Reactions of Deoxy-, Oxy-, and Methemoglobin with Nitrogen Monoxide

MECHANISTIC STUDIES OF THE S-NITROSOTHIOL FORMATION UNDER DIFFERENT MIXING CONDITIONS*

Susanna HeroldDagger and Gabriele Röck

From the Laboratorium für Anorganische Chemie, Eidgenössische Technische Hochschule, ETH Hönggerberg, CH-8093 Zürich, Switzerland

Received for publication, October 8, 2002, and in revised form, December 1, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The reaction between hemoglobin (Hb) and NO· has been investigated thoroughly in recent years, but its mechanism is still a matter of substantial controversy. We have carried out a systematic study of the influence of the following factors on the yield of S-nitrosohemoglobin (SNO-Hb) generated from the reaction of NO· with oxy-, deoxy-, and metHb: 1) the volumetric ratio of the protein and the NO· solutions; 2) the rate of addition of the NO· solution to the protein solution; 3) the amount of NO· added; and 4) the concentration of the phosphate buffer. Our results suggest that the highest SNO-Hb yields are mostly obtained by very slow addition of substoichiometric amounts of NO· from a diluted solution. Possible pathways of SNO-Hb formation from the reaction of NO· with oxy-, deoxy-, and metHb are described. Our data strongly suggest that, because of mixing artifacts, care should be taken to use results from in vitro experiments to draw conclusion on the mechanism of the reaction in vivo.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The interaction of nitrogen monoxide with hemoproteins, in particular with myoglobin and hemoglobin, is currently an area of intense research (1-15). The reaction between NO· and oxyhemoglobin (oxyHb)1 has repeatedly been suggested to represent the main route for NO· depletion in the blood vessels (16-22) and the cause for an increase in blood pressure observed when extracellular hemoglobin-based blood substitutes are administered (18, 23). However, the in vivo relevance of this reaction has recently been questioned (11, 15, 24). Indeed, it has been proposed that in vivo NO· preferentially binds to the very small amount of deoxygenated heme of hemoglobin that is present under physiological conditions (about 1%) to yield an iron(II)-nitrosyl complex (HbFe(II)NO) (15). This proposal is based on the hypothesis that NO· binds R-state hemoglobin at least 100 times faster than T-state hemoglobin (25). It has further been suggested that the "NO group" can be transferred intramolecularly from the heme (HbFe(II)NO) to the conserved cysteine residue beta 93, producing S-nitrosohemoglobin (SNO-Hb) (26). This process has been proposed to be driven thermodynamically, since the S-nitrosocysteine of SNO-Hb is significantly more stable when Hb is in the R-state. When the red blood cells reach hypoxic tissues, O2 is delivered from SNO-Hb, triggering the conversion of Hb to the T-state (26). Because of steric interactions with nearby amino acid residues, the S-nitrosocysteine of SNO-Hb is highly destabilized in the deoxyHb T-state (27). Thus, transnitrosation from SNO-Hb to other thiols within the red blood cell is favored. In particular, it has been suggested that the "NO group" is transferred to a cysteine residue of the band 3 anion exchange membrane protein and finally transported out of the red blood cell by a still unidentified route (11, 24). One of the central points of this hypothetical mechanism is that the delivery of both O2 and NO· to tissues is regulated allosterically; cooperative liberation of NO· and O2, transported under physiological conditions by hemoglobin, should thus allow for very efficient delivery of dioxygen to peripheral respiring tissues (11, 24).

An increasing amount of evidence has recently been published that challenges this fascinating hypothesis. Huang et al. (28) have shown that there is a linear correlation between the oxygen saturation of Hb and the yield of HbFe(II)NO, indicating that the rate of NO· binding to deoxyHb is independent of oxygen saturation. In another work, the same group has shown that the rate constant for NO· binding to R-state hemoglobin is (2.1 ± 0.1) × 107 M-1 s-1, nearly identical to that reported for NO· binding to T-state Hb (2.6 × 107 M-1 s-1) (29). Gladwin et al. (12) found a strong correlation between metHb and plasma nitrate in the venous blood from volunteers inhaling 80 ppm of NO· gas and concluded that the main reaction of NO· with Hb in the red blood cells of arterial blood leads to NO· depletion, not conservation of its bioactivity. In addition, the same group determined that SNO-Hb is present in the low nm range in the blood, but they did not find a significant arterial-venous gradient (30-32). Such a gradient should be found if NO· was released in the capillaries after O2 dissociation. Finally, in a recent paper Gladwin et al. (32) showed that SNO-Hb is intrinsically unstable in the reductive environment of the red blood cells and thus concluded that SNO-Hb cannot accumulate in the red blood cells to form a reservoir of bioactive NO·. Taken together, these results suggest that SNO-Hb cannot play a role in the regulation of blood flow under normal physiological conditions.

In addition, while this work was in progress, three papers have appeared that address the problem of mixing artifacts from the addition of an NO· bolus to oxyHb (33, 34) and oxymyoglobin (oxyMb) (35) solutions. The common conclusion of the authors of the three mentioned papers (33-35) is that the addition of a small volume of a saturated (2 mM) NO· solution to a diluted oxyHb or oxyMb/GSH solution may lead to artifactual generation of high SNO-Hb/HbFe(II)NO or S-nitrosoglutathione (GSNO)/MbFe(II)NO yields, respectively. Thus, some of the high SNO-Hb yields reported in previous in vitro studies (15, 26) may be artifacts arising from the chosen experimental conditions. However, the data reported in these three papers (33-35) are not always consistent, and contrasting mechanisms have been proposed to explain similar observations. In particular, from their studies of the reaction between oxyHb and NO· in the presence of cyanide, Han et al. (33) concluded that, when an NO· bolus is added to an oxyHb solution, SNO-Hb is formed through a pathway that does not include NO+ generated from oxidation of NO· by metHb. In contrast, they suggest that SNO-Hb is possibly formed by reaction of Cys-beta 93 with N2O3. According to their mathematical simulations, formation of N2O3 after the addition of NO· to oxyHb under aerobic conditions is not possible kinetically, and thus they suggested that N2O3 must be present in the NO· stock solution. This suggestion is highly unlikely, since N2O3 rapidly hydrolyzes to nitrite in aqueous solutions. Indeed, it has been shown that the addition of unpurified gaseous NO· (directly from the gas tank) to GSH under anaerobic conditions leads to the formation of GSNO (36). In contrast, GSNO was not detected when a solution of unpurified NO· was added to the GSH solution (under anaerobic conditions) (36). A final observation of Han et al. (33) is that if NO· is delivered with the NO· donor 2-(N,N-diethylamino)diazenolate 2-oxide (DEA), oxyHb is converted exclusively to metHb and nitrate, and no detectable amounts of SNO-Hb are formed.

Joshi et al. (34) also studied the reaction of NO· with oxyHb. From their results, the authors of this paper also concluded that SNO-Hb is generated from the reaction of Cys-beta 93 with N2O3. However, Joshi et al. (34) have proposed that, when NO· is added as a bolus, it is possible that substantial amounts of N2O3 are formed from the reaction of NO· with O2. Nevertheless, this conclusion contrasts with their further observation that the SNO-Hb yields were nearly identical when NO· was added as a bolus or with the NO· donor (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazenium 1,2-diolate (NHMA).

Finally, from their experiments with oxyMb and NO· in the presence of GSH, Zhang et al. (35) also concluded that the addition of a bolus of NO· causes mixing artifacts that facilitate the NO·/O2 reaction. Thus, analogously to Joshi et al. (34), they suggest that GSNO is generated from the reaction of the nitrosating species N2O3 with GSH. This conclusion is supported by the observation that, in contrast to the data reported by Joshi et al. (34) but in analogy to those described in Han et al. (33), the slow addition of NO· with the NO·-donor 2-(N,N-diethylamino)diazenolate 2-oxide to an oxyMb/GSH solution did not result in detectable amounts of GSNO.

In the present work, we carried out a systematic study of the influence of the following factors on the percentage of SNO-Hb generated relative to the amount of NO· added to oxy-, deoxy-, and metHb solutions: 1) the volumetric ratio of the protein and the NO· solutions; 2) the rate of addition of the NO· solution to the protein solution; 3) the amount of NO· added; and 4) the concentration of the phosphate buffer (0.1 versus 0.01 M). For comparison, in some of the reactions, we substituted Hb with equimolar amounts of Mb mixed with 0.5 eq of GSH, to mimic the heme/Cys-beta 93 ratio in Hb. To be able to directly compare our results, we chose to keep the protein concentration constant in most of our experiments (final concentration 50 µM) and generally added 1 or 0.1 eq of NO·. Three different techniques were used to add the NO· solution to the protein solution: the addition of a small volume of a saturated (2 mM) NO· solution (Method 1), the addition of an equivalent volume of a diluted NO· solution (Method 2), either as fast as possible or very slowly, within 1-2 min.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Buffer solutions (0.1 or 0.01 M) were prepared from K2HPO4/KH2PO4 (Fluka) with deionized Milli-Q water and always contained 0.1 mM diethylenetriaminepentaacetic acid (DTPA; Sigma). Sodium nitrite, sodium nitrate, sodium dithionite, potassium hexacyanoferrate(III), potassium cyanide, potassium permanganate, sulfanilamide, N-(1-naphthyl)-ethylenediaminedihydrochloride, ammonium sulfamate, and glutathione were obtained from Fluka. 4,4'-Dithiopyridine (4-PDS) was purchased from Aldrich. GSNO was prepared by the method of Hart (37). Briefly, reduced glutathione was allowed to react with sodium nitrite under acidic conditions at 0 °C, followed by the addition of cold acetone. The resulting precipitate was filtered off, washed, and dried under vacuum in the dark. The pink solid of GSNO, which had similar visible absorption maxima and extinction coefficients as reported by Hart (37), was stored at -80 °C in the dark.

