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

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Analytica Chimica Acta 530 (2005) 317–324 A novel method for determination of zed Ru-X , Wuha ber 200 er 2004 Abstract A novel sp ropose reaction wit fluore coupled dim…


Analytica Chimica Acta 530 (2005) 317–324 A novel method for determination of zed Ru-X , Wuha ber 200 er 2004 Abstract A novel sp ropose reaction wit fluore coupled dim new m of the reacti ended with a detec n grap 5.60× 10−7 terfere seen in biolo − are s © 2004 Else Keywords: Peroxynitrite; Enzymatic method; Hemoglobin; l-Tyrosine; Kinetics 1. Introdu Reactive an importan tosis and n peroxidatio chain destr experiment trite, the re oxide and s trite is con ries of disea reperfusion [5,6]. Eval peroxynitri low concen proaches h as fluoresc fluorescein ∗ Correspon E-mail ad 0003-2670/$ doi:10.1016/j ction oxygen species (ROS) have been implicated as t causative factor in cell damage, including apop- ecrosis. Their proposed actions comprise lipid n, DNA damage, the mitochondrial respiratory uction and protein modifications [1–3]. Recent s have fastened on the importance of peroxyni- action product of the two reactive species nitric uperoxide [4]. Cells injury aroused by peroxyni- sidered to contribute to the pathogenesis of a se- ses, including inflammatory processes, ischemia- , septic shock and neurodegenerative processes uation of the potentially injuring mechanism of te 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 [9–13], such ent 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 been widely employed to monitor peroxynitrite in various biolog- ical systems [14,15]. Generally, the above methods are based on 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 a dramatic increase in fluorescence response. However, though these assays are commonly used, there are some controversy in the literatures concerning the mechanism of peroxynitrite- mediated oxidation of fluorescent indicators [16–18], and the synthesis of these probe molecules is rather difficult and in- convenient [7]. Additionally, the use of organic dyes is very likely to result in environmental pollutants and usually should be avoided. Since peroxynitrite always originates and functions in vivo, 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 [19–22], we consider this kind of method might 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.2004.09.025 on hemoglobin cataly Ju Liang, Zhi-Hong Liu∗, Department of Chemistry, Wuhan University Received 25 May 2004; received in revised form 13 Septem Available online 7 Decemb ectrofluorimetric method for the determination of peroxynitrite is p h hemoglobin as the catalyst and l-tyrosine as the substrate. A new mer of tyrosine, which, instead of nitryl-tyrosine, is likely to be a on is studied and the possible reaction mechanism is also recomm tion limit of 5.00× 10−8 mol L−1 of peroxynitrite. A liner calibratio to 2.10× 10−5 mol L−1, with a correlation coefficient of 0.9983. In gical samples, and also some anions structurally similar to ONOO vier B.V. All rights reserved. peroxynitrite based reaction iu Cai n 430072, China 4; accepted 13 September 2004 d. The method is based on a mimetic enzyme catalyzed scent substance is produced that might probably be the arking substance of ONOO− injury in vivo. Kinetics . The proposed method is simple and highly sensitive h is obtained over the peroxynitrite concentration range nces from some amino acids and metal ions normally tudied. 