[5] Isolation and oxygenation reactions of nitrosylmyoglobins

[5]

NITROSYL MYOGLOBINS

41

levels of superoxide by using the ratio of aconitase activities measured after imposing different treatments X and y13: [O2- condition X] _ [X aconitaseinactive]/ [X aconitaseactive] [02- condition Y] [Y aconitaseinactive]/[Y aconitaSeactive]

(3)

[5] I s o l a t i o n a n d O x y g e n a t i o n R e a c t i o n s of Nitrosylmyoglobins

By ERNST

V . ARNOLD

and D. Scoxr BOHLE

In~oduc~on A remarkable aspect of the mammalian nitric oxide (NO) signal transduction system is that heme proteins are involved in both the biosynthesis of NO from arginine and, in the case of soluble guanylyl cyclase, in its detection. In general, heme proteins have extraordinarily high affinities for nitric oxide, with the most intensively studied systems, either synthetic or natural, being the nitrosyl adducts of hemoglobin (Hb) and myoglobin (Mb). For example, in the case of sheep hemoglobin the affinity for nitric oxide is 3 million-fold greater than for oxygen, 1 a result that is largely attributable to the high rates of geminative recombination of dissociated NO. 2-4 Many studies of heme nitrosyl adducts have been carried out over the past 20 years, largely addressing issues relating to oxygen transport and metabolism by heme proteins. In this period nitric oxide was used as a structural mimic for dioxygen, as both ligands bind in a bent manner to the iron; the important practical difference was that NO forms an odd electron complex and thus is readily detected by electron spin resonance (ESR) spectroscopy. The dioxygen adducts are ESR silent. As a consequence of these studies there exists a large body of literature relating to the spectroscopic characterization of heme protein nitrosyls by a diverse set

1 Q. H. Gibson and F. H. W. Roughton, J. Physiol. (London) 136, 507 (1957). 2 K. A. Jongeward, J. C. Marsters, M. J. Mitchell, D. Magde, and V. S. Sharma, Biochern. Biophys. Res. Commun. 140, 962 (1986). 3 K. N. Walda, X. Y. Liu, V. S. Sharma, and D. Magde, Biochemistry 33, 2198 (1994). 4 j. W. Petrich, J.-C. Lambry, S. Balasubramanian, D. G. Lambright, S. G. Boxer, and J. L. Martin, J. Mol. BioL 238, 437 (1994).

METHODS IN ENZYMOLOGY, VOL. 269

Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

42

EFFECTS OF NITRIC OXIDE IN CELLS AND TISSUES

[5]

of techniques including ESR, 5,6 resonance Raman spectroscopy,7-9 Fourier transform infrared (FTIR) spectroscopy, l° magnetic circular dichroism (MCD) spectroscopy, 11and by electronic spectroscopy (UV-Vis). 12Usually these studies are performed on samples of heme proteins prepared in situ by the method discussed below, and it is significant that despite three decades of crystallographic studies on these heme proteins only a single relatively low-resolution (2.8 ,~) study of a nitrosyl adduct of horse hemoglobin has been described. 13Moreover, the structural analysis for this study was based on a data set of 6591 reflections, with structural deductions being based on the difference Fourier map between nitrosylhemoglobin (HbNO) and methemoglobin (metHb). In addition, Mrssbauer spectroscopy and magnetic susceptibility determinations, physical techniques that are routinely performed on heme proteins but require relatively large amounts of purified protein, have been described only in outline, t4'15 This situation is attributable to the oxygen sensitivity of the nitrosyl adducts of hemoglobin and myoglobin; moreover, we have found that methods in the literature for handling air-sensitive proteins a6 such as nitrosylmyoglobin (MbNO) are generally inadequate for its isolation and purification on the scales required for the above measurements. Therefore, an improved synthesis and purification technique evolved in response to this problem. Particular attention was paid to minimizing chemical modification of the protein structure as well as to developing an anaerobic technique to purify this air-sensitive protein. Herein, we describe this combination of inorganic and biochemical techniques. In the synthesis of MbNO from metmyoglobin (metMb), there must be a reduction step to convert the ferric heme to the ferrous form, E ° = 58.8 mV vs normal hydrogen electrode (NHE) at 250.17 In a reaction termed reductive nitrosylation it is possible to reduce metMb directly with excess nitric oxide, 5 L. C. Dickinson and J. C. W. Chien, J. Am. Chem. Soc. 93, 5037 (1971). 6 R. H. Morse and S. I. Chan, J. Biol. Chem. 255, 7876 (1980). 7 A. E. Yu, S. Hu, T. G. Spiro, and J. N. Burstyn, J. Am. Chem. Soc. 116, 4117 (1994). s j. D. Stong, J. M. Burke, P. Daly, P. Wright, and T. G. Spiro, J. Am. Chem. Soc. 1029 5815 (1980). 9 M. A. Waiters and T. G. Spiro, Biochemistry 21, 6989 (1982). 10 X.-J. Zhao, V. Sampath, and W. Caughey, Biochem. Biophys. Res. Commun. 204, 537 (1994). 11 T. Yamamoto, N. Tsunenori, A. Kaito, and M. Hatano, Bull. Chem. Soc. Jpn. 55, 2021 (1982). 12E. Antonini and M. Brunori, "Hemoglobin and Myoglobin in Their Reactions with Ligands." North-Holland Publ., Amsterdam, 1971. 13j. F. Deatherage and K. Moffat, J. Mol. Biol. 134, 401 (1979). 14 G. Lang and W. Marshall, Proc. Phys. Soc., London 87, 3 (1967) 15 W. T. Oosterhuis and G. Lang, J. Chem. Phys. $0, 4381 (1969). 16B. K. Burgess, D. B. Jacobs, and E. I. Steifel, Biochim. Biophys. Acta 614, 196 (1980). 17 R. J. Crutchley, W. R. Ellis, Jr., and H. B. Gray, J. Am. Chem. Soc. 107, 5002 (1985).

