Resonance Raman in Vitro Detection and Differentiation of the Nitrite

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Resonance Raman in vitro Detection and Differentiation of the Nitriteinduced Hemoglobin Adducts in Functional Human Red Blood Cells Katarzyna M Marzec, Jakub Dybas, Stefan Chlopicki, and Malgorzata Baranska J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b08359 • Publication Date (Web): 11 Nov 2016 Downloaded from http://pubs.acs.org on November 16, 2016

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The Journal of Physical Chemistry

Resonance

Raman

in

vitro

Detection

and

Differentiation of the Nitrite-induced Hemoglobin Adducts in Functional Human Red Blood Cells Katarzyna M. Marzec,1* Jakub Dybas,1,2 Stefan Chlopicki,1,3 Malgorzata Baranska1,2

1

Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University,

Bobrzynskiego 14, Krakow, Poland 2

Faculty of Chemistry, Jagiellonian University, Ingardena 3, Krakow, Poland

3

Department of Experimental Pharmacology, Jagiellonian University Medical College,

Grzegorzecka 16, Krakow, Poland

*Corresponding author: [email protected] (+48 12 6645476); address: Jagiellonian Centre for Experimental Therapeutics (JCET), Bobrzynskiego 14, 30-348 Krakow, Poland

ACS Paragon Plus Environment

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ABSTRACT: This work presents in vitro studies of the content of functional, isolated human red blood cells (RBCs) after treatment with various concentrations of Na14NO2 and Na15NO2 with the use of resonance Raman spectroscopy (RRS) at two different laser excitations supported by absorption spectrophotometry (UV–vis). The products of the reaction between oxyhemoglobin (oxyHb) in isolated RBCs with NaNO2 were analyzed and identified in situ. The metHb–H2O was found as the major product of such reaction; however, additional adducts were also clearly observed. Vibrational analysis allowed identification of the various Hb3+NO2 species: the Fe3+–O–N=O with O-binding mode of nitrite ion to Fe3+ core as well as nitrovinyl adducts with 2-vinyl nitration favored over 4-vinyl nitration. In addition, we were able to visualize in situ the Hb–NO2 species inside functional RBCs with the use of Raman imaging. At lower NaNO2 concentration (below 2 mM) the presence of Fe3+–NO adduct besides metHb–H2O was found as the most plausible.


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1. INTRODUCTION It is known that in anaerobic and acidic conditions, deoxyHb interacts with nitrite ions to produce NO and metHb.1 As a consequence some of the produced NO may interact with deoxyHb and form ferrous–nitrosyl–Hb (Fe2+–NO). In this work, we studied the interaction of RBCs with NO2– in aerobic conditions, in which most of the Hb is in the oxyHb form. It was previously reported that in such conditions after addition of nitrite ions, oxyHb should be transformed to metHb, while nitrite ions change to nitrate ions.2 Previous works suggested that several intermediates may be involved in this complicated, autocatalytic, free-radical chain reaction; however, later studies suggest that H2O2 and NO2 play the most important roles as initiator and autocatalytic propagator species, respectively.3 Some other species, beside metHb such as hemichromes4, HbNO2 5 or Hb3+NO/Hb2+NO 6 have been also reported to be generated from this well-recognized Hb-nitrite reaction. In those studies, the metHb should be understood as the aquomethemoglobin (metHb–H2O) with hematin as a prosthetic group with water and histidine molecules as ligands in axial positions.7,8 The crystal structure of the T-state9 and Rstate human metHb–H2O has been previously reported.10 It is well known that metHb–H2O is obtained as a main product by oxidation of human oxyHb with the use of excess sodium nitrite (NaNO2) or potassium ferricyanide (K3Fe(CN)6).11– 16

However, as was previously determined with the use of UV–vis spectrophotometry, the

products of these reactions are different.14 The UV–vis spectra profile of oxyHb after the treatment with NaNO2 was found to be dependent on the amount of excess sodium nitrite, suggesting the binding not only of water molecule but also nitrite ion to Hb.14 The differences were observed in the resonance Raman spectra (RRS) of the products of such reactions obtained with the abovementioned methods not only for the standard compounds but also for RBCs.17,18


