m criterion, is the slope of the range of the linearity usede standard deviation (n= 9) of the blank solution,be 5.00 108 mol L1. The relative standardR.S.D.) at a 5.60 106 mol L1 peroxynitriten= 7), and is 4.30% (n= 8) at 8 107 mol L1322 J. Liang et al. / Analytica Chimica Acta 530 (2005) 317324Fig. 6. The influence of temperature on the reaction rate. Concentra-tion: l-tyrosine, 5.00 104 mol L1; ONOO, 2.40 105 mol L1; Hb,1.00 107 mol L1; TrisHCl buffer solution, pH 8.50.peroxynitrimethod.In orderstandard cotometry, thbased on itsries of peroparallel detposed enzyfrom bothrelation (r=two meansis comparaFig. 7. The2.10 105 mmol L1; Hb,Table 1Comparison on determination results (mol L1) between this method andstandard spectrophotometrySample no. Standard method This method Relative error (%)1 1.57 105 1.59 105 1.32 1.25 105 1.19 105 5.03 9.19 106 9.28 106 0.94 5.36 106 5.30 106 1.15 6.35 107 6.63 107 4.0r: correlation coefficient between the two methods (0.998).3.5. Interference studiesInterferences from some amino acids, metal ions normallyseen in biological samples, as well as anions structurally sim-ilar to ONOO are considered. At the same time, since alittle amount of H2O2 is possibly remained in the preparedONOO solution [28], and it is also capable of oxidizing thesubstrate as a kind of ROS, it is obligatory to investigate thedisturbance of H2O2 on the system. With the ONOO con-centrationstances unoncennted ato a cthan 5tivityite higroxynto rearatherfromhe iroAnalyis welte, which shows a good reproducibility of thisto demonstrate the accuracy of the new method,ntrol experiments are carried out with spectropho-e standard quantitative method for peroxynitriteabsorbance at 302.0 nm, as the comparison. A se-xynitrite solution with varying concentrations areermined by both spectrophotometry and the pro-matic fluorometric method. The results obtainedmethods, as shown in Table 1, reveal a good cor-0.998) and no statistical difference between theusing a paired t-test. It is seen that the new methodtively precise.ent cpreseleadlessselecis quof pepriorlowscomeHb, t3.6.Itcalibration curve in the range of 5.60 107 tool L1 ONOO. Concentration: l-tyrosine, 2.00 1051.00 107 mol L1; TrisHCl buffer solution, pH 8.50.induced cecell sampleTable 2Tolerance of tForeign substNO2NO3H2O2GlucoseAlbuminGlycineGlutaminePhenylalanineTryptophanCu2+Mn2+Ca2+Na+K+Fe3+fixed at 2.40 106 mol L1, those foreign sub-der study are added into the system with differ-trations. Their influences on the determination ares tolerance, which means the added intruder hashange of the relative fluorescence intensity with%. Results in Table 2 show a fairly satisfactoryof the method. Especially, the tolerance of H2O2h, which may be explained in that the reactivityitrite is 2000 times greater than H2O2 [38], it isct with the reduced substrate. But the method al-low amount of Mn2+ and Fe3+, which might havethe disturbance of them on the catalytic center ofn contained heme.sis of biological samplesl known that ONOO is generated in adriamycin-ll injury. Results of fluorimetric detection of yeasts are shown in Fig. 8. It clearly exhibits the char-he method to some foreign substancesances Tolerance10001000100100040020010010050020086001000100010J. Liang et al. / Analytica Chimica Acta 530 (2005) 317324 323Table 3Standard additions and recovery data of yeast samplesaSample no. ONOO found Recovery (%)123a The final ectivelyFig. 8. The flspectra of the(2,2) the emiamycin treatmacteristic ei.e., the howhereas noever, becauquantitativeternal peroadriamycinTable 3, wminations.4. ConcluA newattention-gity and seleby peroxynis analyzedcells is detessence ofies, and Mquired. Posthe productto direh prodencers frommaticing sulikelynzymach injine resowledONOO in samples(106 mol L1)ONOO added(106 mol L1)2.47 0.652.342.01 0.652.041.40 0.651.17adriamycin concentration for samples 13 are 83, 50 and 17g mL1, respablewhicorescdiffeenzymarkmuchthe eof sutyrosAcknuorescence spectra of biological samples: (1,1) the excitationyeast homogenate with and without adriamycin treatment;ssion spectra of the yeast homogenate with and without adri-ent. Adriamycin: 100g mL1.xcitation and emission spectra in the taken group,mogenate of yeast cells treated with adriamycin,such signal is detected in the control group. How-se of the rather strong background in the samples,results are not obtained yet. With addition of ex-xynitrite standard solutions to cells treated with, the recovery data are obtained and shown inhich are average values of three repeated deter-sionmethod for determination of peroxynitrite, anetting ROS, is built up with satisfactory sensitiv-ctivity. It is based on the oxidation of l-tyrosineitrite in the presence of Hb. Biological sample, and peroxynitrite in adriamycin-treated yeastected with the proposed method. The enzymaticthe redox reaction is revealed by kinetic stud-ichaelisMenten constant Km and Vmax are ac-sible pathway of the reaction and the structure ofare recommended. Peroxynitrite is thought to beThe autfrom the N20275027)Reference[1] R.P. Pate(2000) 1[2] C. Laura[3] P. Sovit1507.[4] D.U. Vic[5] C.M. Bo[6] C. Szabo[7] X.F. Yan[8] H. Posse[9] J. Liang42.[10] D. Kang[11] R.A. Wh1902.[12] S.B. DigJ.S. Bec[13] T. Haya(1999) 3[14] N.W. Koical Biol[15] H. Posse416 (199[16] D. JourdWink, M[17] N.W. Ko(1997) 2(106 mol L1)2.92 93.64.68 97.32.34 88.03.92 96.81.79 87.32.29 89.1.ctly oxidize l-tyrosine under the catalysis of Hb,uces a dimeric substance with strong intrinsic flu-emitted at 410.0 nm. 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Vatassery, Biochem. Pharmacol. 52 (1996) 579.A novel method for determination of peroxynitrite based on hemoglobin catalyzed reactionIntroductionExperimentalReagents and apparatusPreparation of peroxynitrite standard solutionPreparation of NO donor standard solutionMethods for determination of peroxynitriteDetection of peroxynitrite in adriamycin-treated yeast cellsProcedures for dynamic experimentsResults and discussionSpectra characteristics of the system and possible structure of the productKinetics and possible mechanism of the reactionOptimization of determination conditionsAnalytical performances of the methodInterference studiesAnalysis of biological samplesConclusionAcknowledgementsReferences

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