[51

NITROSYL MYOGLOBINS

[MbFein] + I~O [MbFem NOI + [MbFeni_NO]+ °vI2 [MbFe n] + NO2- + 2H + [MbFen ] No [MbFen_NO]

43

(1) (2) (3)

[Eqs. (1)-(3)], TM although these reactions are slower than for hemoglobin) 9 Anaerobic solutions of metMb are required for these preparations, which require long contact times and high nitric oxide pressures at neutral p H . 19 Alternatively, reducing agents such as sodium dithionite, Na28204, 2°-23 [E ° -- -1.12 V at pH 14; Eq. (4)], or ascorbate 24-26 (E ° = -0.08 V) can be used to reduce metMb directly, and the resulting deoxymyoglobin can be nitrosylated with nitric oxide. In the case of dithionite the large overpotential for the reduction leads to fast kinetics,22 kobs = 3.1 × 10 6 M -1 sec -1, so that the rate of reduction with dithionite has little dependence on pH, axial ligand, or cofactor concentration. Unfortunately the practice of using large excesses of dithionite in these reactions is commonplace, and although excess dioxygen will also be removed by this reagent, deleterious side reactions are possible for proteins exposed to this reducing environment. In particular, the dissociative equilibrium shown in Eq. (5), even though small, leads to the generation of reactive radical anions of sulfur dioxide. The potential consequences for protein activity and structure under these conditions are enormous, and some authors have noted these problems in their studies.27

4HO- + $2042----~ 2SO3 - + 2 H 2 0 + 2e-, E ° = -1.12 V at pH14 $2042- ~ 2SO2"-, Kd = 1.4 × 10-9 M at 25°

(4) (5)

To avoid reductive protein modification, a lower potential reducing agent, ascorbate, coupled with nitrite coordination, can simultaneously effect heme reduction and nitrosylation.28 In contrast to the reduction with dithionite, ascorbate reduction of these solutions is slow, k = 3.3 × 102

18 R. W. Romberg and R. J. Kassner, Biochemistry 18, 5387 (1979). 19 K. Keilin and E. F. Hartree, Nature (London) 139, 548 (1937). 20 R. P. Cox and M. R. Hollaway, Eur. J. Biochem. 74, 575 (1977). 21 D. O. Lambeth and G. Palmer, Z BioL Chem. 248, 6095 (1973). 22 E. Olivas, J. A. Dirk De Waal, and R. G. Wilkins, J. Biol. Chem. 252, 4038 (1977). 23 K. Tsukahara and K. Ishida, Bull. Chem. Soc. Jpn. 64, 2378 (1991). 24 K. Tsukahara and Y. Yamamoto, J. Bioehem. (Tokyo) 93, 15 (1983). 25 K. Tsukahara, Inorg. Chim. Acta 124, 199 (1986). 26 K. Tsukahara, T. Okazawa, H. Takahashi, and Y. Yamamoto, Inorg. Chem. 25, 4756 (1986). 27 H. J. Andersen and L. H. Skibsted, J. Agric. Food Chem. 40, 1741 (1992). 28 A. R. Kamarei and M. Karel, J. Food Sci. 47, 682 (1982).