318 J. Liang et al. / Analytica Chimica Acta 530 (2005) 317–324 peroxynitrite and another reducer is studied and applied in determination. Hemoglobin (Hb) is used as the mimetic en- zyme for substituting horseradish peroxidase (HRP). There are two ma vious work conditions ing albume similarity o is capable catalytic ox and enzym our laborat more weigh body it is m organism. Tyrosine As we all with perox as the detec with spectr marking su work, with peroxynitri nitro-tyrosi peroxynitri anism of th The oth since all p organism a is reasonab another pa injured by ular structu new marki the work m on peroxyn 2. Experim 2.1. Reage l-Tyros China), a s by dissolv ter (contai (Lizhu Re lution (1.0 0.3400 g tase (SOD 0.10 mol L pared; 0.20 pH values throughout (4 ◦C). For fluorometric measurements, a Perkin-Elmer LS55 spectrofluorometer equipped with 1 cm quartz cell is used. The excitation and emission wavelength slits are respectively 15.0 a otome riment zer (A ure, b rches, For p recisio Prepa e stan eporte re, 25 nalytic led w and ure is contai moved mol L 2 and d wit the co imatel ite are mine Prepa bout 5 epare 2.00). pared ixed a onor i entrati with Metho a set o × 10− 8.50) �L o der, an the bu eratur n is rec 0.0 nm in reasons for doing so. Firstly, just like in our pre- , it is for the purpose of the mildness of reaction and the minimization of cost. Hb is the respir- n in blood cells of the amniotes, due to the high f its porphyrin (Fe) structure to that of HRP, Hb of being used as a peroxidase substitute in many idation reactions. Its peroxidatic characteristics atic kinetics have been studied systematically in ory during last few years [22–25]. Secondly and tily, since Hb is a kind of nature protein in human uch likely to coexist with peroxynitrite in cell or is chosen as the reduced substrate in this work. have known, based on the nitration of tyrosine ynitrite, 3-nitro-tyrosine has normally been acted ting target for the determination of peroxynitrite ophotometry [26,27]. It is also considered as the bstance of ONOO− injury in vivo. Whereas in this Hb catalyzing, l-tyrosine is directly oxidized by te to produce a fluorescent product other than 3- ne. Thus a new fluorometric method is built up for te determination. The kinetics and possible mech- e enzymatic reaction is also discussed. er notable and important point of the work is that artners of the reaction system are seen in living nd such reaction is very likely to occur in vivo, it ly presumed that the enzymatic reaction may be thway in which the biologic macromolecules are ONOO− and the fluorescent product, the molec- re of which is still in further research, may be a ng substance of ONOO− injury. And we believe ight give a fillip to peroxidase-based researches itrite in future. ental nts and apparatus ine (Tianyuan Biotechnological Co., Wuhan, tock solution (1.00× 10−3 mol L−1) is prepared ing 0.0090 g of l-tyrosine in 50.00 mL wa- ning 1.00 mL 0.10 mol L−1 HCl); hemoglobin agent Co., Shanghai, China), a stock so- 0× 10−4 mol L−1) is prepared by dissolving Hb in 50.00 mL water; Superoxide dismu- , 3000 U mL−1) is purchased from Sigma; A −1 dimethyl sulfoxide (DMSO) solution is pre- mol L−1 Tris–HCl buffer solutions with varying are prepared. Distilled, de-ionized water is used . All the solutions are stored in a refrigerator set at troph expe analy perat resea bath. 3C p 2.2. Th ods r cedu of a distil oxide mixt tion is re 0.30 MnO mine and prox ynitr deter 2.3. A is pr pH = is pre are m NO d conc rately 2.4. In (1.00 (pH = 20.00 in or with temp lutio of 32 nd 5.0 nm. An UVIKON-941 (Kontron Inc.) spec- ter is used for absorbance measurements. Kinetic s are performed on an SX18MV-R stopped-flow pplied Photophysics, UK). The experimental tem- oth for the quantitative determination and kinetic is controlled with a Shimadzu TB-85 thermostat H measurement, a TOA Electronics Model PHS- n pH meter (Shanghai, China) is used. ration of peroxynitrite standard solution dard peroxynitrite is prepared according to meth- d previously [28], to simply describe the pro- .00 mL solution containing 1.00 g sodium nitrite al reagent purity is added with 25.00 mL of ater containing 1.50 mL of 35% hydrogen