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Despite such differences, Raman spectra of those species are still assigned in many works only to the metHb (metHb–H2O),11,12,15–17,19 without mentioning the possibility of the existence of additional Hb adducts (for example, ferricyanide–metHb, nitrite–metHb, aquo‒metHb, nitrosyl‒ metHb). Even so, it was previously reported that only the dialysis of the products obtained in different ways may give the pure fraction of metHb–H2O, which was carried out in the case of crystallographic studies;9,10,14 unfortunately, such a purification method cannot be applied to the isolated RBCs. The composition of Hb species inside RBCs after treatment with different concentrations of NaNO2 is far more complex than pure metHb–H2O and should be properly identified in situ in RBCs, what is a main goal of this work. Herein, this work brings attention to the need of definition of all products, beside metHb–H2O, after RBCs treatment with NaNO2 in many biochemical studies. The appearance of the additional adducts besides metHb–H2O due to the process of its formation should be mentioned as it may have biological consequences on the mode of action of such species. As was previously reported, because NO2– is an important biochemical signaling molecule and a source of the bioavailable NO,20 the addition of NaNO2 to the RBCs cannot be treated just as a way of metHb production. In vitro observation of previously reported, various stable Hb3+NO2 5 and Hb3+NO 6 adducts after NaNO2 treatment in functional RBCs may shed new light on the explanation of a complex autocatalytic reaction between oxyHb and nitrite ions. It was additionally reported on Hb standard that nitrite ion may bind reversibly to ferriheme at physiological conditions.21 Moreover, as it was postulated that the oxyHb–nitrate reaction is crucial for NO release from iron–nitrosyl–Hb,1 it is reasonable to study such interactions in vitro. Even though it has been previously reported that the formation of various nitrosyl heme species


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is pH dependent,22 in this work we had to limit our studies to pH 7.4, typical for functional RBC conditions. The goal of this work is to analyze in situ the content of the functional RBCs in aerobic conditions after treatment with different concentrations of NaNO2 with the use of Raman spectroscopy combined with UV–vis spectrophotometry. The isolated RBCs were kept in a buffer solution (pH 7.4) supplemented with bovine albumin and glucose, which allowed for conditions similar to functional cells. The comparison of the Raman results obtained after treatment of oxyHb with Na14NO2 and Na15NO2 were also carried out. Based on the resonance Raman spectra of RBCs obtained with the use of 488 and 633 nm laser wavelengths, a vibrational analysis of observed Hb species and their changes was performed, and some Hb3+NO2 adducts inside RBCs were visualized with confocal Raman microscopy. Such in situ detection and differentiation of all stable

LSHb

3+

adducts observed inside functional RBCs,

produced after addition of NaNO2 to oxyHb, proofs that some previously reported intermediates like NO2– or NO,23 may actually interact with ferric Hb and form stable adducts like Hb3+–NO2 (metHb–NO2) or Hb3+–NO (metHb–NO) in vitro in functional RBCs.

2. METHODS 2.1 Isolation of RBCs from Whole Human Blood. Human blood samples (around 4 cm3) were collected on heparin anticoagulant from healthy volunteers on the day of the experiment. Within one hour, blood was subjected to triple centrifugations (acceleration: 500g; run time: 10 min; temperature: 21 °C; braking: 0). Supernatant together with buffy coat were removed by aspiration (after each spinning) and packed RBCs were washed in Ringer–Tris buffer solution. The solution was supplemented with bovine albumin and glucose. The purity of the RBC