44

EFFECTS OF NITRIC OXIDE IN CELLS AND TISSUES

[5]

M -~ s e c - I , 29 and the reaction is highly dependent on p H and ligation. This

reaction is the basis for the biochemistry of nitrite preservation of cured meats, in which solutions of 200 ppm sodium nitrite and excess ascorbate or erythorbate are injected or intimately mixed with meat before storage. 24'3° By a controversial and surprisingly poorly understood mechanism nitrite acts as both a preservative by inhibiting bacterial growth, and as a cosmetic agent in that it causes the formation of bright red MbNO from metMb. 3~ A major concern in these studies has been to maximize the lifetime of MbNO and to understand therefore the mechanisms of its aerobic and photooxidation. 32-35In these studies a widely used method for the preparation of MbNO is to treat anaerobic solutions of metMb with a stoichiometric amount of nitrite followed by a 17.6-fold excess of ascorbate at p H 5.5 for s e v e r a l h o u r s . 28 Previous studies have used large excesses of both nitrite and ascorbate and often the nitrosyl myoglobin is used directly without further purification. 31We have found that the presence of excess reductant interferes with our studies of MbNO oxygenation and that the presence of even small amounts of reductant will reduce any metMb back to the ferrous form, which will combine with the oxygen present. Therefore, any residual reductant must be removed in order to avoid this deleterious side reaction. The simplest method to remove excess impurities in the MbNO solution would be to gel filter under anaerobic conditions. This was employed by Anderson and Skibsted, 27 but they observed spectral deviations that were not explained satisfactorily in their kinetic experiments. The problem is to synthesize MbNO efficiently with an absolute minimum of impurities that will affect subsequent kinetics studies with these solutions. It was decided to adapt the anaerobic techniques employed in synthetic inorganic chemistry to this problem. Thus, a stoichiometric amount of sodium nitrite is added to a pre-gel-filtered anaerobic solution of metMb and this is followed by a stiochiometric amount of sodium dithionite. It has been shown that the chemical reduction by either a stoichiometric amount of sodium dithionite or an excess of sodium dithionite that is immediately gel filtered does not affect the oxidation rate of oxymyoglobin.36 Synthesis of MbNO is instantaneous with an absolute minimum of impurities.

29 C. Giulini and E. Cadenas, F E B S Lett. 332, 287 (1993). 3o j. R. Fox and S. A. Ackerrnan, J. Food Sci. 33, 364 (1968). 31 j. B. Fox and J. S. Thomson, Biochemistry 2, 465 (1963). 32 R. F. Kampsehmidt, Food Chem. 3, 510 (1955). 33 M. E. Bailey, R. W. Frame, and H. D. Naumann, J. Agric. Food Chem. 12, 89 (1964). 34 W. M. Urbain and L. B. Jensen, Food Res. 5, 593 (1940). 35 K. A. Walsh and D. Rose, Agric. Food Chem. 4, 352 (1956). 3~ R. E. Brantley, S. J. Smerdon, A. J. Wilkonson, E. W. Singleton, and J. S. Olson, J. Biol. Chem. 268, 6995 (1993).