Nitrogen Monoxide Solutions-- Nitrogen monoxide was obtained from Linde and passed through a NaOH solution as well as a column of NaOH pellets to remove higher nitrogen oxides before use. A saturated NO· solution was prepared by degassing water for at least 1 h with argon and then saturating it with NO· for at least 1 h. When needed, the obtained stock solution (~2 mM) was diluted with degassed buffer in gas-tight SampleLock Hamilton syringes. The final NO· concentrations were measured with an ANTEK Instruments nitrogen monoxide analyzer, with a chemiluminescence detector.

Myoglobin Solutions-- Horse heart myoglobin was purchased from Sigma. metMb was prepared by oxidizing the protein with a small amount of potassium hexacyanoferrate(III). The solution was then purified over a Sephadex G-25 column by using a 0.1 M phosphate buffer solution (pH 7.0) as the eluant. The concentration of the metMb solutions was determined by measuring the absorbances at 408, 502, and/or 630 nm (epsilon 408 = 188 mM-1 cm-1, epsilon 502 = 10.2 mM-1 cm-1, and epsilon 630 = 3.9 mM-1 cm-1) (38). oxyMb was prepared by reducing metMb with a slight excess of sodium dithionite. The solution was purified chromatographically on a Sephadex G-25 column by using a 0.1 M phosphate buffer solution (pH 7.0) as the eluant. The concentration of the oxyMb solutions was determined by measuring the absorbances at 417, 542, and/or 580 nm (epsilon 417 = 128 mM-1 cm-1, epsilon 542 = 13.9 mM-1 cm-1, and epsilon 580 = 14.4 mM-1 cm-1) (38).

Hemoglobin Solutions-- Purified human oxyHb stock solution (57 mg/ml solution of HbA0 with ~1.1% metHb) was a kind gift from APEX Bioscience, Inc. The obtained solution was frozen in small aliquots (0.5-1 ml) and stored at -80 °C. oxyHb solutions were prepared by diluting the stock solution with buffer, and concentrations (always expressed per heme) were determined by measuring the absorbance at 415, 541, and/or 577 nm (epsilon 415 = 125 mM-1 cm-1, epsilon 541 = 13.8 mM-1 cm-1, and epsilon 577 = 14.6 mM-1 cm-1) (38). metHb solutions were prepared by oxidizing oxyHb with a slight excess of potassium hexacyanoferrate(III). The solution was purified chromatographically on a Sephadex G-25 column by using a 0.1 M phosphate buffer solution (pH 7.0) as the eluant. The concentration of the metHb solutions (always expressed per heme) was determined by measuring the absorbances at 405, 500, and/or 631 nm (epsilon 405 = 179 mM-1 cm-1, epsilon 500 = 10.0 mM-1 cm-1, and epsilon 631 = 4.4 mM-1 cm-1) (38). HbFe(II) (deoxyHb) solutions were prepared by thoroughly degassing oxyHb solutions with argon for at least 1 h without causing foaming of the solution. The concentration of the HbFe(II) solutions was determined by measuring the absorbance at 430 and/or 555 nm (epsilon 430 = 133 mM-1 cm-1 and epsilon 555 = 12.5 mM-1 cm-1) (38). metHbCN solutions were prepared by treating metHb with a slight excess of KCN. The solution was purified chromatographically on a Sephadex G-25 column by using a 0.1 M phosphate buffer solution (pH 7.0) as the eluant. The concentration of the metHbCN solutions (always expressed per heme) was determined by measuring the absorbances at 419 and/or 540 nm (epsilon 419 = 124 mM-1 cm-1 and epsilon 540 = 12.5 mM-1 cm-1) (38). SNO-Hb (36 mg/ml with an S-nitroso content of 850 µM; i.e. about 74% of Cys-beta 93 and metHb content of 10.7%) was a kind gift from APEX Bioscience, Inc.

UV-visible Spectroscopy-- Absorption spectra were collected in 1 cm cells on a UVIKON 820 or on an Analytik Jena Specord 200.

General Procedures for the Reactions of Different Forms of Hemoglobin and Myoglobin with NO·-- All reactions were carried out in phosphate buffer (0.1 or 0.01 M), pH 7.2, at room temperature. For Method 1, 2 ml of a protein solution (mostly ~50 µM in 0.1 M phosphate buffer, pH 7.2, containing 0.1 mM DTPA) were placed in a 5-ml round bottom flask, which was then closed with a rubber septum. If anaerobic conditions were required, the solutions were thoroughly degassed with argon for 45-60 min. Depending on the equivalents of NO· required (mostly 1, 0.5, or 0.1 eq) 5-50 µl of a NO·-saturated solution were rapidly added to the oxyHb solution under constant stirring by using a Hamilton gas-tight syringe. For the experiments under aerobic conditions, particular care was taken to add the NO· solution at the bottom of the flask to avoid any contact of NO· with the oxygen present in the head space of the flask. Alternatively, the reaction was carried out in a sealable cell for anaerobic applications (Hellma). In this case, the required amount of a saturated NO· solution (5-50 µl) was added to 2 ml of a protein solution while vortex-mixing. Method 2 used the same procedure as described for Method 1 with the difference that the volumetric ratio of the protein and the NO· solutions was always 1. In a typical experiment, 2 ml of a 100 µM protein solution (in 0.1 M phosphate buffer, pH 7.2, containing 0.1 mM DTPA) were mixed under constant stirring with 2 ml of a diluted NO· solution (10-100 µM depending on the required equivalents). The diluted NO· solutions were prepared by mixing in a gas-tight SampleLock Hamilton syringe the NO·-saturated solution with the required amounts of degassed buffer. In most cases, each experiment was carried out by adding the NO· solution in two different ways: fast (in one shot as fast as possible) or slow (as slowly as possible, within 1-2 min).

Reaction of oxyHb with NO·-- The reactions were carried out under aerobic conditions according to Method 1 or 2. After the addition of the NO· solution, the reaction mixtures were stirred for 10 min and then analyzed for S-nitrosated Cys-beta 93 and free Cys-beta 93, and, in some cases, a UV-visible spectrum was recorded. A control experiment was carried out to check that no artifactual S-nitrosation was taking place under the acidic conditions of the Saville assay. For this purpose, after treating the oxyHb solution with NO·, 100 µl of a 10 mM N-ethylmaleimide (NEM) solution in H2O were added and allowed to react for 20 min. Finally, the amount of S-nitrosated Cys-beta 93 was quantified.

A series of experiments was carried out in the presence of an excess KCN. For this purpose, a solution containing oxyHb and either 10 or 100 eq of KCN (relative to the oxyHb concentration) was treated with 1 eq of NO· according to Method 1 exactly as described above for the reaction in the absence of KCN.

Reaction of deoxyHb with NO·-- Most of the reactions were carried out in a sealable cell for anaerobic applications. The oxyHb solutions were thoroughly degassed for about 30 min, until the recorded UV-visible spectrum indicated the complete formation of deoxyHb. Alternatively, the reactions were carried out in round bottom flasks under anaerobic conditions according to Method 1 or 2. After the addition of the NO· solution, the reaction mixtures were stirred for 10 min. Then the flasks or the cells were opened, and the solutions were analyzed for S-nitrosated Cys-beta 93 (under aerobic conditions) immediately and/or after 10 min. In a control experiment, the S-nitrosated Cys-beta 93 content was determined under anaerobic conditions by carrying out the analysis in a sealable cell with thoroughly degassed Saville reagents. A series of experiments was carried out by adding an excess KCN before exposing the reaction mixtures to air. For this purpose, to 2 ml of deoxyHb (50 µM in 0.1 M phosphate buffer) we first added 100 µl of a NO·-saturated solution and, immediately after, 100 µl of a 10 mM KCN solution in water. The reaction mixture was stirred for 10 min, exposed to air, and, after 10 more min, analyzed with the Saville assay.

Reaction of Mixtures of oxyHb and deoxyHb (1:1 and 3:1) with NO·-- An oxyHb solution (2 ml, 50 µM in 0.1 M phosphate buffer, pH 7.2, containing 0.1 mM DTPA) was placed in a sealable cell and degassed for ~30 min, until the recorded UV-visible spectrum indicated the formation of deoxyHb. Then 110 or 130 µl of an O2-saturated H2O solution were added until the recorded UV-visible spectrum matched a spectrum calculated for a 1:1 or a 3:1 mixture, respectively. The concentration of the two hemoglobin species present in solution was also controlled by using the equation given in Ref. 39. The solutions were then treated with a concentrated NO· solution according to Method 1. The reaction mixtures were stirred for 10 min. Finally, the cell was opened, and the solution was immediately analyzed for S-nitrosated Cys-beta 93 under aerobic conditions.