per- 0.40 mL of 96% sulphuric acid. The resulting promptly poured into 50 mL of aqueous solu- ning 2.50 g sodium hydroxide. The excess H2O2 by 0.40 g MnO2. The product is diluted by −1 NaOH and filtrated to eliminate the excess placed in a thermostat. The product is deter- h UV–vis spectrophotometry (λmax = 302.0 nm) ncentration of peroxynitrite is found to be ap- y 4.00–7.00× 10−4 mol L−1. Aliquots of perox- monitored spectrophotometrically to accurately the concentration before each experiment. ration of NO donor standard solution 0.00 mL of reduced GSH (2.00× 10−2 mol L−1) d in citric acid buffer (1.00× 10−3 mol L−1, About 50.00 mL of KNO2 (2.00× 10−2 mol L−1) in NaCl solution (9.0%). The above two solutions nd the pH is adjusted to 2.00. A yellow solution of s obtained and stored in a refrigerator (4 ◦C). The on of this NO donor solution is determined accu- UV–vis spectrophotometry (λmax = 338.0 nm). ds for determination of peroxynitrite f 10 mL-colorimetric tubes, 0.20 mL of l-tyrosine 3 mol L−1), 5.00 mL of Tris–HCl buffer solution , a series of different amount of ONOO− and f hemoglobin (1.00× 10−4 mol L−1) are added d then the reaction solution is diluted to the mark ffer solution. The reaction solution is kept at room e 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) 317–324 319 2.5. Detection of peroxynitrite in adriamycin-treated yeast cells When y are harvest subsequent (pH = 8.5). same volum group with group are p cubated for centrifugat (pH = 8.5), nized comp 10 min at 1 for analysis •OH and S are introdu ble interfer perimental and control additions a various con 2.6. Proce Stopped quire kine amount of stream con a 20�L c sion wavel is set to 1 tation/emis within the rate (repre of l-tyrosin Further, an plotted so t are obtaine 3. Results 3.1. Spectr structure o The sub the maximu 225.0 and ONOO− w decreased new fluores sion wavele tra of both It is seen t (a) (1) The excitation spectra of the product, (2) the emission spectra product; (b) (1) the excitation spectrum of l-tyrosine, (2) the emission um 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 the ct has two excitation peaks, which locates at 250.0 and nm, respectively. Consider that strong violet light may bly result in the decomposing of the compounds in the ion system, the relatively long wavelength, 320.0 nm is n 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 absorbance d 428.0 nm.Thus it is safely excluded the production nitro-tyrosine in this reaction. As has been proved in lit- res, peroxidase-catalyzed oxidation of substrates having ar molecular structure to that of tyrosine, i.e., p-cresol, east culture enters the stationary phase, cells ed by centrifugation for 3 min at 5000 rpm and ly washed twice with 0.20 mol L−1 Tris–HCl The same amounts of cells are suspended in the e of buffer in closed screw-cap cuvettes. A taken adriamycin (100�g mL−1) added and a control repared synchronously. Both groups are then in- 20 h with shaking at 28 ◦C. Cells are harvested by ion and washed twice with 0.20 mol L−1 Tris–HCl transferred to a glass homogenizer and homoge- letely on ice. The homogenate is centrifuged for 2000 rpm under 4 ◦C. The suspension is taken . DMSO (0.10 mol L−1), a specific scavenger for OD (3 U mL−1), 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 group group are detected fluorimetrically. For standard nd recovery experiments, yeast cells treated with centration of adriamycin are taken as samples. dures for dynamic experiments -flow fluorescence analysis is performed to ac- tic parameters. One stream containing various l-tyrosine, buffer and Hb is mixed with the other taining ONOO− and buffer in the flow cell with ubage. The slit width of excitation and emis- ength are both set at 2.5 nm and the dead time ms. The fluorescence at 320.0/410.0 nm (exci- sion) is recorded under a constant temperature