nitrosylation of manganese(ii) tetrakis(n-ethylpyridinium-2-yl)porphyrin: a simple and sensitive...

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NITRIC OXIDE: Biology and ChemistryVol. 4, No. 5, pp. 526–533 (2000)doi:10.1006/niox.2000.0303, available online at http://www.idealibrary.com on

Nitrosylation of Manganese(II) Tetrakis(N-ethylpyridinium-2-yl)porphyrin: A Simple and Sensitive SpectrophotometricAssay for Nitric Oxide1

Ivan Spasojevic, Ines Batinic-Haberle, and Irwin Fridovich2

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

P

Received February 28, 2000, and in revised form May 23, 2000

Reaction between NO• and manganese tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnIIITE-2-PyP51)was investigated at 25°C. At high excess of NO• (1.5

M) the reaction with the oxidized, air-stable formnIIITE-2-PyP51 (5 mM), proceeds very slowly (t1/2 >

0 min). The presence of excess ascorbate (1 mM)roduces the reduced form, MnIITE-2-PyP41, whicheacts with NO• stoichiometrically and in the timef mixing (k > 1 3 106 M21 s21). The high rate oformation and the stability of the product, MnIITE-

2-PyP(NO)41 ({Mn(NO)}6), make the reaction out-compete the reaction of NO• with O2. Our in vitromeasurements show a linear absorbance responseupon addition of NO to a PBS, pH 7.4, solution con-taining an excess of ascorbate over MnIIITE-2-PyP51.Thus, the observed interactions can be the basis of aconvenient and sensitive spectrophotometric assayfor NO•. Also, it may have important implicationsor the in vivo behavior of MnIIITE-2-PyP51 which is

currently exploited as a possible therapeutic agentfor various oxygen-radical related disorders. © 2000

Academic Press

1 This work was supported by grants from the National Insti-tutes of Health, the Council for Tobacco Research, U.S.A., theNorth Carolina Biotech Collaborative Funding Assistance Pro-gram, Duke Comprehensive Cancer Center (Grant P30 CA14236), and Aeolus/Incara.

2 To whom correspondence should be addressed at Department
of Biochemistry, Box 3711, Duke University Medical Center,Durham, NC 27710. Fax: (919) 684-8885.
526

Key Words: nitric oxide; nitrosylation; manganeseporphyrin; MnTE-2-PyP; ascorbate.

Nitric oxide (NO)3 is produced in biological sys-tems by synthases that oxidize L-arginine toL-citrulline. NO• serves numerous purposes includ-ing relaxation of smooth muscle (1), neurotransmis-sion (2), and host defense (3). Assays for NO• arethus important and several have been devised. Oneof these depends upon measurement of the chemilu-minescence that accompanies the oxidation of NO•

by ozone (4) and another upon the conversion ofoxyhemoglobin to methemoglobin by NO• (5). In ad-dition there is a fluorescence assay based upon theNO•-dependent conversion of diaminonaphthaleneto 2,3-naphthotriazole (6) and an electrochemicalassay (7, 8). We now describe a rapid reaction of NO•

with a Mn(II) cationic porphyrin (see structure inScheme I) that yields a semistable product andwhich is accompanied by a large change in visibleabsorbance. This reaction can be the basis of a con-

3 Abbreviations used: NO, nitric oxide; MnIII/IITE(M)-2(4)-yP51/41, manganese (III/II) 5,10,15,20-tetrakis(N-ethyl(methyl)-

pyridynium-2(4)-yl) porphyrin (2-ortho isomer and 4-para isomer,E and M denote N-ethylated and N-methylated analogues);{Mn(NO)}6, Enemark-Feltham formalism for describingmetal-NO link (15); A 22, ascorbate dianion; A•2, ascorbate radi-cal; NOC-9, H C-N[N(O)NO]-(CH ) -NH1-CH , zwitterionic
3 2 6 2 3
NO•-releasing compound, t 1/ 2 ; 3 min at 25°C; PBS, phosphate-buffered saline.