[5]

NITROSYL MYOGLOBINS

45

Even with this minimum level of contamination, it is often desirable to remove all impurities and have only MbNO and buffer. The following is a description of just such a method. Materials Horse heart myoglobin and Sephadex are obtained from Sigma (St. Louis, MO). Sodium nitrite (Sigma) and sodium dithionite (Aldrich, Milwaukee, WI) are used without further purification. Phosphate buffer solutions or Tris-HCl buffer solutions are used in all experiments. 37 Apparatus A dual-vacuum nitrogen manifold is used to control the atmosphere in the reaction flasks. The double-tube manifold as well as Schlenk glassware can be obtained commercially or can be fabricated by a glassblower, as A modified Schlenk flask with two ports is an ideal container for anaerobic solution storage. The nitrogen line on the manifold is vented to either an oil bubbler or a mercury bubbler with the overpressure controlled by a three-way stopcock. The stirred cell is fitted with a modified cellulose membrane with a molecular weight cutoff of 10,000 (Cole-Parmer, Chicago, IL). Although stainless steel cannulas are widely used, we have found that the Teflon cannulas work just as well and have several advantages over the stainless steel cannulas. Teflon tubing (1.6-mm o.d.; Cole-Parmer) is a useful substitute because it is easy to ascertain if it is dirty, it is readily manipulated, and it is inexpensive. Method of Degassing One hundred milligrams of horse heart metmyoglobin is dissolved in 2 ml of p H 7.0 phosphate buffer and carefully pipetted onto a Sephadex G-50 column prior to use. Ultrasonication can assist the dissolution. The single dark band is collected and assayed by U V - V i s spectroscopy at 502 nm for concentration (e = 10.2 m M -I cm-1). 12 The brown solution is transferred into a Schlenk flask for degassing. The quickest and surest method for creating an anaerobic solution is a series of vacuum degassing and nitrogen purges. The technique involves freezing the solution in the Schlenk flask by immersion into a Dewar flask of liquid nitrogen. Once frozen, a vacuum is pulled until it is fully evacuated. The vacuum line is closed and the 37D. D. Perrin, Aust. J. Chem. 16, 572 (1963). 38D. F. Shriver, "The Manipulation of Air Sensitive Compounds." McGraw-Hill, New York, 1979.

46

EFFECTSOFNITRICOXIDEINCELLSANDTISSUES

[5]

solution is gently thawed with a heat gun. After it is thawed, nitrogen gas is reintroduced and the system is briefly purged with a flow of nitrogen, which is then vented through a mercury bubbler. Once the purge is complete, the nitrogen line is closed and the solution is frozen and evacuated again. Three repetitions of this technique results in an anaerobic solution ready for the preparations below. Method of Preparation and Isolation of Nitrosylmyoglobin Nitrosylation of metmyoglobin is efficiently done by treating it with stoichiometric amounts of sodium nitrite and sodium dithionite under anaerobic conditions. Anaerobic solutions of these solid reagents are required and prepared as follows. After the appropriate buffer solution is vacuum degassed in a Schlenk flask, 0.087 g of sodium dithionite and 0.034 g of sodium nitrite are placed in separate 5-ml volumetric flasks, which are then fitted with septa. One end of the cannula is inserted through the septum of the Schlenk flask and the other is inserted into the septum of the volumetric flask, which is then vented to an oil bubbler (Fig. 1). The whole system is purged for at least 20 min with a gentle flow of nitrogen and then exactly 5 ml of the buffer solution is transferred over to the volumetric flask to obtain 0.1 M anaerobic solutions of each reagent. This technique can be

I[ ~ e f l o n

V" a C ~ n e ~ . . ~

Cannula . . To Oil Bubbler

Septum ~

~itn~rOge~jed~O2e~

~

~

Septum

5mL /

N

~/ Volumetric Flask QuartzCuvette ESRTube Solution FIG.1. Cannulaand Schlenktechniquesfor the transferof solutionsanaerobically.

[5]

NITROSYLMYOGLOBINS

47

adapted for transfers to any of the receiving vessels shown in Fig. 1. After the purge, the solution can be transferred through the cannula into the receiving flask. Alternatively, a gas-tight syringe can be used to withdraw degassed buffer and inject it into a nitrogen-purged flask. Once the anaerobic sodium nitrite and sodium dithionite solutions are ready, a microliter gas-tight syringe is used to inject a stoichiometric amount into the degassed myoglobin solution prepared above. The injections can either go through the septum or, with the modified Schlenk flask, through the valve. It is important to have the Schlenk flask under a positive pressure of nitrogen and then inject the nitrite followed quickly by the dithionite. A swirl of the Schlenk flask produces a beautiful red MbNO solution with a minimum of impurities. To purify the material further, an ultrafiltration or stirred cell can be used. A stirred cell utilizes a membrane to separate material on the basis of molecular weight while under a considerable pressure of nitrogen. Anaerobic conditions must be maintained in the stirred cell. Most importantly, all solutions must be anaerobically introduced into the stirred cell. The best method is to open the relief valve and flush the cell with nitrogen for 15 min and afterward, while under a slight positive pressure of nitrogen, remove the relief valve so that the Teflon cannula tubing can be inserted into the cell from the Schlenk flask (Fig. 2). While maintaining the degassed buffer solution to be introduced into the cell under a greater nitrogen