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fraction, collected from the bottom of the tube after the third centrifugation, was verified. The samples with white blood cells count exceeding 200/mm3 were subjected to another centrifugation. The Ringer–Tris buffer was added to the washed RBC fraction to obtain the hematocrit of about 0.04% for UV–vis and resonance Raman spectroscopy (RRS). All of the experiments were conducted within 8 h of collecting the blood samples. 2.2 Chemicals and Solutions. The Ringer–Tris buffer solution was prepared also ex tempore with the following composition: 140.5 mM NaCl, 2 mM CaCl2, 4.7 mM KCl, 1.2 mM MgSO4, 21 mM Tris base, 5.5 mM glucose and 76 µM bovine albumin. All reagents were dissolved in distilled water and filtered through a 0.22 µm pleated filter; pH was adjusted to 7.35–7.45 using 1 M hydrochloric acid. To obtain pure deoxyHb, fresh sodium dithionate (from Sigma-Aldrich CAS 7775‒14‒6) was added to oxyHb to final concentration of 10 mM, and buffer with RBCs was equilibrated with gaseous N2 (continuous nitrogen flow across the buffer). An aqueous solution of hematin porcine (hematin standard), purchased from Sigma-Aldrich (CAS 15489‒90‒4), was prepared ex tempore by dissolving in water. Sodium nitrite, Na14NO2 and Na15NO2, were purchased from Sigma-Aldrich (CAS 7632‒00‒0 and 68378–96–1, respectively) and added to isolated RBCs in different molar concentrations. 2.3 Experimental Methods. The RRS spectra of isolated, functional RBCs were recorded using a WITec confocal CRM alpha 300 Raman microscope. The spectrometer is equipped with an air-cooled solid-state laser operating at 488 nm and 633 nm and a CCD detector, which was cooled to –60 °C. The lasers are coupled to the microscope via optical fiber with a diameter of 50 µm. A water-immersive Nikon Fluor (60×/1.00 W) objective was used. The spectral resolution was equal to 3 cm–1. The monochromator of the spectrometer was calibrated using a radiation


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spectrum from a calibrated xenon lamp (Witec UV light source). In addition, the standard alignment procedure (a single point calibration) was performed before measurement with the use of the Raman scattering line produced by a silicon plate (520.5 cm–1). RBC samples were diluted 1:1000 (v:v) with buffer solution and put into a glass-bottom dish with CaF2 plate. RBCs were measured in Ringer–Tris buffer solution equilibrated with air in the OKOlab microscope cooling/heating incubator (H101–UP chamber) placed on the microscope scan table. To obtain the pure deoxyHb spectrum, nitrogen was introduced to the gas chamber to replace air, and additionally treated with sodium dithionate (6‒8 mM) under constant flow of N2. Measurements were carried out for deoxyHb, oxyHb and oxyHb after treatment with NaNO2. The spectra were always recorded at least one hour after sample preparation to allow the sample to stabilize. In the case of single addition of NaNO2, measurements were started one hour after administration; in cumulative addition, measurements were done after one hour of each administration, so the last spectra were recorded approximately eight hours after the experiment started (including half hour for each measurement). Raman spectra were averaged from at least 600 single Raman measurements obtained from different RBCs at each sample (one Raman spectrum – one red blood cell). Spectra were acquired in the line scan mode with the integration time of 3 s and one scan of each point (each RBC). The laser intensity in the focus spot was equal to 9 µW (as previously reported, higher laser power can cause the photo/thermal dissociation of oxyHb24). In addition, the higher laser intensity in the focus spot was used to study the photo/thermal dissociation of adducts present in the mixture with metHb. Raman measurements and data analysis were performed using WITec software (WITec Project Plus 2.10), Opus 7.2 and Origin 9.1. All averaged Raman spectra were


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preprocessed (cosmic spike removal and background subtraction), normalized and presented in the range 300–1700 cm–1. Absorption spectra (UV–vis) were obtained on a Perkin Elmer double beam spectrophotometer Lambda 950 in the range of 350–700 nm using a cuvette of 1 cm path length. All samples containing RBCs were diluted 1:1000 (v:v).

3. RESULTS AND DISCUSSION The UV–vis absorption spectra of heme derivatives are characterized by the strong Soret band (or B band) at around 410–440 nm and Q bands (mainly Qv and Q0 or α and β, respectively) in the region of 500–600 nm.16,25,26 The UV–vis spectra of ferric HS heme derivatives contain an additional band at around 600–650 nm originating from a charge-transfer (CT) process.16 Figure 1A presents the UV–vis spectrum typical for pure oxyHb, deoxyHb, hematin and metHb–H2O. The UV–vis spectrum of aqueous hematin solution has H2O molecule bound to Fe3+ of heme, but due to the absence of the histidine linkage, it gives a UV–vis spectrum different from metHb– H2O standard and cannot be treated as a model of metHb. Such a spectrum exhibits a Q band in the region typical for five-coordinate species (similar to deoxyHb) and CT band strongly redshifted compared with metHb–H2O. As is seen in the UV–vis spectrum of metHb–H2O standard, formation of metHb can be proven by the occurrence of the CT band in the UV‒vis spectra of RBCs at around 629 nm,3 as well as the presence of the QIV band at around 498 nm.27 Observation of the Qv and Q0 bands at around 540 and 570 nm, respectively, confirms the presence of the ferric HS six-coordinate species,21 and are observed at 544/578 nm and at 539/575 nm for oxyHb and metHb–H2O, respectively.