initial 10 s after sampling, and the initial reaction sented as dFI/dt) under different concentrations e are calculated from such kinetic curves (FI–t). initial rate versus tyrosine concentration curve is hat the Michaelis–Menten constant Km and Vmax d. and discussion a characteristics of the system and possible f the product strate, l-tyrosine has its inherent fluorescence with m excitation and emission wavelength located at 305.0 nm, respectively. After being oxidized by ith Hb catalyzing, the fluorescence of l-tyrosine greatly or disappeared completely, meanwhile a cent 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 complete Fig. 1. of the spectr ONOO and t terfer produ 320.0 proba react chose ment In lishe tyros sorba troph uct i aroun of 3- eratu simil 320 J. Liang et al. / Analytica Chimica Acta 530 (2005) 317–324 Fig. 2. Relat der different 2.40× 10−5 m pH 8.50, 22 ◦ would alwa early resea by H2O2 w alyzing to p mation of t donors–inh dard soluti peroxynitri cates that th product. 3.2. Kineti With dy catalytic re fluorescenc concentrati tial reactio concentrati is discover centration level, whic reactions; w reaction tu finally, wh higher, i.e. initial rate reaction ha that Hb, th sine, the s sponding t enzyme-ba tyrosine by typical enz of Michaelis–Menten constant Km and Vmax are acquired as 2.27 mmol L−1 and 3.34 �FI s−1, respectively. udies have shown that the oxidation of various substrates roxyn t path rates; e spec rate o e prese ine by fic sca efore m is fo not co larly, a d to th lso sh D, pr e fluor tion o but no e resu erning and ty lows. methe roxyn te rad n, tyr obin i uct is f . Optim s is w ionship between the reaction rate and reaction time un- concentrations of l-tyrosine. Concentration: ONOO−, ol L−1; Hb, 2.00× 10−6 mol L−1; Tris–HCl buffer solution, C. ys yield polymers of the substrate [31,32]. And an rch reported that l-tyrosine was directly oxidized ith manganese-tetrasulfonatophthalocyanine cat- roduce coupled dimer [33]. In this work, the for- he dimer of tyrosine is further verified with NO ibitors of dimer [34]. With introducing the stan- on of NO donor to the reaction mixture before te, the fluorescence signal disappears, which indi- e NO donor restrains the formation of the dimeric cs and possible mechanism of the reaction namic experimental approach, the kinetics of this action is studied. By recording the FI–t (relative St by pe feren subst activ subst In th tyros speci ture b syste does Simi adde tem a of SO of th oxida self, abov conc trite as fol form of pe bona izatio mogl prod icals 3.3. A e intensity versus time) curves under different ons of tyrosine, the relationship between the ini- n rate (represented as dFI/dt) and the substrate on is thus obtained, which is shown in Fig. 2. It ed that the initial rate is proportional to the con- of tyrosine at comparatively lower concentration h accords with the kinetic property of first-order ith the concentration of tyrosine increasing, the rns to be the so-called mixed order reaction. And en the concentration of tyrosine becomes even , up to 4.90× 10−4 mol L−1 in this system, the does not increase any longer, in other words, the s turned into a zero-order reaction due to the fact e enzyme, has already been saturated by tyro- ubstrate. All the experimental results are corre- o the Michaelis–Menten’s law, which describes sed kinetic behaviors, indicating the oxidation of peroxynitrite in the presence of Hb is a quite ymatic reaction. According to Fig. 2, the values of peroxyn which is ra termination with buffer solutions w Na2HPO4– the highest tem is inve Tris–HCl b seen the de pH value u within the p with pH 8. reported th say, in a sli of peroxyn indicating to yield a h consistent itrite (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 the r hydroxylates and nitrates aromatic compounds. nt work, the possible pathway