•~

•

VentedNitrogen /

-°ell -in

,Nitrogen Line

/

, ~ ~

Modified Schlenk

ReliefValv

,

f"

StirrcdCdl

"'] J

FIG. 2. Purification of MbNO by anaerobic ultrafiltration with a stirred cell.

48

EFFECTS OF NITRIC OXIDE IN CELLS AND TISSUES

[5]

pressure, push the tip of the cannula into the solution and it will transfer 3 to 4 ml through the cannula into the cell. Reassemble the cell and pressurize to 40 psi or to the standard operating pressure. This pushes the buffer through the cell. Be careful not to push it all through and allow the membrane to dry. When there is 0.5 ml left in the cell, introduce 10 ml of MbNO by following the same protocol. Push 6 ml or at least 50% of the MbNO through the cell and then redilute to at least 10 ml with fresh degassed buffer solution. Repeat this procedure at least three times in order to ultrafilter MbNO. To get the MbNO out of the cell, simply pressurize the cell to a higher nitrogen overpressure than in the degassed receiving Schlenk flask and cannula the solution into the Schlenk flask. This solution can be used as is or lyophilized under high vacuum to give a red powdered sample of MbNO. The purity and concentration of the MbNO solution so obtained can be assayed by UV-Vis spectroscopy where, at pH 7.0 and room temperature, the millimolar extinction coefficients at/~max(e) for the peaks are 543 (11.6) and 575 (10.5). I2 Impurities, if present, are likely to be metMb from oxidation of MbNO. This can be ascertained by the presence of peaks at 502 and 630 nm, which are not present in the MbNO spectrum.

Apparatus for Kinetics Studies The kinetics of MbNO oxygenation were followed by monitoring the absorbance change between the wavelengths 390 and 680 nm by means of an HP-8452A spectrophotometer (Hewlett-Packard, Palo Alto, CA) fitted with a thermostatted cell holder held at 37° by a model 9105 Fisher Scientific (Pittsburgh, PA) isotemperature standard circulator. Pseudo-first-order kinetics with respect to the heme were obtained by injection of 30 cm 3 of 99.99% (v/v) pure oxygen into the MbNO solution.

Kinetics Calculations Raw spectroscopic data were imported into the Specfit package 39 and subjected to factor analysis by singular value decomposition (SVD) to determine the principal number of unique orthogonal eigenvectors needed to describe the complete set of spectra. 4° The statistically significant non39 "SPECFIT, A Program for Global Least Squares Fitting of Equilibrium and Kinetics Systems using Factor Analysis and Marquardt Minimization," Version 2.3. Spectrum Software Associates, Chapel Hill, NC, 1993. 40 E. R. Malinowske, "Factor Analysis in Chemistry," 2nd ed. Wiley (Interscience), New York, 1991.

[5]

NITROSYL MYOGLOBINS

49

noise eigenvectors are then back-transformed by SVD to give noise-filtered data. These can either be used for the evolving factor analysis to estimate the concentration profiles and component spectra, or they can be globally fitted by a Marquardt least-squares algorithm to a particular model. Method A long-necked quartz cuvette is degassed for 10 min with a nitrogen purge as previously described and filled to a marked volume, 3 ml, with the appropriate degassed buffer. A set volume of MbNO is introduced into the cuvette by calibration of the Teflon cannula. The solution is shaken and put in the thermostatted cell holder to equilibrate to the experimental temperature. A U V - V i s spectrum can be taken to ascertain MbNO concentration, about 10 -5 M, or this can be performed later. Once the solution is equilibrated to the desired temperature (at least 5 min), oxygen gas (30 ml) is injected with a long needle that reaches to the bottom of the cuvette, which not only introduces the oxygen but also provides adequate mixing. Kinetics of Nitrosylmyoglobin Oxygenation As shown in Fig. 3 the oxygenation of MbNO by oxygen exhibits isosbestic behavior. This has generally been interpreted as being due to two colored

0.,'.