1089-8603/00 $35.00Copyright © 2000 by Academic Press

All rights of reproduction in any form reserved.

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venient and sensitive spectrophotometric assay forNO•.

EXPERIMENTAL

The H2T-2-PyP and H2T-4-PyP were purchasedfrom Mid-Century Chemicals (Chicago, IL). Mn(III)5,10,15,20-tetrakis (N-ethylpyridynium-2-yl) por-

hyrin (MnIIITE-2-PyP51) and Mn(III) 5,10,15,20-tetrakis (N-ethylpyridynium-4-yl) porphyrin(MnIIITE-4-PyP51) (available from Mid-CenturyChemicals) were prepared by the N-ethylation ofH2T-2-PyP and H2T-4-PyP and subsequent metalla-tion as described previously (9). The following areanalytical data for MnIIITE-4-PyP51. The uv/visdata: l, nm (log e) 5 767 (3.33), 560 (4.15), 462.55.18), 399 (4.70), 377.5 (4.69). Anal. Calcd for the

nIIITE-4-PyPCl5 3 11.5 H2O (C48H67N8O11.5Cl5Mn):C, 49.18; H, 5.76; N, 9.56; Cl, 15.12. Found: C, 49.23,H, 5.58; N, 9.60; Cl, 15.05. The metal-centered re-duction potential determined as described for its

SCHEME I. Structures of MnIII/IITE-2-PyP51/41 (A) and MnIITE--PyP(NO)41 (B).

NITROSYLATI

N-methyl analogue (9) is E 1/ 2 5 175 mV vs NHE.All measurements were done at room temperature

Copyright © 2000 by Academic Press. All right

(25 6 0.1°C) in phosphate-buffered saline (PBS), pH7.4, obtained from Gibco-BRL to which 0.1 mMEDTA was added. NO• gas was passed seriallythrough 6 M NaOH, H2O, and then a column ofNaOH pellets and was finally collected into argon-purged PBS. The NO•-saturated PBS solution at25°C was determined to be 1.5 mM in NO• (10). TheNONOate NOC-9, from Calbiochem, was dissolvedin argon-purged 10 mM NaOH to 10 mg/ml (;100

M). This stock solution and dilutions thereof weretored at 220°C. Sodium L-ascorbate (991%) was

from Aldrich Chemical Company and 0.1 M stocksolutions of it were made in argon-purged PBS andwere stored at 220°C. Argon (ultrapure) and oxygen

ere from National Welders Supply Company. Ex-eriments were performed in a 2-mL volume, 1-cmath-length quartz cuvette cell, equipped with a se-um stopper through which additions could be madey injection from Hamilton gas-tight syringes. PBSolutions were purged with argon for 30 min prior tose. Spectra were recorded with a Shimadzu UV-501-PC spectrophotometer.Oxygen consumption was measured in a water-

acketed cell equipped with Clark electrode (Pt/.01-mm PTFE membrane).

RESULTS

Reaction of NO• with MnIII/IITE-2-PyP51/41

When Mn(III) porphyrin, MnIIITE-2-PyP51 (5 mM),as added to NO•-saturated (;1.5 mM) PBS there

was a gradual change in spectrum. The slowness ofthis process (t 1/ 2 > 60 min), as well as the literaturedata (11) which indicated that NO• binds morestrongly to divalent metalloporphyrins, suggestedthat NO• was first acting as a reductant and thatanother molecule of NO• was then binding axially tothe reduced metal center. This led us to the priorreduction of MnIIITE-2-PyP51 with ascorbate (Eq.[1]) followed by the addition of NO• (Eq. [2]). Figure1 shows that the spectrum of 5 mM MnIIITE-2-PyP51

(spectrum 1) was changed to that of MnIITE-2-PyP41

(spectrum 2) upon addition of 0.5 mM ascorbate andthat subsequent addition of 15 mM NO• causes afurther hypsochromic shift in the Soret band (spec-

527MnTE-2-PyP

trum 3). These spectral changes were complete inthe time of mixing of the reactants. The numerical

s of reproduction in any form reserved.