Reaction of metHb with NO·-- The reactions were carried out both under aerobic and under anaerobic conditions according to Method 1 or 2. After the addition of the NO· solution, the reaction mixtures were stirred for 30 min, degassed for 30-60 min (only for the anaerobic experiments), and analyzed for S-nitrosated Cys-beta 93 under aerobic conditions.

Reaction of a Mixture of metHb and oxyHb with NO·-- Solutions containing twice the amount of Hb (50 µM oxyHb and 50 µM metHb) were mixed under aerobic conditions with 50 or 5 µM NO· according to either Method 1 or Method 2 (fast and slow). The reaction mixtures were stirred for 30 min and then analyzed for S-nitrosated Cys-beta 93.

Reaction of a Mixture of metMb and oxyHb with NO·-- To 2 ml of a solution containing 50 µM oxyHb and 50 µM metMb, 1 or 0.1 eq of NO· were added from a saturated solution (Method 1) under aerobic conditions. The reaction mixture was stirred for 30 min and then analyzed for S-nitrosated Cys-beta 93.

Reaction of metHbCN with NO·-- The addition of NO· was performed under strictly anaerobic conditions according to Method 1 or 2. After 10 min, the solution was thoroughly degassed for 1 h and analyzed for S-nitrosated Cys-beta 93 under aerobic conditions.

Reaction of metMb with NO· in the Presence of GSH-- metMb was mixed with 0.5 eq of GSH (relative to the metMb concentration), treated with NO·, and analyzed exactly as described above for the reaction of metHb with NO·.

Reaction of oxyMb with NO· in the Presence of GSH-- oxyMb was mixed with 0.5 eq of GSH (relative to the oxyMb concentration), treated with NO·, and analyzed exactly as described above for the reaction of oxyHb with NO·.

Analysis of the S-Nitrosothiol Content with the Saville Assay-- The S-nitrosothiol content was determined by the method of Saville (40). Our NO· solutions always contained variable amounts of nitrite (variable amounts between 0.1 and 1 eq, relative to the NO· concentration). Thus, since the analysis of the S-nitrosothiol content is carried out under acidic conditions, we first eliminated excess nitrite by the addition of ammonium sulfamate (40). For the analysis of the S-nitrosothiol content, two different sets of reagents were prepared. Diluted reagents include solution A (1 mM ammonium sulfamate in 0.5 M HCl), solution B (1% sulfanilamide in 0.5 M HCl), solution C (1% sulfanilamide and 0.2% HgCl2 in 0.5 M HCl), and solution D (0.02% N-(1-naphthyl)-ethylendiaminedihydrochloride in 0.5 M HCl. Concentrated reagents include solution A (10 mM ammonium sulfamate in 0.5 M HCl), solution B (7% sulfanilamide in 0.5 M HCl), solution C (7% sulfanilamide and 1% HgCl2 in 0.5 M HCl), and solution D (0.2% N-(1-naphthyl)-ethylendiaminedihydrochloride in 0.5 M HCl). Analyses with the diluted reagents were carried out as follows. Two protein samples (500 µl) were mixed with an equivalent volume of solution A and allowed to react for 10 min to destroy the nitrite present in the reaction mixture. Solution B (500 µl) was then added to one of the samples, and the same amount of solution C was added to the other. After 5 min, when the formation of the diazonium salt was complete, 500 µl of solution D were added to each of the two samples. After 5 min, when color formation indicative of the azo dye was complete, the absorbance of 540 nm was read spectrophotometrically. Analyses with the concentrated reagents were carried out analogously by mixing 1 ml of two protein solutions with 0.1-0.2 ml of solution A, 0.1 ml of solution B or C, respectively, and 0.1 ml of solution D. The amount of S-nitrosothiol was quantified as the difference in absorbance between the samples containing solution C and solution B. The extinction coefficient was determined by measuring a series of calibration curves by using either the diluted or the concentrated reagents. S-Nitrosoglutathione and SNO-Hb standard solutions gave an average value of 51 mM-1 cm-1 for the extinction coefficient at 540 nm. In general, the diluted reagents were used for samples containing S-nitrosothiol concentrations higher than 0.3 µM, whereas the concentrated reagents were utilized when the amounts of S-nitrosothiols were lower. However, several SNO-Hb concentrations (higher than 0.3 µM) were determined with both the diluted and concentrated reagents, and the results were always in very good agreement.

Analysis of the Sulfhydryl Content-- The amount of free sulfhydryl groups was measured by reaction with 4,4'-dithiodipyridine (PDS) (41). This analytical method allows for the determination of the amount of free Cys-beta 93, the only reactive cysteine residue of hemoglobin. In brief, the protein sample to be analyzed (500 µl) was mixed with an equal volume of 4-PDS (10 mM in 0.1 M phosphate buffer, pH 7.2, containing 0.1 mM DTPA). After incubation for 20 min at room temperature, when the product 4-thiopyridone had completely formed, the absorbance of the sample was measured spectrophotometrically at its maximum (324 nm). The free Cys-beta 93 content was quantified as the difference in absorbance between this solution and that of a protein sample (500 µl) mixed with an equivalent volume of buffer. The extinction coefficient of 4-thiopyridone was determined by measuring a calibration curve of 5-10 glutathione standard solutions (epsilon 324 = 21 mM-1 cm-1) and was comparable with that described in the literature (epsilon 324 = 19.8 mM-1 cm-1 (41)). To measure the total amount of thiols in hemoglobin (six cysteine residues/tetramer), the analysis was carried out with 1% SDS added to the buffer (42).

Statistics-- The experiments reported here were carried out at least in triplicate on independent days. The results are given as mean values of at least three experiments plus or minus the corresponding standard deviation.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Detection Limits of SNO-Hb with the Saville Method-- To validate our methodology used to quantify low amounts of SNO-Hb with the Saville assay, a series of calibration curves were measured to determine the limit of detection of S-nitrosothiols under our experimental conditions. With the concentrated Saville reagents (see "Experimental Procedures" for details), it was possible to measure accurately only standard solutions containing more than 0.5 µM GSNO. This result is in agreement with a recent report of Gladwin et al. (32), who showed that quantification of SNO-Hb and SNO-albumin within the concentration range 0.5-7 µM gave comparable results with the Saville and the I<UP><SUB>3</SUB><SUP>−</SUP></UP> chemiluminescence assays.2 This detection limit is set by the absorbance values obtained with very low S-nitrosothiol concentration, which are lower than 0.1 and, thus, cannot be measured accurately. However, under our experimental conditions, the reaction solutions to be analyzed with the Saville assay always also contained ~50 µM hemoglobin. Thus, at 540 nm, the wavelength corresponding to the absorbance maximum of the azo dye quantified in the assay, our solutions had a basal absorbance of about 0.3-0.4. Consequently, in the presence of 50 µM hemoglobin, GSNO concentrations as low as 0.05 µM could still be measured accurately. Indeed, as shown in Fig. 1, an excellent correlation was found in the range of 0.05-5 µM GSNO by measuring the lower concentrations (0.05-0.1 µM) in the presence and the higher concentrations (2.5-5 µM) in the absence of 50 µM oxyHb.


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Fig. 1.   Validation of the Saville assay for the quantification of very low amounts of S-nitrosothiols in the presence of 50 µM oxyHb. Standard GSNO solutions were analyzed in the absence (2.5-5 µM) or in the presence (0.05-1 µM) of 50 µM oxyHb. Inset, enlargement of the lowest GSNO concentrations, all measured in the presence of 50 µM oxyHb.

Our NO· solutions were usually contaminated with nitrite (variable amounts between 0.1 and 1 eq, relative to the NO· concentration). Thus, before carrying out the Saville assay, we always eliminated excess nitrite by the addition of ammonium sulfamate (40) to avoid a high background absorbance arising from the reaction of nitrite with the Saville reagents and to prevent nitrite-mediated acid-catalyzed formation of S-nitrosothiol compounds. Ammonium sulfamate reduces nitrite to dinitrogen, but it has recently been observed (43) that nitrite cannot be removed completely with this reagent. However, since the Saville assay is based on the measurement of an absorbance difference, incomplete removal of nitrite does not interfere with our measurements. In addition, it has recently been argued that the reaction of ammonium sulfamate with nitrite, which also takes place under acidic conditions, might lead to artifactual formation of S-nitrosated thiols (44). As a control, we thus mixed glutathione (50 µM) with nitrite (10 mM) and then destroyed it by the addition of a 5-fold excess of ammonium sulfamate (equivalent volume of a 50 mM solution in 0.5 M HCl). In our hands, this reaction does not lead to detectable amounts of S-nitrosoglutathione, and analysis of the free sulfhydryl group of glutathione confirmed that no reaction had taken place. Finally, as a further control, we determined the SNO-Hb yield of the reaction between oxyHb and NO· after the addition of ~500 µM N-ethylmaleimide (NEM) prior to the Saville assay. Also in this case, no difference was observed between the samples analyzed with or without prior blockage of the free thiols with N-ethylmaleimide.