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The UV–vis spectrum of RBCs before addition of NaNO2 was typical for oxyHb species. The addition of nitrite ions caused the appearance of the band at around 360 nm attributed to the free NO2- ions and changes of the UV–vis profile typical for oxyHb due to formation of the novel Hb species. Their formation was dependent on sodium nitrite concentration, on the method of its addition to the solution (a single administration or cumulative addition) as well as on the measurement time from the moment of nitrite ion addition. The single addition of sodium nitrite from the level of 15 mM concentration was much more toxic for RBCs than cumulative addition of the same dose (Figures 1C and D). In the case of a single addition, the 60 mM NO2– and higher sodium nitrite doses resulted in destruction of the porphyrin ring inside hemoglobin which results in the disappearance of the Soret band (at around 420 nm) and Q bands (in the region of 540-580 nm) typical for porphyrin structure. On the other hand, cumulative addition doses up to 100 mM did not evoke such an effect. The differences of single versus cumulative addition could be due to inability of methemoglobin reductase activity in functional RBCs to reduce metHb to oxyHb upon single exposure to a high concentration of sodium nitrite.


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Figure 1. UV‒vis absorption spectra obtained from the (A) isolated RBCs typical for the deoxyHb, oxyHb and aqueous solution of hematin and metHb standards; (B–D) isolated RBCs with a majority of oxyHb treated with cumulative or single addition of different concentrations of NaNO2. Spectra were normalized to the Soret band. The normalized absorbance is increased in the region of 475‒700 nm 3× for A, 5× for B–D and 30× for brown (60 mM/60 min) and black (100 mM/60 min) spectra in D. The time of the UV–vis spectra collection after NaNO2 addition is given in the legends.


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Table 1. Peak Position for Absorption Bands (nm) Presented in Figure 1 for the Different Hb Species Soret QIV Qv (β) Q0 (α) CT deoxyHb 434 – 559 – oxyHb 419 – 544 578 – hematin 398 – 558 657 metHb 406 498 539 575 629 cumulative addition: 416 494 544 578 – oxyHb + NaNO2 (300 µM) oxyHb + NaNO2 (2 mM) 416 494 544 578 633 oxyHb + NaNO2 (15 mM) 409 495 544 578 634 oxyHb + NaNO2 (60 mM) 411 496 543 574 633 oxyHb + NaNO2 (100 mM) 414 – 542 568 629 single addition: 416 493 545 579 636 oxyHb + NaNO2 (300 µM) 412 497 544 578 635 oxyHb + NaNO2 (2 mM) 419 493 547 574 632 oxyHb + NaNO2 (15 mM) 422 497 549 571 631 oxyHb + NaNO2 (60 mM) oxyHb + NaNO2 (100 mM) – 500 564broad 631broad

The UV–vis bands seen in the spectrum of oxyHb treated with a single administration of 300 µM – 60 mM NaNO2 originate mainly from metHb–H2O and either from residues of oxyHb or from other Hb adducts. Confirmation of the existence of other than the oxyHb form of Hb can be seen mainly in the change in the intensity of Qv and Q0. The increase in the ratio of the intensity of the α to β bands suggests the additional formation of less stable adducts than oxyHb metalloporphyrin.28 Such changes of the UV–vis profile indicate that besides metHb–H2O, additional adducts different from oxyHb are observed inside isolated RBCs after treatment with low concentrations of NaNO2. It was previously reported that the UV–vis spectrum of the Hb with Fe3+NO core, which has lower stability than oxyHb,29 also has a similar UV–vis profile to oxyHb with the Q bands slightly shifted toward lower wavelengths,30 which supports the hypothesis of formation of such an adduct. However, the addition of such a low concentration of NaNO2 to RBCs should result only in slight changes in the UV–vis profile and the exact conformation of the produced Hb adducts cannot be defined just with the use of UV‒vis