2se

i

fs givenale.

values of the millimolar absorptivities derived fromFig. 1 are given in Table I.

The nitrosylation reaction was performed using aNO• donor (NONOate NOC-9) as well. When 3.13mM MnIIITE-2-PyP51 plus 1.0 mM ascorbate wastreated with small aliquots of the NOC-9, with a10-min incubation following each addition to allowfor complete release of NO• by decomposition of theNONOate, there was a linear decrease in absor-bance at 438 nm obeying the simple relation DA 438 5

De 438 3 [NO•]. This is shown in Fig. 2 which docu-ments linearity in the range 0.16–1.2 mM NO•. Allspectra taken 10 min after each addition of NONO-ate pass through set of isosbestic points (inset in Fig.) which tells that each spectrum is a compositepectrum of only two absorbing species that are in

FIG. 1. Absorption spectra. Spectrum 1 is that of 5 mM MnIIITEorm, MnIITE-2-PyP41, was recorded after addition of excess asco

after subsequent addition of excess NO• (15 mM). The ordinate ispectra in the region 500–620 nm with an expanded ordinate sc

528 SPASOJEVIC, BATINIC-

quilibrium in the solution, MnIITE-2-PyP41 andMnIITE-2-PyP(NO)41.


The Effect of Oxygen

Because of the reaction of NO• with O2, it wasmportant to investigate the effect of O2 upon the

yield of the nitrosylated Mn(II) porphyrin complex.Figure 3 presents the absorbance at 420 nm as afunction of time after addition of NOC-9 to the mix-ture of MnIIITE-2-PyP51 and 1 mM ascorbate. Insolutions equilibrated with 1% O2 there is a gradualincrease in absorbance at 420 nm over 10 min, dueto the release of NO from NOC-9 followed by analmost imperceptible decline over the following 25min. When the solutions were equilibrated with air(;20% O2) the maximum absorbance reached was atrifle less, followed by the slightly faster rate ofsubsequent decline as shown in Fig. 3. These effects

P51 in argon-purged PBS at pH 7.4. Spectrum 2 of fully reduced0.5 mM) while spectrum 3 of MnIITE-2-PyP(NO)41 was obtained

in millimolar absorptivities. The inset presents the absorption

RLE, AND FRIDOVICH

-2-Pyrbate (

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of O2 were more pronounced in solutions equili-brated with 100% O2.


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The Effect of Ascorbate

An increased concentration of ascorbate (10 mM)substantially improves the stability of the MnIITE--PyP(NO)41 complex formed under 20% O2 (pre-

sumably by preventing the reoxidation of MnIITE-2-PyP41), although it does not completely eliminate asmall loss (;10%) due to the reaction of NO• with O2

during the 10-min release of NO• from NONOate(Fig. 3). From Fig. 3 it is also apparent that ascor-bate does not react with NO• at any significant level.

DISCUSSION

The rate of nitrosylation of MnIIITE-2-PyP51 byNO• was limited by its prior reduction by NO•.When the reduction was achieved with ascorbate(Eq. [1]) (12) (E 0( A 22/A•2) 5 119 mV), the nitrosy-lation reaction was rapid (Eq. [2]). Nitrosylation ofmetalloporphyrins has previously been explored andwas reported to stabilize the divalent state of themetal (Mn(II) or Fe(II)) (11). Other than a few stud-

TABLE I

Characteristic Millimolar Absorptivities, e, Takenfrom Spectra in Fig. 1

nm

Millimolar absorptivity, e, mM21 cm21

MnIIITE-2-PyP51

(1)MnIITE-2-PyP41

(2)MnIITE-2-PyP(NO)41

(3)

21.6 20.9 75.2 233.920.0 21.1 70.3 229.530.4 24.6 129.3 129.8

438.0 36.8 180.6 49.4438.4 37.9 180.9 46.9449.3 104.3 104.2 13.4455.0 144.6 59.2 7.4540.0 8.3 5.2 21.3542.2 9.1 5.8 22.0552.6 12.2 11.2 11.2560.6 13.0 15.9 5.9564.2 12.7 17.3 5.4565.6 12.4 17.4 5.5577.0 8.1 11.2 11.6

Note. The numbers in bold are emax values at their correspond-ng wavelengths (peaks), the numbers in italic are from isosbesticoints (a wavelength at which two compounds have the samebsorptivity), and underlined value is a valley. Conditions as in

Fig. 1.