of the oxidation of peroxynitrite is studied. DMSO (0.10 mol L−1), a venger for •OH, is introduced to the reaction mix- the addition of peroxynitrite, and the signal of the und to be almost unchanged, indicating that •OH ntribute to the fluorescence increase of the system. specific scavenger for O2•−, SOD (3 U mL−1), is e reaction system, and the fluorescence of the sys- ows no changes compared with that in the absence oving that O2•− does not mediate the generation escent product. The above results suggest that the f tyrosine must have raised from peroxynitrite it- t its decomposed reactive species. Combined the lts of kinetic studies with other reported opinions the mechanism of the reaction between peroxyni- rosine [35–37], the reaction pathway is proposed Firstly, hemoglobin is oxidized by peroxynitrite to moglobin, which will promote the isomerization itrite. 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 dimeric ormed through the polymerization of tyrosyl rad- ization of determination conditions ell known, in an acid circumstance, the majority itrite will present as its conjugate acid, ONOOH, ther unstable and easy to decompose. Thus the de- is performed under alkaline conditions controlled solutions. Having studied several kinds of buffer ith similar pH range, i.e., Tris–HCl, NH3–NH4Cl, KH2PO4 and so on. Tris–HCl is preferred due to sensitivity. Then the pH dependence of the sys- stigated over the range from pH 7.20 to 9.10 using uffer solution, which is shown in Fig. 3. It can be tection signal (FI) of the system increases with the p to 8.40, and there exists a platform on the curve H range of 8.40–8.80. Hence the Tris–HCl buffer 50 is chosen in the subsequent experiments. It is at peroxynitrite has a pKa value of 6.80, that is to ghtly alkaline environment (pH 8.50), about 85% itrite will present in the form of ONOO− [27], that it is mainly ONOO− who oxidized tyrosine ighly fluorescent product in this system, which is with the oxidizing nature of ONOO−. J. Liang et al. / Analytica Chimica Acta 530 (2005) 317–324 321 Fig. 3. The influence of pH on the system. Concentration: l- tyrosine, 2.00× 10−5 mol L−1; ONOO−, 1.00× 10−5 mol L−1; Hb, 1.00× 10−7 mol L−1; Tris–HCl 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 en alyst is alw mance of t priate amo cent produ rescence in ing till 2.00 concentrati cence sign matic reac becomes p Fig. 4. The in tration: l-tyro Tris–HCl buff Fig. 5. The i Concentration Tris–HCl buff sitivity as of hemoglo tion. e con a very n of su e inco conce e catal d reac mptio tyrosi ystem he det × 10− llowi e tem zymatic 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- × 10−7 mol L−1, as is shown in Fig. 4. With Hb on higher than 2.00× 10−7 mol L−1 the fluores- al decrease instead, which is often seen in enzy- tions. Besides, the reproducibility of the signal oor with even higher Hb. Considering the sen- Th ways tratio of th high to th signe consu of l- the s that t 2.00 the fo Th fluence of the concentration of Hb on the system. Concen- sine, 2.00× 10−5 mol L−1; ONOO−, 2.40× 10−5 mol L−1; er solution, pH 8.50. the charact tal tempera rate in the r Fig. 6 as a ature of thi 3.4. Analy The cali above men from 5.60× coefficient tion limit, in which ‘m and ‘Sb’ th is found to deviation ( is 3.80% ( nfluence of the concentration of l-tyrosine on the system. : ONOO−, 2.40× 10−5 mol L−1; Hb, 1.00× 10−7 mol L−1; er solution, pH 8.50. well as the reproducibility, 2.00× 10−7 mol L−1 bin 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 because mpleteness of reactions. Meanwhile, improperly ntration of substrate will be extremely detrimental yst and also contribute to the blank. For the de- tion completed and the signal fully obtained, less n of substrate is certainly better. The influence ne concentration on the fluorescence intensity of is also studied in this work. It