Since a large excess of ammonium sulfamate was used to remove the nitrite contaminations, it is likely that part of it was still present during the Saville assay. It has been reported that the reaction of nitrite with sulfanilamide proceeds at a significantly faster rate, and, thus, nitrite liberated from S-nitrosothiols can be analyzed quantitatively also in the presence of ammonium sulfamate (40). Nevertheless, we checked that this observation was true also under our experimental conditions. For this purpose, we prepared S-nitrosoglutathione solutions (~4 µM) containing different amounts of nitrite (0, 1, 10, and 100 µM) and analyzed them with the Saville assay after the addition of the usual excess of ammonium sulfamate. In all cases, the amount of S-nitrosothiol found corresponded to the GSNO content of the original solution.

Reaction of oxyHb with NO·-- It has been proposed that the reaction of oxyhemoglobin with NO· leads to significant nitrosation of its cysteine residue beta 93 (~40% relative to the amount of added NO·) and thus to the formation of considerable amounts of SNO-hemoglobin (15). This hypothesis does not fit with our observation that nitrate is formed quantitatively from the reaction of oxyHb with 1 eq of NO· (45). Thus, we determined the amount of SNO-Hb generated after the addition of 1, 0.5, and 0.1 eq of NO· to an oxyHb solution. The reactions were carried out in a closed vial under aerobic conditions at room temperature in 0.1 M phosphate buffer, pH 7.2, in the presence of 0.1 mM of the metal chelator DTPA. As summarized in Table I, when 1, 0.5, or 0.1 eq of NO· from a saturated (2 mM) solution were added to a 50 µM oxyHb solution (Method 1) and allowed to react for 10 min, as expected, the yield of nitrosated Cys-beta 93 decreased continuously. However, when the SNO-Hb yields are expressed relative to the NO· concentration, it becomes apparent that these relative yields of SNO-Hb increase with decreasing amounts of NO· added. The relative SNO-Hb yields are very low and span from 1.5 to 5% of NO· added (1 or 0.1 eq), respectively. The sum of the concentration of the nitrosated Cys-beta 93 and that of unreacted Cys-beta 93, determined separately by reaction with 4-PDS (41), was always in good agreement with the total amount of Cys-beta 93. In a typical experiment, when 53 µM NO· were allowed to react with 53 µM oxyHb (total Cys-beta 93 concentration 26.5 µM), we found 0.8 µM nitrosated Cys-beta 93 and about 26 µM free Cys-beta 93. Taken together, these data suggest that only the cysteine residue Cys-beta 93 is nitrosated and that the other two less exposed cysteine residues of Hb, Cys-alpha 104 and Cys-beta 112, are not modified.

                              
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Table I
Amount of nitrosated Cys-beta 93 formed from the reaction of oxyHb with different amounts of NO· in 0.1 M and (in parenthesis) in 0.01 M phosphate buffer, pH 7.2: Influence on the concentration of the added NO· solution

To avoid artifacts derived from inhomogeneous mixing of different volumes of two solutions (33-35) with significantly different concentrations (2 ml of a 50 µM oxyHb solution and 5-50 µl of a 2 mM NO· solution), we repeated the same reactions by mixing equivalent volumes of oxyHb and NO· solutions having similar concentrations (2 ml of a 100 µM oxyHb solution and 2 ml of a 10-100 µM NO· solution; Method 2). Surprisingly, as shown in Table I, the yields of nitrosated Cys-beta 93 (relative to the amount of NO· added) were almost identical to those obtained by adding NO· directly from the saturated solution.

Interestingly, higher yields of nitrosated Cys-beta 93 were obtained when the reaction was carried out by adding very slowly (within 1-2 min) a diluted NO· solution to a oxyHb solution at a volume ratio of 1:1. As summarized in Table I, for all three amounts of NO· added (1, 0.5, or 0.1 eq) the yields of SNO-Hb were always larger than those obtained by the fast addition of a diluted NO· solution. The highest yield of SNO-Hb obtained, expressed relative to the amount of NO· added, was 14 ± 1%, for the slow addition of 0.1 eq of NO·.

As a control, to check that the dilution and/or slow addition of the NO· solution did not lead to artifactual SNO-Hb generation due to O2 contamination of the NO· solution, we carried out a series of reactions between GSH and NO· under anaerobic conditions. In the absence of O2, it is known that thiols and NO· do not generate nitrosothiols (46-48). Thus, this reaction can be utilized to confirm that we do not have O2 contamination in the system under our experimental conditions. 1 eq of NO· was added to a thoroughly degassed 50 µM (final concentration) GSH solution either directly from a saturated solution (Method 1) or after dilution at a volumetric ratio of 1:1 fast or slow (Method 2). After 30 min, the reaction mixtures were degassed to remove the unreacted NO· and finally analyzed with the Saville assay under aerobic conditions, in the absence or in the presence of 50 µM oxyHb. As expected, independently of the way the NO· solution was added, no detectable amounts of GSNO were formed. Thus, we can exclude that the differences between the SNO-Hb yields obtained when the NO· solution was added fast or slowly are due to O2 contamination during the addition of the NO· solution.

Since it has recently been argued that the phosphate concentration influences the nature of the reaction between oxyHb and NO· (15), we determined the yields of SNO-Hb generated by the fast and slow addition of a diluted NO· solution to oxyHb in a 0.01 M phosphate buffer at pH 7.2. As summarized in Table I (data in parenthesis), when the reactions were carried out in the diluted buffer, the nitrosation yields were higher than those obtained in 0.1 M phosphate buffer under the same experimental conditions.

The reaction between oxyHb and 1 eq of NO· (in 0.1 M phosphate buffer, pH 7.2), added with the three different methods described above, was also followed by UV-visible spectroscopy. As shown in Fig. 2A, the slow addition of a diluted NO· solution led to the formation of ~90-95% metHb. In contrast, the fast addition of either a saturated or a diluted NO· solution gave a slightly lower metHb yield, about 80%.


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Fig. 2.   UV-visible spectra of the products of the reaction of 1 eq of NO· with oxyHb, deoxyHb, and oxyHb/deoxyHb mixtures. A, the addition of NO· (50 µM, final concentration) to oxyHb (50 µM, final concentration) from a saturated solution (Method 1), and fast or slow from a diluted solution (Method 2). B, the addition of NO· (50 µM, final concentration) to a 3:1 mixture of oxyHb/deoxyHb (50 µM, final protein concentration) from a saturated solution (Method 1). C, the addition of NO· (50 µM, final concentration) to a 1:1 mixture of oxyHb/deoxyHb (50 µM, final protein concentration) from a saturated solution (Method 1). D, a deoxyHb solution (46 µM in 0.1 M phosphate buffer (thick line 1) was treated under anaerobic conditions with 1 eq of NO· (from a saturated solution, Method 1) to generate HbFe(II)NO (thick dotted line 2). The cell was exposed to air, and a spectrum was recorded immediately (spectrum 3). Spectra 4-10 were measured at time intervals of 10 min, for a total of 70 min.

Finally, to find out whether metHb played a role for the generation of SNO-Hb from oxyHb and NO·, we carried out a series of experiments in the presence of CN-. For this purpose, we added 1 eq of NO· from a saturated solution (Method 1) to a solution containing 50 µM oxyHb and 500 µM KCN. Under these conditions, the SNO-Hb yield was 1.1 ± 0.1% (expressed relative to the amount of NO· added), not significantly lower than that observed under the same conditions in the absence of added CN- (1.5 ± 0.1%). The slow addition of NO· from a diluted stock solution (Method 2, slow) to 50 µM oxyHb in the presence of 50 µM KCN led to the formation of 5 ± 1% SNO-Hb, approximately the same amount generated under the same conditions in the absence of added CN- (5.9 ± 0.4%). For both methods, the addition of 10 times more CN- (5 mM) did not lead to a significant change in the relative SNO-Hb yield. Since it has recently been noted that CN- interferes with the Saville assay (32), we mixed GSNO (5 µm) with 0, 1, and 10 eq of KCN and measured the amount of S-nitrosothiols. In our hands, no interference was observed; indeed, the values were within 4% error (5.0 ± 0.2 µM) without showing any clear dependence on the amount of cyanide added.

Reaction of deoxyHb with NO·-- Since it has been proposed that in vivo NO· reacts with the small amount of deoxygenated hemoglobin present in the red blood cells to generate SNO-Hb (15), we also determined the yield of SNO-Hb obtained when deoxyHb was allowed to react with NO· both in 0.1 and 0.01 M phosphate buffer, pH 7.2. The deoxyHb solutions were incubated for 10 min with NO· (added in different ways), oxygenated, and immediately subjected to the Saville assay under aerobic conditions. The results summarized in Table II show that the rapid addition of 1 eq of NO· (from a concentrated solution, Method 1) to a 50 µM deoxyHb solution (in 0.1 M phosphate buffer) yielded approximately the same amount of SNO-Hb as that obtained by the analogous addition of NO· to oxyHb. In contrast, we found about 1.5-2 times higher relative SNO-Hb yields (relative to the amount of NO· added) when we added 0.1 eq of NO·. Almost identical yields were obtained by the fast addition of a diluted NO· solution (Method 2). Interestingly, in contrast to the reaction with oxyHb, no significant increase in the SNO-Hb yields was observed when the diluted NO· solution was added very slowly (Table II). Finally, for the reactions with deoxyHb, no significant differences were detected when all of the reactions were carried out in 0.01 M phosphate buffer (Table II; data in parenthesis). The observation that the method of NO· addition does not influence the SNO-Hb yields suggests that formation of SNO-Hb takes place only after oxygenation of the reaction solutions, which is from the reaction of HbFe(II)NO with O2. Indeed, the rapid addition of 1 eq of NO· to a deoxyHb solution, followed by Saville analysis under strictly anaerobic conditions yielded only ~0.2% SNO-Hb.