NITROSYLATI

ies (13, 14), most of the earlier work was based onwater-insoluble metalloporphyrins (11, 15–25); thus

c


the rate of reaction of NO• with Mn(II) tetraphenyl-porphyrin in tetrahydrofuran was reported to be3.3 3 107 M21 s21 at 25°C (26). This high rate con-stant is in accord with the rapid and stoichiometricreaction of NO• with the water-soluble MnIITE-2-PyP41 observed by us. Preliminary measurementsby a stopped-flow method, assuming the validity ofEq. [2], yielded a rate constant of ;1 3 106 M21 s21

at 25°C.

MnIIITE-2-PyP51 1 A 22 N MnIITE-2-PyP41 1 A •2

[1]nIITE-2-PyP41 1 NO• N MnIITE-2-PyP~NO! 41

[2]

The affinity of MnIITE-2-PyP41 for NO• was highenough and the De438 was large enough to provide alinear response to NO• in the range 0.16–1.2 mM. O2

could interfere by oxidizing the NO• (Eq. [3]) (27).However, this process was slow in the range of NO•

concentrations explored here and equilibration ofsolutions with 0–5% O2 would not seriously inter-

FIG. 2. Calibration curve in the range [NO•] 5 0.16–1.2 mM.MnIII/IITE-2-PyP51/41] 5 3.13 mM; [ascorbate] 5 1.0 mM; in 2.0 ml

of argon-purged PBS. Aliquots (2.5 mL) of 130 mM NO• (65 mMOC-9) were added and absorbance at 438 nm was recorded 10in after each addition of NOC-9, which was long enough for its

529MnTE-2-PyP

omplete decomposition with release of NO•. The inset representsspectra taken 10 min after each addition of NOC-9.


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24

ai

HABE

fere provided that absorbance was recorded within10 min after mixing the reactants (Fig. 3). Even at20% of dissolved oxygen a correction can be easilymade through a calibration procedure under well-defined conditions.

4NO• 1 O2 1 2H2O N 4H1 1 4NO22 [3]

The possibility that the effect of ascorbate on thestability of MnIITE-2-PyP(NO)41 ({Mn(NO)}6) (2, 15)complex (Fig. 3) is due to deoxygenation of the solu-tion was further explored in oxygen consumptionexperiments with Clark electrode. Figure 4 demon-strates a very slow oxygen consumption by 0.1 Mascorbate in a closed air-equilibrated PBS solutionat pH 7.4 in agreement with the literature (28).However, when 5 mM MnIIITE-2-PyP51 was added tothe solution a catalytic (;10 turnovers in 2 min)increase in O2 consumption was observed. This ob-servation raises the question: which product of thereaction in Eq. [1], MnIITE-2-PyP41 or A•2, is morereactive toward O2 and responsible for the observed

2 consumption (i.e., Eqs. [4] and [5] or Eqs. [6] and[7])?

II 41 III 51 •2

FIG. 3. Effect of % O2 upon NO• measurement. [MnIII/IITE-2-PyP51/41] 5 6.13 mM; [ascorbate] 5 1.0 mM; in 2 ml of PBSequilibrated with the indicated percentage of O2. After addition of-mL aliquot of 1.3 mM NO• (0.65 mM NOC-9) the absorbance at20 nm was recorded during the following 30 min.