is shown in Fig. 5 ection signal (FI) reaches to the maximum at about 5 mol L−1 of l-tyrosine, which is then used during ng experiments. perature dependence of the reaction rate is one of eristics of enzymatic reactions and the experimen- ture is always precisely controlled. The reaction ange of 4–50 ◦C was obtained, which is shown in typical bell-mouthed curve. The optimal temper- s Hb catalyzed reaction is located at 22 ◦C. tical performances of the method bration curve for peroxynitrite obtained from the tioned determination procedure is linear ranging 10−7 to 2.10× 10−5 mol L−1 with a correlation 0.9983, 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 used e standard deviation (n= 9) of the blank solution, be 5.00× 10−8 mol L−1. The relative standard R.S.D.) at a 5.60× 10−6 mol L−1 peroxynitrite n= 7), and is 4.30% (n= 8) at 8× 10−7 mol L−1 322 J. Liang et al. / Analytica Chimica Acta 530 (2005) 317–324 Fig. 6. The influence of temperature on the reaction rate. Concentra- tion: l-tyrosine, 5.00× 10−4 mol L−1; ONOO−, 2.40× 10−5 mol L−1; Hb, 1.00× 10−7 mol L−1; Tris–HCl buffer solution, pH 8.50. peroxynitri method. In order standard co tometry, th based on its ries of pero parallel det posed enzy from both relation (r= two means is compara Fig. 7. The 2.10× 10−5 m mol L−1; Hb, Table 1 Comparison on determination results (mol L−1) between this method and standard spectrophotometry Sample no. Standard method This method Relative error (%) 1 1.57× 10−5 1.59× 10−5 −1.3 2 1.25× 10−5 1.19× 10−5 5.0 3 9.19× 10−6 9.28× 10−6 −0.9 4 5.36× 10−6 5.30× 10−6 1.1 5 6.35× 10−7 6.63× 10−7 −4.0 r: correlation coefficient between the two methods (0.998). 3.5. Interference studies Interferences from some amino acids, metal ions normally seen in biological samples, as well as anions structurally sim- ilar to ONOO− are considered. At the same time, since a little amount of H2O2 is possibly remained in the prepared ONOO− solution [28], and it is also capable of oxidizing the substrate as a kind of ROS, it is obligatory to investigate the disturbance of H2O2 on the system. With the ONOO− con- centration stances un oncen nted a to a c than 5 tivity ite hig roxyn to rea rather from he iro Analy is wel te, which shows a good reproducibility of this to demonstrate the accuracy of the new method, ntrol experiments are carried out with spectropho- e standard quantitative method for peroxynitrite absorbance at 302.0 nm, as the comparison. A se- xynitrite solution with varying concentrations are ermined by both spectrophotometry and the pro- matic fluorometric method. The results obtained methods, as shown in Table 1, reveal a good cor- 0.998) and no statistical difference between the using a paired t-test. It is seen that the new method tively precise. ent c prese lead less selec is qu of pe prior lows come Hb, t 3.6. It calibration curve in the range of 5.60× 10−7 to ol L−1 ONOO−. Concentration: l-tyrosine, 2.00× 10−5 1.00× 10−7 mol L−1; Tris–HCl buffer solution, pH 8.50. induced ce cell sample Table 2 Tolerance of t Foreign subst NO2− NO3− H2O2 Glucose Albumin Glycine Glutamine Phenylalanine Tryptophan Cu2+ Mn2+ Ca2+ Na+ K+ Fe3+ fixed at 2.40× 10−6 mol L−1, those foreign sub- der study are added into the system with differ- trations. Their influences on the determination are s tolerance, which means the added intruder has hange of the relative fluorescence intensity with %. Results in Table 2 show a fairly satisfactory of the method. Especially, the tolerance of H2O2 h, which may be explained in that the reactivity itrite is 2000 times greater than H2O2 [38], it is ct with the reduced substrate. But the method al- low amount of Mn2+ and Fe3+, which might have the disturbance of them on the catalytic center of n contained heme. sis of biological samples l known that ONOO− is generated in adriamycin- ll injury. Results of fluorimetric detection of yeast s are shown in Fig. 8. It clearly exhibits the char- he method to some foreign substances ances Tolerance 1000 1000 100 1000 400 200 100 100 500 200 8 600 1000 1000 10 J. Liang et al. / Analytica Chimica Acta 530 (2005) 317–324 323 Table 3 Standard additions and recovery data of yeast samplesa Sample no. − − ONOO− found Recovery (%) 1 2 3 a The final ectively Fig. 8. The fl spectra of the (2,2′) the emi amycin treatm acteristic e i.e., the ho whereas no ever, becau quantitative ternal pero adriamycin Table 3, w minations. 