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spectrophotometry. On the other hand, a cumulative addition of NaNO2 up to 100 mM concentration produced a different UV–vis profile without the QIV band and with more intense Qv than Q0 band, which has been previously defined as the spectrum originating from metHb– NO2 adducts.3 To detect and study further additional heme adducts induced by nitrite in situ in RBCs, RRS was applied. RRS is a powerful tool to investigate heme-containing molecules. The use of 488 nm excitation wavelength allows for observation of intense marker bands of the oxidation and spin states31 as well as high-resolution imaging of the RBCs with the use of a confocal Raman system.24 On the other hand, such a short wavelength can be applied for detection of oxyHb or other Hb adducts in functional RBCs only when very low power is applied, which requires the collection of vast numbers of individual Raman spectra from different RBCs (around 1200 single spectra had to be averaged, each collected with integration of 3 s from different RBCs). The laser intensity in the focus spot for RBCs measured with 488 nm excitation should be up to around 9 µW to be able to observe pure oxyHb, based on our previous studies on blood smears.24 Higher laser power can cause the photo/thermal dissociation of oxyHb to deoxyHb; however, such an effect is slightly attenuated in the case of immersive measurements carried out in this study due to absorption of some laser energy by the buffer solution. It was previously shown that to enhance selectively the Fe3+ axial-ligand vibrations (for example, for metHb–H2O, metHb–N3– and metHb–F–), excitation in the 550–650 nm region of the CT band of metHb should be applied.32 Therefore, fully to analyze changes in RBCs induced by nitrite ion, we have additionally used 633 nm excitation with laser power in the focus spot up to around 0.5 mW.


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Figure 2. The average Raman spectra measured from isolated, deoxygenated and oxygenated human RBCs as well as after exposure of RBCs with high oxyHb content to different NaNO2 concentrations (cumulative addition). Each average spectrum is obtained from the range of the 600–1200 single spectra recorded from different RBCs with the use of 488 nm laser excitation and with the laser intensity in the focus spot approximately equal to 9 µW. The RRS were


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recorded at the same conditions as UV–vis spectra presented in Figure 1C. For clarity, the Raman intensities in the region of 400-800 cm-1 were scaled by factor of 5 comparing to the region of 1200-1700. Similar to the case of the UV–vis results, the changes observed in RRS after nitrite ion addition depended on sodium nitrite concentration, on the method of its addition to the solution (a single administration or cumulative addition) as well as on the measurement time from the moment of nitrite ion addition. Figure 2 presents the averaged Raman spectra obtained with 488 nm excitation from functional RBCs in air (originating from oxyHb) and treated with sodium dithionate under continuous nitrogen flow (originating from deoxyHb) as well as gradually treated with different NaNO2 concentrations at the same conditions as the UV–vis spectra presented in Figure 1C. All spectra were collected from functional RBCs kept in buffer (pH 7.4) at room temperature. Table 2 gathers an assignment of the most important Raman bands presented in Figure 2. The oxidation state marker band, ν4, is located at around 1378 cm‒1 for the ferric ion, or at 1356 cm‒1 for the ferrous ion.33,34 The Raman spectrum of RBCs treated with sodium dithionate (6‒8 mM) under constant flow of N2 (Fig. 2, red) originates mainly from deoxyHb (HSHbII) and is characterized mainly by bands appearing at 1357, 1551, 1564 and 1604 cm‒1, assigned to ν4, ν11, ν2 and ν19 modes, respectively. The oxidation state marker band, ν4 is at a position typical for FeII, whereas the weak intensity of ν37 and ν3 located at 1470 cm‒1, suggest

HSFe.