Mn TE-2-PyP 1 O2 N Mn TE-2-PyP 1 O2

[4]


MnIITE-2-PyP41 1 O2•2 1 2H1 N

MnIIITE-2-PyP51 1 H2O2 [5]

A •2 1 O2 N A 1 O2•2 [6]

A •2 1 O2•2 1 2H1 N A 1 H2O2. [7]

Ascorbate radical ( A•2), like ascorbate dianionitself ( A 22), has been shown to react very slowlywith O2 (29) and is not expected to react directlywith O2 in the oxygen consumption observed in thepresence of Mn-porphyrin. According to the litera-ture, there is no evidence of O2

•2 production duringthe “autooxidation” of ascorbate (29, 30). Actually,this is expected to be generally valid for any smallmolecule that is capable of reducing O2 to O2

•2 (E 0 5160 mV) (31) (Eq. [6]). Namely, in an excess of

educing agent, according to the reduction potentialor H2O2/O2

•2 couple of 1890 mV (31, 32), the subse-uent and immediate reduction of O2

•2 to H2O2 be-

FIG. 4. The effect of ascorbate, MnIII/IITE-2-PyP51/41 and NO• onO2 consumption. In a closed cell equipped with Clark electrode,ir-equilibrated PBS solution was treated consecutively with des-gnated amounts (final concentrations) of ascorbate, MnIIITE-2-

RLE, AND FRIDOVICH

PyP51, NO• (saturated in PBS, not from NONOate), and finallywith another portion of MnIIITE-2-PyP51.


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comes a highly favorable process (Eq. [7]) (33). Thereactions in Eqs. [4]–[7] are written on the ground ofsuch reasoning. Addition of 200 mM (as well as 5mM) Cu/Zn SOD had no effect on the rate of oxygenconsumption in the same experiment as in Fig. 4while in the same experiment catalase caused anincrease in percentage O2 signal (data not shown).Furthermore, the presence of catalase does not af-fect the rate of O2 consumption which means that itis neither O2

•2 nor H2O2 but O2 that is responsible forreoxidation of MnIITE-2-PyP41 to MnIIITE-2-PyP51

and that this is the reaction (Eq. [4]) by which theobserved O2 consumption is taking place.

Along this line, in our next experiment, the orthoisomer MnIIITE-2-PyP51 was replaced by para iso-

er MnIIITE-4-PyP51. The para isomer has a 153mV more negative reduction potential (this work,E 1/ 2 5 175 mV vs NHE) than the ortho isomerE 1/ 2 5 1228 mV vs NHE, Ref. 9). This makes the

para isomer less prone to reduction, but once re-duced to MnIITE-4-PyP41 it becomes a stronger re-ducing agent than the ortho isomer, MnIITE-2-PyP41. Indeed, the acceleration of O2 consumption inhe presence of MnIIITE-4-PyP51 was more profound

FIG. 5. The comparison between ortho and para isomer,nIIITE-2-PyP51 and MnIIITE-4-PyP51, respectively, in their cat-

lytic effect on O2 consumption in presence of ascorbate. In aclosed cell equipped with Clark electrode, air-equilibrated PBSsolution containing either 10 mM ortho isomer MnIIITE-2-PyP51

(solid line) or 10 mM para isomer MnIIITE-4-PyP51 (dashed line)as treated consecutively with designated amounts (final concen-

rations) of ascorbate.

NITROSYLATI

than in the case of MnIIITE-2-PyP51 (Fig. 5). Thatmeans that it is Mn(II) porphyrin and not A•2 spe-


cies (which is less efficiently produced by MnIIITE-4-PyP51) that reacts with O2. MnIIITE-4-PyP51 wassubstantially faster even at 1 mM ascorbate, whereonly a fraction of MnIIITE-4-PyP51 is in its reducedform MnIITE-4-PyP41, while under the same condi-tions MnIIITE-2-PyP51 is completely reduced toMnIITE-2-PyP41. This means that the reaction de-

icted by Eq. [1] is in a rapid preequilibrium for theate-determining reaction of Mn(II) porphyrin with