1

c.) ct~

0.;

0 ¢-~

<

I 0.1

450

500 550 600 Wavelength (rim)

650

FIG. 3. Observed changes in Q-band region for the oxygenationof MbNO at 37° and pH 7. Spectra taken at 3-min intervals.

50

EFFECTS OF NITRIC OXIDE IN CELLS AND TISSUES

HbNO + 0 2 HbNO Hb + 0 2 4NO + 02 + 2H20

k=l.3xlO-3sec 1

metHb + + NO3> Hb + NO ) HbO2 ~ 4NO2- + 4H + HbO2 + NO2~ metHb + + N O 3 MbNO + O2 ~ {MbNO----O2} ---* metMb + + NO3)

[5]

(6) (7) (8) (9) (10) (11)

SCHEME I. Proposed mechanisms of hemoglobin oxygenation. (Taken from Kon et aL41 and Andersen and Skibsted. 27)

species, MbNO and metMb, which follow first-order A ~ B kinetics. In human hemoglobin, the rate of conversion of HbNO to metHb at 37 ° has been estimated to be 1.3 × 10 -3 sec -1 [Eq. (6) in Scheme I]. 41 It was proposed that the rate-limiting step in this reaction was the dissociation of HbNO into Hb and NO followed by heme oxygenation to give HbO2 [Eqs. (7) and (8) in Scheme I]. It was then suggested that nitric oxide is converted to nitrite [Eq. (9), Scheme I] and this is then oxidized by HbO2 to give the products metHb and nitrate [Eq. (10), Scheme I]. There are two problems associated with this proposed mechanism; one is a matter of detail in the later steps of the reaction, but the other casts doubt on the key denitrosylation step [Eq. (7), Scheme I]. Studies have redetermined the rate of nitric oxide oxidation by HbNO and found it to be a rapid bimolecular reaction, unlike the direct oxygenation of NO shown in Eq. (9) (Scheme I), which is a termolecular reaction that is bimolecular in nitric oxide concentration. 42 Thus at transient low concentrations of nitric oxide the amount of nitric oxide oxidized in Eq. (9) (Scheme I) will be relatively small. More difficult to reconcile is the requirement for the denitrosylation of HbNO in Eq. (7) (Scheme I); the rate of nitric oxide dissociation from HbNO is slow, kd~ = 2.2 x 10 -s sec i and kd¢ = 4.8 × 10 -5 sec -1, at least three orders of magnitude slower than the rate shown in Eq. (6) (Scheme I). 12 In a more recent, thorough study of myoglobin oxygenation, the reported first-order rate of conversion of MbNO to metMb is 5.1 × 10 -4 sec -1 at 300.27 In this case, it was suggested that there is an initial preequilibrium with oxygen, which associates with the heme pocket in the protein before undergoing a ratelimiting addition to the metal-bound nitrosyl to yield the products metMb and NO3- [Eq. (11), Scheme I]. Both of these studies determined that nitrate is the ultimate product of these reactions, but a word of caution needs to be sounded: excess ascorbate was present in each case and the 41 K. Kon, N. Maeda, T. Suda, and T. Shiga, Jpn. Soc. Air Pollut. 15, 401 (1980). 42 R. S. Lewis and W. M. Deen, Chem. Res. Toxicol. 7, 568 (1994).

[5]

NITROSYL MYOGLOBINS

51

authors found that this complicated the interpretation of the spectroscopic results. Moreover, under the conditions of excess reductant it is also difficult to quantify the partition between nitrite and nitrate by-products due to the reactions in Eq. (10) (Scheme I). In spite of these problems the conclusion of the myoglobin study is that the addition of oxygen to myoglobin occurs while the nitric oxide is bound to the metal and not dissociated. k1

A

k2

~B

~C

(12)

The oxygenation of MbNO is poorly described by a single exponential rate term anticipated for a simple first-order transformation that follows A ~ B behavior. Instead, the results are better fitted by a biexponential term that is consistent with either the generation of an intermediate as a kinetic scheme that follows an A ~ B ~ C law, or with three species connected by two different rates, A ~ C and B ~ C. As shown in Fig. 4 the observed data are best fitted by three species connected by two processes with kl = 4.635 x 10 4 see 1 and k2 = 7.848 x 1 0 - 4 s e c -1 at 30 ° and p H 7.0 [Eq. (12)]. Of these two options our results, in particular the predicted