33,35

Moreover,

there is no sign of an Fe–O2 stretching vibration band at around 572 cm‒1, which proves the absence of oxyHb. However, it is important to mention that Benko et al. have put in doubt the validity of the assignment of this Fe–O2 band as a stretching mode, suggested by Brunner,36 and


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they assigned it to a bending mode.37 On the other hand, in most studies the Fe–O2 stretching modes are still mainly assigned to the band at around 570 cm‒1, while bending is at 418 cm‒1.38 The Raman spectrum of RBCs recorded in air originates mainly from the oxyHb (LSHbIII– O2‒). This is confirmed by the presence of a strong ν10 band at 1640 cm‒1 and blue-shifts of both oxidation and spin-state marker bands, ν4 to 1379 cm‒1 (Fe3+), ν37 to 1586 cm‒1 and ν3 to 1507 cm‒1 (LSFe). The ν(Fe–O2) band at 572 cm‒1 provides additional evidence for the oxyHb as well as the absence of photo/thermal dissociation of O2 because of the laser power.24 Addition of the nitrite ions to oxyHb suggests formation of other Fe3+ oxidation state Hb species as ν4 is observed at around 1372-1376 cm‒1 after addition of all nitrite ion concentrations. The band at 572 cm‒1 ν(Fe–O2) disappears at 300 µM NO2–, which suggests disappearance of oxyHb and formation of other Hb species. The ν10 band may be treated as an additional marker for Fe3+ state in hemes or rather the marker of the presence of the ligand connected to the Fe3+ as it corresponds to a C=C double bond in the periphery of the porphyrin ring structure and is sensitive to out-of-plane motions of the heme.24,39

Changes suggest the appearance of metHb

(Fe3+–H2O), but depending on the sodium nitrite concentration, additional changes in RRS are observed, which can be assigned to formation of various, beside to metHb, adducts. We may observe changes typical for low NO2– concentration (300 µM ‒ 2 mM) and typical for high NO2– concentration (15‒100 mM), which are described below. Hb Adducts Observed at High Nitrite Ion Concentrations– metHb–H2O, nitrovinyl, Fe3+–ONO and Fe3+–NO Species. It is clearly seen that when the concentration of NO2– added to RBCs in the oxyHb state reached 15 mM NaNO2, an intense band at around 1322 cm–1 appeared and gradually increased with increasing concentration of NO2–. The band at 1322 cm–1, which originates from symmetric νsym(NO2) stretches typical for a nitroaromatic group, was


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previously assigned mainly to species where nitrite ion was connected to the nitrovinyl group of the heme—for synthesized myoglobin nitrito heme complexes22 and leghemoglobin.40 Moreover, this band is shifted in the RRS of RBCs treated with 100 mM

15

NO2– to the doublet at around

1292 and 1309 cm–1. Such a doublet and its intensity may suggest the existence of two different conformations of the bonded 15NO2– with similar abundances. This is in agreement with previous studies, which suggested that some of the nitrite ion adducts of Hb exist in the trans and cis conformations for the α and β subunits, respectively.40,41 The formation of the Hb nitrovinyl species can also be supported by the appearance of the band at around 442 cm–1, which originates from the 2-vinyl bending of the Cβ–Ca=Cb.22 As the band at around 410 cm–1, which is assigned to the 4-vinyl bending of the Cβ–Ca=Cb, is much weaker than the band at 442 cm–1, we may assume that 2-vinyl nitration is favored over 4-vinyl. Even though such regiospecificity was previously observed and studied for complexes of human Hb,42 this is the first time that such changes are confirmed for Hb studied in vitro in functional RBCs. The Raman imaging of the monolayer of functional RBCs treated with 100 mM NaNO2 presented in Figure 3 allowed us to confirm that NO2– is bonded to heme inside human RBCs. The integration of the band at around 1322 cm–1 confirms that the highest concentration of nitrite ions is observed inside RBCs. The k-means clustering (KMC) proves that the highest concentration of band originating from nitrite ion is connected with these imaged areas, which have the highest Hb concentration, and moreover it is absent for buffer solution. Such results show that Hb–NO2 adducts can be formed inside functional RBCs at pH 7.4 without significant influence on the RBCs’ integrity (see the photograph of monolayer RBCs in Figure 3A). Moreover, no presence of Heinz bodies or other inclusions on the RBCs’ surface is observed, which proves that in the case of the cumulative addition of nitrite ions, they are systematically


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embedded into the Hb structure and even such high concentrations as 100 mM do not damage RBCs. Of course, this may be connected with the fact that functional RBCs are able to neutralize some of nitrite ions with the use of methemoglobin reductase, which is impossible in the case of a single addition of a high concentration of sodium nitrite (see UV–vis results, Figures 1C, D).