2.How does the interaction with O2 affect the ability

of MnIITE-2-PyP41 to bind NO•? The reduced form ofthe manganese porphyrin, MnIITE-2-PyP41, is ex-pected to react with O2 from the solution to formhydrogen peroxide (33, 34) (Eqs. [4] and [5]); pre-sumably the intermediate O2

•2 is being quickly re-duced to H2O2 by another molecule of MnIITE-2-PyP41 (Eq. [5]) or by ascorbate radical (Eq. [7]). Therate-limiting, one-electron reaction with O2 (Eq. [4])for the para-methylated analogue, MnIITE-4-PyP41,proceeds with a rate constant of 1.1 3 106 M21 s21

(34). Assuming an outer-sphere mechanism to beoperative, which predicts that the rate of oxidationwill decrease by an order of magnitude for each 120mV positive shift in the reduction potential (35), weestimate the rate constant for ortho-ethylated com-pound MnIITE-2-PyP41 (with its 153 mV more posi-tive potential than MnII/IIITE-4-PyP41/51) (Ref. 9 andthis work) to be ;8 3 104 M21 s21. On the otherhand, MnIITE-2-PyP41 reacts with NO• (Eq. [2])with the rate constant of k ; 1 3 106 M21 s21 (thiswork). According to these values and under aerobicconditions (Fig. 3) (6.13 mM MnIII/IITE-2-PyP51/41, 1.3mM NO•, 240 mM O2), the reaction with O2 wouldprevail because of the excess of O2 and no MnIITE-2-PyP(NO)41 complex would be formed. However, asFig. 4 demonstrates, this is not the case becauseNO• binds stoichiometrically to MnIITE-2-PyP41 (22)lessening its redox-cycling power.

The most frequently used oxyhemoglobin (oxyHb)assay (5) was specifically designed to allow NO•

determination in the presence of oxygen, which re-stricts its use under anaerobic and under low oxygenconcentration conditions. On the contrary, themethod presented herein is ideally suited for anaer-obic and low oxygen concentration work. Also, in

531MnTE-2-PyP

contrast to oxyhemoglobin assays, our method is pHindependent. Thus, having the same sensitivity, the


PP

1

1

1

1

1

1

1

HABE

method fully complements the existing oxyhemoglo-bin assay. The metalloporphyrin required, MnIIITE-2-PyP51, is commercially available (Mid-CenturyChemicals) and does not require any purificationbefore preparation of aqueous stock solutions whichare indefinitely stable at room temperature and areinsensitive to air. In our lab we use this methodroutinely to quickly and conveniently check thefreshness of NONOates and NO•-saturated aqueousstock solutions.

CONCLUSIONS

Nitric oxide reacts very rapidly with MnIITE-2-yP41 forming a semistable complex MnIITE-2-yP(NO)41. This property may be utilized as a sim-

ple and sensitive spectrophotometric assay for NO•

in solutions equilibrated with 0–1% O2. Even 5% O2

would not introduce unacceptably high levels of er-ror, especially at higher ascorbate concentrations,but in the case of higher % O2 a calibration proce-dure should be applied under well-controlled condi-tions. Under aerobic conditions, in the presence ofascorbate, MnII/IIITE-2-PyP41/51 catalytically scav-enges O2 producing H2O2. Added NO• efficientlyhalts the catalytic oxygen consumption by binding tothe MnIITE-2-PyP41.

The MnIIITE(M)-2-PyP51 is a very efficient O2•2

scavenger in vitro and in vivo (9). Thus, besidesproviding a new way of measuring NO•, the ob-served favorable and fast nitrosylation of MnIITE-2-PyP41 and the interaction of MnIITE-2-PyP41 andMnIITE-2-PyP(NO)41 with O2 bring a new light intothe possible diverse biological effects of MnIIITE(M)-2-PyP51 and related compounds (9, 36–44).

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533MnTE-2-PyP

44. Spasojevic, I., Ines-Batinic-Haberle, and Fridovich, I., unpub-lished data.


nitrosylation of manganese(ii) tetrakis(n-ethylpyridinium-2-yl)porphyrin: a simple and sensitive...

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