                              
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Table II
Amount of nitrosated Cys-beta 93 formed from the reaction of deoxyHb with different amounts of NO· in 0.1 M and (in parenthesis) in 0.01 M phosphate buffer. pH 7.2: Influence on the concentration of the added NO· solution and on the time elapsed before analysis

We thus investigated whether allowing HbFe(II)NO to react with O2 for longer times (10 min or 1 h) had an influence on the amount of SNO-Hb produced. As summarized in Table II (10 min), when we carried out the Saville analysis 10 min after oxygenation of the reaction solutions, we found ~10-20% higher SNO-Hb yields, both in 0.1 and 0.01 M (data in parenthesis) phosphate buffer. However, because of the large errors associated with some of these data (up to 20%), this small increase cannot be considered significant. When 1 h elapsed prior to the Saville assay, no significant further increase in the SNO-Hb yields was observed. In a separate experiment, we determined that no loss of nitrosated Cys-beta 93 in SNO-Hb is observed within 1 h under the conditions of our experiment. Finally, if the solution was allowed to stand overnight, the amount of SNO-Hb slightly decreased, probably because of the instability of the S-nitrosocysteine.

The nitrosyl complex HbFe(II)NO is known to slowly oxidize to metHb (49). To find out the extent of oxidation that takes place under our experimental conditions, we also carried out a parallel UV-visible spectroscopic analysis of the reaction solution. As expected, the addition of 1 eq of NO· to a deoxyHb solution (thick line 1 in Fig. 2D) led to the quantitative formation of HbFe(II)NO (thick dotted line 2 in Fig. 2D). Oxygenation of this solution causes the slow decay of HbFe(II)NO to metHb (spectra 3-10 in Fig. 2D). The solution measured immediately after opening the vial (spectrum 3 in Fig. 2D) contained ~90% HbFe(II)NO and 10% metHb. After 10 min (spectrum 4 in Fig. 2D), the reaction mixture consisted of ~85% HbFe(II)NO and 15% metHb. After 60 min (spectrum 9 in Fig. 2D) ~50% of HbFe(II)NO had been oxidized to metHb.

To find out whether metHb played a role in the reaction that leads to nitrosation of Cys-beta 93 when HbFe(II)NO is oxygenated, we added KCN prior to opening the reaction flask. Thus, we prepared a deoxyHb solution, added 1 eq of NO· from a saturated solution, added 10 eq KCN from a thoroughly degassed solution, and allowed it to react for 10 min. Then the reaction mixture was exposed to air and analyzed immediately with the Saville assay. Interestingly, the relative SNO-Hb yield (expressed relative to the amount of NO· added) was 1.7 ± 0.2%, almost identical to the corresponding relative yield obtained in the absence of CN- (1.5 ± 0.1%).

Reaction of Mixtures of oxy- and deoxyHb (1:1 and 3:1) with NO·-- Since in vivo hemoglobin is present as a mixture of the oxy and deoxy forms, depending on the dioxygen concentration, we also measured the SNO-Hb yields obtained by addition of a NO· solution to mixtures of deoxy- and oxyHb. The percentages of oxygenated molecules were determined by measuring a UV-visible spectrum immediately prior to the addition of the NO· solution and by comparing it to a spectrum calculated from those of pure deoxy- and oxyHb. In addition, the relative concentration of the two hemoglobin species was controlled by using the equations described by Benesh et al. (39). Thus, since it was essential to measure a spectrum of the reaction mixture, the reactions had to be carried out in sealable cells, and consequently it was only possible to add NO· from a concentrated saturated solution (Method 1). The results of the experiments in 0.1 M buffer show that the rapid addition of 1 or 0.1 eq of NO· to a 3:1 mixture of oxy- and deoxyHb (total protein concentration 50 µM) yielded 1.8 ± 0.3 and 5.6 ± 0.8% SNO-Hb (relative to NO·), respectively. In 0.01 M phosphate buffer, the yields were almost identical, 1.9 ± 0.5 and 6.0 ± 0.4% (relative to NO·), respectively. As for the reactions only with deoxyHb, slightly, but not significantly, larger yields were obtained when after the addition of 1 or 0.1 eq of NO· the solutions were allowed to react for 10 min with air prior to the S-nitrosothiol content analysis (2.1 ± 0.2 and 7.0 ± 0.1% in 0.1 M phosphate buffer and 2.3 ± 0.3 and 8.0 ± 0.8% in 0.01 M phosphate buffer, respectively).

Analogous experiments were carried out in 0.1 M phosphate buffer with a 1:1 mixture of oxy- and deoxyHb. The rapid addition of 1 or 0.1 eq of NO· (from a concentrated solution, Method 1) to a 1:1 mixture of oxy- and deoxyHb (total protein concentration 50 µM) yielded 1.5 ± 0.2 and 10.0 ± 0.6% SNO-Hb (relative to NO·), respectively. Only slightly larger yields (2.0 ± 0.3 and 12.4 ± 0.7% SNO-Hb, respectively) were obtained when the solutions were allowed to react for 10 min with air prior to the S-nitrosothiol content analysis.

The reactions between the 3:1 or the 1:1 oxyHb/deoxyHb mixtures and 1 eq of NO· were followed by UV-visible spectroscopy. As shown in Fig. 2B and 2C, the products of these reactions corresponded approximately to a 3:1 and a 1:1 mixture of metHb/HbFe(III)NO, respectively.

Reaction of metHb with NO·-- In contrast to O2 and CO, NO· binds not only to deoxyHb but also to the oxidized iron(III) form of hemoglobin. Fourier transform infrared spectroscopy and resonance Raman data indicate that the resulting nitrosyl complex, HbFe(III)NO, formally has a linear HbFe(II)(NO+) character, which means that partial charge transfer from NO· to the iron(III) occurs during the bonding process (50-52). HbFe(III)NO is not very stable, and in the presence of an excess of NO· it undergoes reductive nitrosylation to finally generate the reduced hemoglobin nitrosyl complex, HbFe(II)NO (Equation 1). This reaction has recently been shown to take place also within the red blood cells (33). In the presence of substrates such as thiols, amines, and phenols, it has been reported that the corresponding myoglobin complex MbFe(III)NO can act as a nitrosating species (53).


<UP>HbFe</UP>(<UP>III</UP>)<UP> + NO<SUP>⋅</SUP> ⇄ HbFe</UP>(<UP>III</UP>)<UP>NO ↔ HbFe</UP>(<UP>II</UP>)(<UP>NO<SUP>+</SUP></UP>) (Eq. 1)

 <LIM><OP><ARROW>→</ARROW></OP><UL><UP>NO<SUP>⋅</SUP></UP></UL></LIM><UP> HbFe</UP>(<UP>II</UP>)<UP>NO + NO<SUB>2</SUB><SUP>−</SUP></UP>
To find out whether HbFe(III)NO also acts as a nitrosating agent, we mixed NO· with metHb both under anaerobic and aerobic conditions and determined the amount of SNO-Hb generated. The addition of 1 or 0.1 eq of NO· to 50 µM metHb (in 0.1 M phosphate buffer) under argon led to the formation of SNO-Hb yields significantly larger than those obtained under the same conditions with oxy- and deoxyHb (Table III). No significant difference was observed when NO· was added fast either from a saturated (Method 1) of from a diluted solution (Method 2, fast). Interestingly, in analogy to the reactions with oxy- and deoxyHb, the addition of 0.1 eq of NO· to metHb led to significantly larger relative SNO-Hb yields (expressed relative to the amount of NO· added) than those generated by the addition of 1 eq of NO·. In addition, similarly to the reactions with oxyHb, significantly larger amounts of SNO-Hb (approximately 2 times larger) were generated when a diluted NO· solution was added slowly (Method 2, slow). Surprisingly, when 1 eq of NO· was added to metHb under aerobic conditions, only slightly larger SNO-Hb amounts were generated (relative to the amount formed under anaerobic conditions), independently of the method of NO· addition (Table III, data in parenthesis). In contrast, the addition of 0.1 eq of NO· under aerobic conditions generated approximately twice the amount of SNO-Hb formed under anaerobic conditions, independently of the method of NO· addition (Table III, data in parenthesis). Thus, the highest relative SNO-Hb yields (expressed relative to the amount of NO· added) were obtained when 0.1 eq of a diluted NO· solution were added slowly to the metHb solution (23 ± 3 and 42 ± 6% under anaerobic and aerobic conditions, respectively).