2.00

t-

O oo

1.5

I

~

I

'

2000

Time

I

~

I

4000

'

I

'

6000

(sec)

FIG. 4. Observed (A) and calculated changes in the absorbance at 420 n m for the oxygenation of M b N O under the conditions described in Fig. 3. Single exponential first-order best fit for an A-to-B process is marked with a dotted line (-.--) and the biexponential fit for two consecutive first-order reactions, A to B to C, is shown as a solid line (--).

52

EFFECTS

OF NITRIC OXIDE

IN CELLS AND TISSUES

a

b

c

d

[5]

50,000

0 390

I

I

410

430

450

I

520

I

'

I

620

Wavelength (nm) F]o. 5. Predicted spectra for the Soret band (a and c) and the Q-band region (b and d) from an SVD/global fit analysis for the kinetic species responsible for the spectral changes described in Fig. 3. (a) and (b) are those for kl = 0.00281(4) sec 1 and k2 = 0.00134(1) sec -1, while for (c) and (d) the rates are reversed, k1 = 0.00134(1) sec -1 and k2 = 0.00281(4) sec -1. The predicted spectra that remain the same in (a) and (b), and in (c) and (d), and match known spectra are MbNO, the starting material (A) is depicted by open squares (t3), and the product metMb is shown as filled squares (11). The predicted spectra for the intermediate are shown by open diamonds (O).

spectral components shown in Fig. 5, indicate that there is one species identical to MbNO at the beginning of the reaction and one final species, spectroscopically identical to metMb, at the end of the reaction. We can therefore rule out the possibility of the kinetics being due to A ~ C and B ~ C. Prior workers have noticed similar kinetic behavior: the formation of metHb does not parallel the decay of HbNO even though the final conversion is stoichiometric. 43 Also in the forementioned study of MbNO oxygenation [Eq. (11), Scheme I], plots of nitrate formation vs time indicate quite clearly an induction period at the beginning of the reaction. A recognized problem with the analytical solutions to the kinetics of two consecutive 43 G. D. Case, J. S. Dixon, and J. C. Schooley, Environ. Res. 20, 43 (1979).

[5]

53

NITROSYL MYOGLOBINS

C 2

O v t"

.o

O ¢.. O

O

0

'

0

I

'

I

'

i

'

I

2000 4000 Time (sec)

'

I

'

6000

FIG. 6. Predicted concentrations for M b N O (A), m e t M b (C), and intermediate (B) for kl = 0.00134(1) sec -1 and k2 = 0.00281(4) sec -1.

reactions is that there is an ambiguity in assigning the pair of specific values from these solutions to the individual processes, kl and k2 in Eq. ( 1 2 ) . 44-46 For example, either kl = 1.34(1) x 1 0 -3 s e c -1 and k2 = 2.81(4) x 1 0 -3 s e c -1 or kl = 2.81(4) × 1 0 -3 sec -t and k2 = 1.34(1) x 10-3 sec -1, both given equally adequate solutions in terms of a least-squares fit. To solve this problem there are two approaches involving either redetermination of these rates at various temperatures 44 or examination of the predicted spectra based on the SVD approach. 39 In Fig. 5 the predicted spectra for the three kinetically active species that give rise to the spectra in Fig. 3 are shown for the Soret and Q-band regions for both possible orderings of rates. Note that the known components in this system, MbNO and metMb, are both present, with the third intermediary species shown in Fig. 5 by the trace with the open diamonds. For the ordering of kl > k2 in Fig. 5a and b the bands of the intermediate represent a hybrid or average of the MbNO and metMb, while for a slow first reaction followed by a faster second reaction, k2 > kl (Fig. 5c and d), the spectrum is markedly different and closely 44 D. J. B e n t o n and P. M o o r e , J. Chem. Soc. A, 3179 (1970). 45 N. W. Alcock, D. J. B e n t o n , and P. M o o r e , Trans. Faraday Soc. 66, 2210 (1970). 46j. H. E s p e n s e n , " C h e m i c a l Kinetics and Reaction M e c h a n i s m s . " McGraw-Hill, N e w York, 1981. 47 j. S. Beckman, J. Chen, H. Ischiropoulos, and J. P. Crow, Methods Enzymol. 233, 229 (1994).