4. Conclu A new attention-g ity and sele by peroxyn is analyzed cells is det essence of ies, and M quired. Pos the product to dire h prod ence rs from matic ing su likely nzyma ch inj ine res owled ONOO in samples (×10−6 mol L−1) ONOO added (×10−6 mol L−1) 2.47 0.65 2.34 2.01 0.65 2.04 1.40 0.65 1.17 adriamycin concentration for samples 1–3 are 83, 50 and 17�g mL−1, resp able whic oresc diffe enzy mark much the e of su tyros Ackn uorescence spectra of biological samples: (1,1′) the excitation yeast homogenate with and without adriamycin treatment; ssion spectra of the yeast homogenate with and without adri- ent. Adriamycin: 100�g mL−1. 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 in hich are average values of three repeated deter- sion method for determination of peroxynitrite, an etting ROS, is built up with satisfactory sensitiv- ctivity. It is based on the oxidation of l-tyrosine itrite in the presence of Hb. Biological sample , and peroxynitrite in adriamycin-treated yeast ected with the proposed method. The enzymatic the redox reaction is revealed by kinetic stud- ichaelis–Menten constant Km and Vmax are ac- sible pathway of the reaction and the structure of are recommended. Peroxynitrite is thought to be The aut from the N 20275027) Reference [1] R.P. Pate (2000) 1 [2] C. Laura [3] P. Sovit 1507. [4] D.U. Vic [5] C.M. Bo [6] C. Szabo [7] X.F. Yan [8] H. Posse [9] J. Liang 42. [10] D. Kang [11] R.A. Wh 1902. [12] S.B. Dig J.S. Bec [13] T. Haya (1999) 3 [14] N.W. Ko ical Biol [15] H. Posse 416 (199 [16] D. Jourd Wink, M [17] N.W. Ko (1997) 2 (×10−6 mol L−1) 2.92 93.6 4.68 97.3 2.34 88.0 3.92 96.8 1.79 87.3 2.29 89.1 . ctly oxidize l-tyrosine under the catalysis of Hb, uces a dimeric substance with strong intrinsic flu- emitted at 410.0 nm. The new product obviously 3-nitro-tyrosine, the recognized product in non- reactions, which is traditionally thought of as the bstance of peroxynitrite injury of cells. Since Hb is to coexist with peroxynitrite within living body, tic reaction might probably be another pathway ury to cells or organism, especially proteins with idual. gements hors gratefully acknowledge financial support ational Natural Science Foundation of China (No. . s l, D. Moellering, M.U. Joanne, Free Radical Biol. Med. 28 780. , B.A. Freeman, Nutrition 17 (2001) 161. j, M.R. Gerald, J. Chem. Soc., Perkin Trans. 998 (1998) tor, H. Wiseman, B. Halliwell, FEBS Lett. 369 (1995) 131. ulanger, J. Mol. Cell. Cardiol. 31 (1999) 39. , Brain Res. Bull. 41 (1996) 133. g, X.Q. Guo, Y.B. 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A novel method for determination of peroxynitrite based on hemoglobin catalyzed reaction Introduction Experimental Reagents and apparatus Preparation of peroxynitrite standard solution Preparation of NO donor standard solution Methods for determination of peroxynitrite Detection of peroxynitrite in adriamycin-treated yeast cells Procedures for dynamic experiments Results and discussion Spectra characteristics of the system and possible structure of the product Kinetics and possible mechanism of the reaction Optimization of determination conditions Analytical performances of the method Interference studies Analysis of biological samples Conclusion Acknowledgements References


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