                              
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Table III
Amount of nitrosated Cys-beta 93 formed from the reaction of metHb with different amounts of NO· in 0.1 M phosphate buffer, pH 7.2, under anaerobic and (in parenthesis) under aerobic conditions: Influence on the concentration of the added NO· solution

Reaction of oxyHb with NO· in the Presence of Added metHb or metMb-- In a further set of experiments, we carried out the reaction of oxyHb with NO· under aerobic conditions in the presence of 1 eq metHb (relative to the oxyHb concentration). In all cases, that is when 1 or 0.1 eq of NO· were added according to Method 1 or Method 2 (fast and slow) the SNO-Hb yields (expressed relative to the amount of NO· added) were higher than those obtained from the corresponding experiments in the absence of metHb (Table IV). The difference between the yield obtained in the presence and in the absence of added metHb was more significant in the experiment with 0.1 eq of NO·. In analogy to the reaction between oxyHb and NO·, the SNO-Hb yields did not depend on the concentration of the added NO· solution when it was added fast (Method 1 and Method 2 fast). In contrast, when the diluted NO· solution was added slowly, the SNO-Hb yields were significantly larger.

                              
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Table IV
Amount of nitrosated Cys-beta 93 formed from the reaction of a mixture of oxyHb and metHb (50 µM each) with different amounts of NO· in 0.1 M phosphate buffer, pH 7.2, under aerobic conditions: Influence on the concentration of the added NO· solution

A similar reaction was carried out between oxyHb and NO· in the presence of 1 eq of metMb (relative to the oxyHb concentration). Interestingly, when NO· was added from a saturated solution (Method 1) we also obtained a significant increase in the relative SNO-Hb yields, compared with that obtained in the absence of added metMb. Indeed, the addition of 1 or 0.1 eq of NO· (relative to the oxyHb concentration) to a mixture of 50 µM oxyHb and 50 µM metMb under aerobic conditions led to the formation of 2.6 ± 0.1 and 10 ± 2% SNO-Hb (expressed relative to the amount of NO· added).

Reaction of metHb with NO· in the Presence of Added oxyMb-- Finally, we carried out the "inverse" reaction and added under aerobic conditions 1 or 0.1 eq (relative to the metHb concentration) of NO· from a saturated solution (Method 1) to a solution of 50 µM metHb and 50 µM oxyMb. In this case, we obtained 5 ± 1 and 15 ± 3% SNO-Hb (relative to the amount of NO· added) for the addition of 1 or 0.1 eq of NO·, respectively. Interestingly, these relative yields are only 30-40% lower than those obtained under the same (aerobic) conditions in the absence of added oxyMb.

Reaction of oxyMb and metMb with NO· in the Presence of GSH-- To determine the efficiency of oxyMb and metMb (and possibly oxyHb and metHb) to nitrosate an external thiol, we carried out a set of experiments analogous to those described above between NO· and oxyHb or metHb, with the only difference being that Hb was replaced by an equimolar amount of Mb plus 0.5 eq of GSH. This ratio was chosen to mirror the Cys-beta 93/heme ratio in hemoglobin. As summarized in Table V, when oxyHb was replaced by oxyMb/GSH, we observed the same trends and similar GSNO yields (relative to the amount of added NO·) as those discussed above for the reaction of NO· with oxyHb. In particular, higher yields were obtained when NO· was added slowly from a diluted solution, and higher relative GSNO (expressed relative to the amount of added NO·) was obtained from the reaction with 0.1 eq of NO·. Thus, the highest relative GSNO yield, 16 ± 6% expressed relative to the amount of NO· added, was obtained when 0.1 eq of a diluted NO· solution were added slowly to the oxyMb/GSH solution. When metHb was replaced by metMb/GSH, again the same trends in the GSNO yields were observed, depending on the way the NO· solution was added. However, most GSNO yields were ~2-3 times lower than the corresponding SNO-Hb yields (Table V, data in parenthesis). Thus, the highest relative GSNO yield, 13 ± 4% expressed relative to the amount of NO· added, was obtained when 0.1 eq of a diluted NO· solution were added slowly to the metMb/GSH solution.

                              
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Table V
Amount of GSNO formed from the reaction of oxyMb (50 µM) or (in parenthesis) from the reaction of metMb (50 µM) with different amounts of NO· in the presence of GSH (25 µM) in 0.1 M phosphate buffer pH 7.2: The experiments with oxyMb were carried out under aerobic conditions and those with metMb under anaerobic conditions

Reaction of metHbCN with NO·-- It has recently been proposed that beside interacting with the heme and with Cys-beta 93, NO· can occupy hydrophobic sites within hemoglobin (8). To test whether noncovalent bonding of NO· played a role in our reactions, we prepared a thoroughly degassed metHbCN solution, added either 1 or 0.1 eq of NO· from a saturated solution (Method 1), and allowed it to interact for 10 min. No changes were observed in the UV-visible spectrum of metHbCN, suggesting that, as expected, NO· cannot displace the strong cyanide ligand from metHb (data not shown). Then we thoroughly degassed the reaction mixture, opened the reaction vial, and finally immediately carried out the Saville assay under aerobic conditions. Interestingly, we found 0.9 ± 0.2% and 4.0 ± 0.3% SNO-Hb (expressed relative to the amount of NO· added) for the experiments with 1 and 0.1 eq of NO·, respectively. Waiting 10 min or 1 h after opening the reaction vials prior the Saville assay did not lead to a significant increase in the measured relative SNO-Hb yields (~1.1% after 1 h). Moreover, when 1 h elapsed after the addition of NO·, prior to degassing and immediate analysis of the reaction mixture, no significant change was detected in the SNO-Hb yields (~1% relative to the amount of NO· added). The slow addition of 1 eq of NO· from a diluted solution (Method 2, slow) under the same conditions also led to approximately the same SNO-Hb yield (1% relative to the amount of NO· added). Finally, when 50 eq of NO· were added from a saturated solution (Method 1), we obtained 1.2 and 1.3% SNO-Hb (relative to the amount of NO· added) after 10 min or 1 h, respectively. Taken together, our results suggest that in all of these experiments it is conceivable that NO· is trapped in a hydrophobic pocket of the protein and is not displaced by degassing. Interestingly, Cys-beta 93 is surrounded by hydrophobic residues. Thus, after opening the reaction vials, O2 could also rapidly diffuse into this pocket, react with NO·, and lead to the formation of SNO-Hb.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reactions of NO· with metHb or metMb/GSH-- Among the three Hb forms studied, metHb is the species most likely to be capable of nitrosating a thiol by interaction with NO·. It has been shown that metHb binds NO· and generates HbFe(III)NO, a nitrosyl complex that has formally HbFe(II)(NO+) character (50-52), and could thus act as a nitrosating agent. The corresponding myoglobin complex MbFe(II)(NO+) has been shown to undergo NO+ transfer and, among others, react with thiols to generate S-nitrosothiols (53, 54). Here we show that reaction of metHb with NO· leads to nitrosation of Cys-beta 93. Our results indicate that the percentage of SNO-Hb generated relative to the amount of NO· mixed with metHb depends on how the NO· solution is added and is higher when substoichiometric amounts of NO· are added. Maximal relative yields of SNO-Hb are obtained when 0.1 eq of NO· are added very slowly to metHb from a diluted solution (Method 2, slow). The higher relative SNO-Hb yields obtained when 0.1 eq of NO· were mixed with metHb, compared with the amount of SNO-Hb generated by the addition of 1 eq of NO·, can be rationalized as follows. The association rates of NO· to the beta  and alpha  subunits of metHb are 6.4 × 103 M-1 s-1 and 1.71 × 103 M-1 s-1, respectively (55). The dissociation rates of NO· from HbFe(III)NO are 1.5 s-1 and 0.65 s-1, for the beta  and the alpha  subunits of Hb, respectively (55). Thus, under the conditions of our experiments, all of these reactions have a half-life on the order of seconds, and the system reaches equilibrium within a few seconds. The equilibrium constants, calculated from the given association and dissociation rates, indicate that the affinity for NO· of the beta  subunit is ~1.6 times larger than that of the alpha  subunit. In addition, the percentage of the associated species HbFe(III)NO generated by mixing 50 µM metHb with 50 or 5 µM NO·, expressed relative to the total amount of NO· added, is 13 and 16%, respectively. Thus, when smaller amounts of NO· are added to metHb, a larger fraction of NO· is present as HbFe(II)(NO+) and may consequently lead to higher relative SNO-Hb yields. However, since the difference in the relative SNO-Hb yields obtained from the reaction with 50 or 5 µM NO· is about 10%, a value significantly larger than the difference between the HbFe(II)(NO+) concentrations (3%), this mechanism may represent only one of the pathways responsible for the higher SNO-Hb yields.

The higher SNO-Hb yields obtained when the diluted NO· solution is added slowly to the metHb solution, compared with the amount of SNO-Hb generated by rapid addition, is difficult to explain. The argumentation outlined above suggests that when NO· is added slowly as a diluted solution, at the beginning there will be a large excess of protein versus NO· and thus a large relative amount of NO· bound to the protein. The fraction of NO· present as HbFe(II)(NO+) will decrease continuously by further addition of the NO· solution. However, since the time needed to add the NO· solution is only about 1-2 min but the half-life of the hydrolysis of HbFe(II)(NO+) is about 10 min (1.1 × 10-3 s-1) (56), this reasoning cannot explain the higher yields obtained when NO· solutions are added slowly.