54

EFFECTS OF NITRIC OXIDE IN CELLS AND TISSUES

coco LnM--N"

[5]

0-0% ~a~~ LnM--N

N--MLn

o// /

\

LnM--N

N - - M L n --~ 2 LnM--NO 2

oo// SCHEME II. Peroxynitrite intermediates in metallonitrosyl oxygenation reactions. M, Co, Ni; L., PR3, pyridines, and imidazoles.

resembles a ferric form of myoglobin. We conclude that the second ordering with kl = 1.34(1) × 10-3 sec -1 and k2 = 2.81(4) × 10 -3 sec -1 is the correct assignment of rates and is most consistent with the mechanism described below (Eq. 13). Mechanism of Nitrosylmyoglobin Oxygenation To explain our kinetics we suggest that there is an initial attack by electrophilic oxygen on the nitrogen of the nitrosyl to form a peroxynitrite species coordinated to the iron [Eq. (13)]. (~0=0 MbFeII

N ".

O--Okl )

MbFeIn__N /

k2

MbFeiii +

[ONOOl ;O3(13)

This initial reaction is followed by either dissociation of peroxynitrite or a coordination sphere rearrangement to give nitrate. Equation (13) differs from Eq. (11) in that we suggest there is a direct new nitrogen-oxygen bond in this intermediate. There is precedent for this mechanism from early oxygenation studies on nonheme metallonitrosyls (Scheme II). 48'49In these studies of low molecular weight metallonitrosyl complexes, L,M(NO), the peroxynitrite intermediates are not protected by a large polypeptide as in MbNO, and they therefore undergo rapid bimolecular reactions with L,M(NO) to give nitrite complexes. This latter pathway is clearly not possible for MbNO. Particularly important data that support this hypothesis 48 R. Ugo and S. Bhaduri, J. Chem. Soc., Chem. Commun., 694 (1976). 49 S. G. Clarkson and F. Basolo, Inorg. Chem. 12, 1528 (1973).

[6]

ENDOTHELIAL

NOS

EXPRESSION

55

include the nature of the predicted spectra by SVD in Fig. 5: consistent with the formation of the N-bound peroxynitrite complex, metMb{N (=O)OO}, the intermediate spectrum is clearly a ferricmyoglobin and it is similar to the reported spectra for metMb(NO2). 5° If the intermediate in Eq. (13) is an N-bound peroxynitrite complex, then the physiological consequences of this reaction are numerous. These results suggest that in addition to the recognized superoxide-mediated pathway for the generation of peroxynitrite, 47 heme proteins may mediate a constitutive or non-cytokine-activated biosynthetic pathway for the formation of this reactive cytotoxin. Studies are underway to characterize further the intermediate in this reaction and to determine if peroxynitrite so generated is released before isomerizing or if the isomerization occurs in the ligand sphere of the heme. Acknowledgment We gratefully acknowledge the NIH (grant GM53828-01), the Alzheimer's Association, the Arthritis Foundation, the American Heart Association, and the Research Corporation (Cottrell Scholarship to D. S. B.) for their generous support of this research. 50 L. J. Young and L. M. Siegel, Biochemistry 27, 2790 (1988).

[6] E n d o t h e l i a l

Nitric Oxide Synthase Heterologous Systems

Expression

in

By LISA J. ROBINSONand THOMAS MICHEL Introduction The availability of nitric oxide synthase (NOS) cDNAs and antibodies has facilitated the study of enzyme expression in a variety of heterologous cell types and in cell-free systems. These expression systems can be exploited for the analysis of enzyme structure-function relationships, as well as the study of NOS biosynthesis and metabolism. The various heterologous expression systems each require that the relevant NOS cDNA be cloned into a plasmid or viral construct possessing specific features appropriate for the given expression system. Furthermore, to characterize the expressed protein using immunoblot or immunoprecipitation analyses, it may be helpful to develop specific polyclonal or monoclonal NOS antibodies, or to utilize commercially available NOS antibodies, as indicated by the specific applications. For the most part, the experimental principles and methodologies METHODS IN ENZYMOLOGY, VOL. 269

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