Interestingly, when NO· is added to metHb under aerobic conditions the relative SNO-Hb yields are significantly higher only in the experiments with 0.1 eq of NO·, whereas they are approximately unchanged in those with 1 eq of NO·. The reaction of NO· with dioxygen in aqueous solution is believed to proceed according to the following mechanism (Equations 2-4) (57).
<UP>2NO<SUP>⋅</SUP> + O<SUB>2</SUB> → 2NO<SUP>⋅</SUP><SUB>2</SUB></UP> (Eq. 2)

<UP>NO<SUP>⋅</SUP><SUB>2</SUB></UP>+<UP>NO<SUP>⋅</SUP></UP>→<UP>N<SUB>2</SUB>O<SUB>3</SUB></UP> (Eq. 3)

<UP>N<SUB>2</SUB>O<SUB>3</SUB></UP>+<UP>H<SUB>2</SUB>O</UP>→<UP>2H</UP><SUP>+</SUP>+<UP>2NO</UP><SUP><UP>−</UP></SUP><SUB><UP>2</UP></SUB> (Eq. 4)

<UP>4NO<SUP>⋅</SUP></UP>+<UP>O<SUB>2</SUB></UP>+<UP>2H<SUB>2</SUB>O</UP>→<UP>4H<SUP>+</SUP></UP>+<UP>4NO</UP><SUP><UP>−</UP></SUP><SUB><UP>2</UP></SUB> (Eq. 5)
The overall stoichiometry of the reaction is given in Equation 5, and the rate of NO· consumption is determined by the rate law 4k[NO·][O2], with k = 2 × 106 M-2 s-1 at 25 °C (57). The calculated half-lives for aqueous 50 and 5 µM NO· solutions under aerobic conditions are ~10 and 100 s, respectively. Thus, when 1 eq of NO· (50 µM) is added to metHb under aerobic conditions, it will rapidly be consumed by its reaction with O2. However, since it takes significantly longer for 0.1 eq of NO· (5 µM) to autoxidize, in particular when added slowly, a larger amount of NO· will bind to metHb and possibly generate SNO-Hb before all of the NO· will be consumed. Nevertheless, under aerobic conditions, NO· has been shown to nitrosate thiols also in the absence of hemoglobin. The mechanism of nitrosation of thiols by oxygenated NO· solutions has been studied by different groups (36, 47, 48). Goldstein et al. (47) showed that under limiting concentrations of NO· ([RSH]0 >=  [NO·]0/2) NO·2 derived from Equation 2 is the only species responsible for nitrosation via Equations 6 and 7. In our reactions between metHb and 0.1 eq of NO·, the conditions of limiting NO· are satisfied, and this alternative nitrosating pathway may explain the higher relative SNO-Hb obtained under aerobic conditions. Nevertheless, since the thiol group is within the protein and not free in solution as in the experiments of Goldstein et al. (47), the mechanism of the reaction may still be different.
<UP>NO<SUB>2</SUB><SUP>⋅</SUP></UP>+<UP>RS<SUP>−</SUP></UP>→<UP>NO</UP><SUP><UP>−</UP></SUP><SUB><UP>2</UP></SUB>+<UP>RS<SUP>⋅</SUP></UP> (Eq. 6)

<UP>NO<SUP>⋅</SUP></UP>+<UP>RS<SUP>⋅</SUP></UP>→<UP>RSNO</UP> (Eq. 7)

<UP>N<SUB>2</SUB>O<SUB>3</SUB></UP>+<UP>RS<SUP>−</SUP></UP>→<UP>RSNO</UP>+<UP>NO</UP><SUP><UP>−</UP></SUP><SUB><UP>2</UP></SUB> (Eq. 8)
At higher NO· concentrations, N2O3, generated from Equation 3, will directly nitrosate the thiols according to Equation 8. Parallel hydrolysis of N2O3 (Equation 4), catalyzed by inorganic phosphates (47), will limit the S-nitrosothiol yields. The rate constant for the S-nitrosation of GSH by N2O3 (Equation 8) has been determined as 6.6 × 107 M-1 s-1 (at pH 7.4 and 23 °C) (48). Thus, in the presence of 50 µM GSH, the observed rate constant of the reaction is 3.3 × 103 s-1. As a comparison, in the presence of 0.1 or 0.01 M phosphate buffer, the observed rate constant for the hydrolysis of N2O3 (Equation 4) is 6.4 × 104 or 6.4 × 103 s-1, respectively. Taken together, these rate constants suggest that when metHb and NO· are allowed to react under aerobic conditions, N2O3 generated from the concurrent reaction of NO· with O2 may also contribute to SNO-Hb formation. In particular, in the experiments with 50 µM NO· (1 eq), large amounts of NO· will be consumed by its reaction with O2, and SNO-Hb may mainly be generated by reaction of Cys-beta 93 with N2O3. However, Kharotonov et al. (36) have shown that the rate of the reaction of N2O3 with proteins is 1-2 orders of magnitude lower than that of the reaction with low molecular weight thiols such as cysteine of glutathione. In this case, the hydrolysis of N2O3 would represent the major reaction pathway. With the data obtained up to now it is not possible to distinguish between these two nitrosation pathways, since, paradoxically, Nedospasov et al. (58) have recently reported that cysteine residues of bovine serum albumin are nitrosated more rapidly than GSH and Cys. In conclusion, to obtain a better understanding of the mechanism of nitrosation of Cys-beta 93 by NO· and metHb under aerobic and anaerobic conditions, the nitrosation rate of Cys-beta 93 by N2O3 should be determined.

The lower yields of GSNO obtained from the comparable reaction between metMb and NO· in the presence of GSH can be rationalized in two ways. First, MbFe(II)(NO+) has been shown to be more stable toward hydrolysis than HbFe(II)(NO+) (56), and thus, since the reaction times of the experiments with HbFe(II)(NO+) and MbFe(II)(NO+) were identical, the slower reaction rate may explain the lower relative GSNO yields. Alternatively, hemoglobin may possess a facilitated pathway for intramolecular S-nitrosation of Cys-beta 93. Indeed, the x-ray of SNO-Hb (27) shows that the nitrosated Cys-beta 93 is located on the proximal side of the heme, at a distance of only ~10 Å from the iron center. Interestingly, one of the hydrophobic xenon-binding sites in myoglobin is located in the proximal cavity very close to where Cys-beta 93 is found in hemoglobin (59). Recent crystallographic analyses of reaction intermediates of photolyzed MbFe(II)CO showed that one of the docking sites occupied by the photodissociated CO is the above mentioned xenon-binding site on the proximal site of the heme (60, 61). Taken together, these data suggest that also in hemoglobin there may be a pathway for the transfer of a ligand from the distal to the proximal side. Thus, HbFe(II)(NO+) may react with H2O and generate H2NO2+ or HNO2, which may rapidly react with Cys-beta 93 prior to dissociation to nitrite and H+, a pathway possibly facilitated by the hydrophobic environment of Cys-beta 93.

Reactions of NO· with deoxyHb-- It has previously been shown that upon oxygenation of partially nitrosylated hemoglobin, the percentage of SNO-Hb generated relative to the amount of NO· mixed with deoxyHb increases with a decreasing amount of NO· added (26). At a NO·/Hb ratio of 0.1, about 20% of the added NO· was found bound to Cys-beta 93 (26). Under very similar conditions, we found only ~10% SNO-Hb. To explain this discrepancy and to get a better understanding of the mechanism of this reaction, we determined the relative SNO-Hb yields under different mixing conditions and at different reaction times. First, we confirmed that nitrosation of Cys-beta 93 takes place only after the addition of O2. Indeed, the addition of 1 eq of NO· to deoxyHb, followed by Saville analysis under strictly anaerobic conditions, leads to the detection of only traces of SNO-Hb. These small amounts of SNO-Hb are probably generated because of O2 contamination during the numerous steps of the Saville analysis. Moreover, the SNO-Hb yields obtained after oxygenation of the reaction mixtures containing fully or partially nitrosylated Hb did not depend significantly on the procedure used to add the NO· solution (Method 1 or 2, fast or slow). This result suggests that SNO-Hb formation does not take place during the addition of the NO· solution.

As for the reactions with metHb, when 0.1 eq of NO· were added to the deoxyHb solution and then exposed to O2, the relative SNO-Hb yields were significantly larger than those obtained by the addition of 1 eq of NO·. MbFe(II)NO has been shown to react with O2 at a very slow rate (5.1 × 10-4 s-1 at pH 5.8 and 30 °C (62)). The mechanism of this reaction has been proposed to proceed in two steps. An initial electrophilic attack of O2 on the nitrogen atom of the nitrosyl to form a peroxynitrite species coordinated to the iron of metMb via the nitrogen atom, is followed by dissociation of peroxynitrite and/or rearrangement to nitrate (62). Alternatively, it has recently been suggested that the reaction may rather proceed first by dissociation of NO·, followed by rapid binding of O2 to deoxyMb and subsequent NO·-mediated oxidation to metMb and nitrate (63). The corresponding oxidation of HbFe(II)NO has not been studied yet, but it is conceivable that it proceeds via a similar mechanism. The addition of 1 or 0.1 eq of NO· to deoxyHb solutions, followed by oxygenation of the reaction mixtures, yields Hb(Fe(II)NO)4 or a mixture of Hb(FeO2)4 and Hb(FeO2)3(Fe(II)NO), respectively. Thus, the difference in the relative SNO-Hb yields obtained by the addition of 1 or 0.1 eq of NO· may reflect the different mechanism and/or rate of oxidation of these two species. A possible pathway for SNO-Hb formation consists of NO· dissociation from Hb(Fe(II)NO)4 or Hb(FeO2)3(Fe(II)NO) and subsequent reaction with O2, followed by nitrosation of Cys-beta 93 by NO·2 or N2O3. The experiments carried out in the presence of an excess cyanide suggest that metHb is not involved in the mechanism of SNO-Hb formation. In addition, since the rate of hydrolysis of N2O3 is dependent on the phosphate concentration, the observation that the SNO-Hb yields generated from the oxygenation of fully or partially nitrosylated deoxyHb solutions do not depend on the phosphate buffer concentration may suggest that if N2O3 is generated, it may largely remain within the protein.

Reactions of NO· with oxyHb or oxyMb/GSH-- The reaction of oxyHb with NO· has been proposed to lead to a significant generation of SNO-Hb (15). In particular, Gow et al. (15) showed that reaction of 48 µM oxyHb with 1.2 µM NO· leads to the generation of 400 nM SNO-Hb (i.e. 40% relative to the amount of NO· added). Under very similar experimental conditions, we found lower SNO-Hb yields, which varied significantly depending on the way the NO· solution is added to the oxyHb solution. In particular, the highest yields (up to 14%, relative to the amount of NO· added) were obtained by adding the NO· very slowly from a diluted solution. In agreement with Han et al. (33), we have shown that under our experimental conditions metHb is not involved in the generation of SNO-Hb, since the addition of cyanide leaves the relative SNO-Hb yields unchanged, independently of the procedure used to add the NO· solution. Thus, SNO-Hb is likely to be generated by reaction of the Cys-beta 93 with N2O3, generated from the reaction of NO· and O2. As proposed by Joshi et al. (34), it is possible that when NO· is added as a bolus of a concentrated solution, a high local NO· concentration favors the formation of large amounts of N2O3. The fast addition of a more diluted NO· solution may still generate high local NO· concentrations and thus lead to similar SNO-Hb yields. However, the low SNO-Hb yields suggest that most NO· generates metHb and nitrate. The observation that the SNO-Hb yields obtained from the reaction of oxyHb with NO· in 0.01 M phosphate buffer are higher than those obtained when the same reaction is carried out in 0.1 M phosphate buffer supports the hypothesis that N2O3 is responsible for the nitrosation of Cys-beta 93. Indeed, we have previously shown that the second order rate constant (per heme) for the reaction of oxyHb and NO· is nearly identical when the reaction is carried out in 0.1 or 0.001 M phosphate buffer, pH 7.0 (89 ± 3 × 106 and 87 ± 2 × 106 M-1 s-1, respectively) (45). However, the rate of hydrolysis of N2O3 to nitrite is accelerated by the presence of phosphate (47). Thus, it is conceivable that in the experiments described by Gow et al. (15), the higher SNO-Hb obtained, in particular when the reaction was carried out in 0.01 M phosphate buffer, is due to larger amounts of N2O3 generated under their experimental conditions.

When NO· is added very slowly from a diluted solution, the conditions of limiting NO· may favor the generation of SNO-Hb from the reaction of Cys-beta 93 with NO·2 via Equations 6 and 7. Since the relative SNO-Hb yields are higher when lower amounts of NO· are added, our results suggest that the nitrosation pathway via NO·2 is more efficient than that via N2O3. Indeed, when only 0.1 eq of NO· are added as a bolus of a concentrated solution, nitrosation of Cys-beta 93 may also take place via reaction with NO·2 and thus explain the higher relative SNO-Hb yields. Interestingly, our experiments with mixtures of oxy- and deoxyHb showed that similar SNO-Hb yields are obtained also when Hb is present as a 3:1 mixture of oxy- and deoxyHb.

Reactions of oxyHb with NO· in the Presence of Added metHb and metMb-- The observation that the SNO-Hb yields are higher when oxyHb is mixed with NO· in the presence of metHb is difficult to rationalize based on kinetic arguments. In fact, our data suggest that, despite the fact that the rate constant for NO· binding to metHb is about 4 orders of magnitude lower than that for the NO·-mediated oxidation of oxyHb (kbeta  = 6.4 × 103 M-1 s-1 and kalpha  = 1.71 × 103 M-1 s-1 (55) and 8.9 × 107 M-1 s-1 (45), respectively), some NO· reacts with metHb even in the presence of equimolar amounts of oxyHb. In addition, the higher SNO-Hb yields measured when oxyHb is mixed with NO· in the presence of metMb suggest that the reaction between NO· and metMb can lead to the nitrosation of Cys-beta 93 of Hb. This observation is again surprising, since the rate constant for the reaction between metMb and NO· is 2 orders of magnitude lower than that for the NO·-mediated oxidation of oxyHb (1.9 × 105 M-1 s-1 (64, 65) and 8.9 × 107 M-1 s-1 (45), respectively). Taken together, our data indicate that some NO· reacts with metMb even in the presence of oxyHb and generates SNO-Hb. Finally, reactions of metHb and NO· in the presence of oxyMb suggest that despite the fact that from the values of the second order rate constants NO· would be expected to be rapidly scavenged by oxyMb, NO· still reacts with metHb to generate SNO-Hb.

Conclusions-- We have previously shown that the reactions of NO· with oxyHb and oxyMb proceed via peroxynitrito-metHb and -metMb intermediates (45, 66). In addition, we have demonstrated that the iron(III) forms of the proteins are the major final protein forms generated and that only traces of 3-nitrotyrosine (less than 0.1%) are also formed. The reaction of an excess of peroxynitrite with thiols is known to yield only trace amounts of S-nitrosothiols (typically 1-2%) (67), probably from a side reaction rather than from a direct nitrosation (69). Thus, it is very unlikely that the peroxynitrito-metHb or -metMb complexes are the species responsible for the nitrosation of Cys-beta 93.

The data discussed in this work further support the hypothesis (33-35) that, because of the very large rate constant of the reaction between oxyHb and NO·, the yield of SNO-Hb strongly depends on the procedure used to add the NO· solution to the oxyHb solution. The low yields of S-nitrosated Cys-beta 93 observed in our experiments are in contrast to the much larger amounts found by Stamler and co-workers (15, 70) under analogous conditions. This discrepancy may be due to different procedures used to carry out the reactions. Thus, care should be taken to use the results obtained from in vitro experiments to draw conclusions on the mechanism of the reaction of oxyHb and NO· in vivo.

Possible pathways for SNO-Hb generation under our experimental conditions are reactions of Cys-beta 93 (a) with N2O3 generated from the reaction of NO· with O2; (b) with NO·2, present in larger concentrations under conditions of limiting NO·, followed by reaction with NO·; or (c) with HbFe(II)(NO+) or MbFe(II)(NO+), generated from the reaction of NO· with metHb or metMb. In addition, it has recently been shown that NO· may also occupy noncovalent binding sites in hemoglobin (8). In analogy to a synthetic model in which NO· is bound tightly and reversibly between two cofacial aromatic groups (71, 72), it is conceivable that NO· may be trapped in hemoglobin by aromatic amino acid residues (8). Interestingly, two aromatic amino acids, Tyr-beta 145 and Phe-beta 103, are located at a distance of 4-5 Å from the sulfur atom of Cys-beta 93 and could thus facilitate noncovalent NO· binding. Moreover, it has been shown that myoglobin has four sites within the globin in which hydrophobic xenon molecules can bind (59). These cavities have been proposed to represent important components of the diffusion pathways of the ligands from the solution to the heme and vice versa (73). Interestingly, the so-called xenon-binding site 1 is located on the side of the proximal histidine very close to the position of Cys-beta 93 in the corresponding hemoglobin structure. Our experiments with metHbCN support the hypothesis that noncovalent binding of NO· followed by reaction with O2 may represent an additional pathway for SNO-Hb formation. However, because of the experimental difficulties associated with the study of these reaction, with the data obtained up to now it is not possible to draw any conclusions on the mechanism of these reaction in vivo.

    ACKNOWLEDGEMENT

We thank APEX Bioscience, Inc. for the supply of purified human hemoglobin and SNO-Hb.

    FOOTNOTES

* This work was supported by the Swiss National Science Foundation and ETH Zürich.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Laboratorium für Anorganische Chemie, ETH Hönggerberg, HCI H 215, CH-8093 Zürich, Switzerland. Fax: 411-632-10-90; E-mail: herold@inorg.chem.ethz.ch.

Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M210275200

2 The I<UP><SUB>3</SUB><SUP>−</SUP></UP> chemiluminescence assay is based on the I<UP><SUB>3</SUB><SUP>−</SUP></UP>-mediated reduction of S-nitrosothiols under acidic conditions and the subsequent quantification of NO· with a NO· analyzer with chemiluminescence detection.

    ABBREVIATIONS

The abbreviations used are: oxyHb, oxyhemoglobin; deoxyHb, deoxyhemoglobin; metHb, iron(III)hemoglobin; Hb, hemoglobin; SNO-Hb, hemoglobin with the cysteine residue beta 93 nitrosated; HbFe(II)NO, iron(II)-nitrosyl complex; GSNO, S-nitrosoglutathione; 4-PDS, 4,4'-dithiopyridine; DTPA, diethylenetriaminepentaacetic acid; Mb, myoglobin; metMb, iron(III)myoglobin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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