Figure 3. (A) Microphotographs of the functional RBCs in buffer treated with 100 mM NaNO2 buffer solution with the labeled investigated area. Integration maps were made for the (B) band at 1322 cm–1 originating from the N–O stretching of the NO2 group indicating the presence of Hb–NO2, (C) bands in the region of around 1500–1700 cm–1 corresponding to the Hb signal in RBCs and (D) bands of buffer solution in the region of around 3000–3800 cm–1. The yellow color corresponds to the highest relative intensity of the integrated band. (E) The KMC results with the average Raman spectra for three main classes including RBCs rich in Hb–NO2 (red–the highest concentration, pink–lower concentration of Hb–NO2) and buffer class (black). The


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Raman map was obtained with the use of 488 nm laser excitation and with the laser intensity in the focus spot approximately equal to 9 µW. Some additional changes as well as previous studies on standard Hb complexes41 suggest that formation of nitrovinyl species is contingent upon the O binding of nitrite ion to Fe ion of heme for Hb.40 The variations of the nitrite ion binding mode in different heme species as well as preference of the O-binding mode for Hb is connected mainly with the changes in the distal pocket of heme.43 The band near 1580–1590 cm–1 assigned to the ν37 mode, postulated to be a LS state marker34, suggests the increase of the LS character with the increase of the NO2– concentration originating from binding of the ligand to the Fe3+ core. It was previously shown from magnetic moment and Raman measurements that the population of the HS state in the case of pure aquometHb–H2O is close to 90%, while for NO2– derivatives of metHb, such population decreased to around 40%,44 which is in agreement with our results. Another spin-state marker, which is shifted toward lower wavenumber values during conversion from LS to HS, is the ν3 mode.33,45 This band is observed at around 1470 cm–1 for the HS ion and at 1507 cm–1 for LS iron ion.45 Unfortunately, those bands, which are visible for deoxyHb (HS) and oxyHb (LS) are very weak in the case of oxyHb treated with nitrite ions. The weak band observed especially for 100 mM NO2– at 1479 cm–1 can be assigned to the ν(N=O) stretching of the NO2 ̶ group bonded to the Fe3+.22 The assignment of this band to –O– N=O unit bound to the Fe3+ was previously proven with the use of isotope labeling.22 It was also previously reported that the bending vibration of the NO2– bonded to the metal ion should be observed at a lower frequency than bending of the free NO2–, which is observed at around 825 cm–1.46 Therefore the band at 761 cm–1, which increases parallel with the increase of the nitrite ion concentration was assigned to the bending vibration of the NO2– bonded to Fe3+ core. This


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insensitive to the isotope labeling of 15N band, which is observed as a shoulder of the typical Hb band at around 757 cm–1 (ν15), also supports the existence of the Fe3+–O–N=O adduct. In addition, to support results with the use of 488 nm excitation and study in detail the axial-ligand stretches, the use of 633 nm excitation was applied to enhance such vibrational modes.

Figure 4. The average Raman spectra measured from isolated RBCs with high oxyHb content treated with 100 mM concentrations of Na14NO2 and Na15NO2 (cumulative addition) with the difference spectrum. Each average spectrum is obtained from around 600 single spectra recorded from different RBCs with the use of 633nm laser excitation and with the laser intensity in the focus spot approximately equal to 0.5 mW.


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As shown in Figure 4, the biggest changes presented in the difference spectrum between Hb–14NO2 and Hb–15NO2 originate, similar to that observed for 488 nm excitation, from the presence of the nitrovinyl derivatives. The band at around 1318 cm–1 originating from Ca=Cb‒ 14

NO2– vibrations is shifted for Ca=Cb‒15NO2– to the doublet at around 1287 and 1305 cm–1. The

633 nm excitation allowed also for insight into the bands at 410 cm–1 and 436 cm–1, which can be assigned to the 4-vinyl and 2-vinyl bending of the Cβ‒Ca=Cb‒14NO, respectively. For Hb‒15NO2, those bands are slightly split to doublets, which supports the existence of two different conformations of the bonded nitrite ion. In addition, the comparison presented in Figure 4 suggests that the ν2 mode assigned to the strong vinyl Ca=Cb stretching band is also shifted due to isotope labeling. Moreover, the region between 500 and 600 cm–1 (which is still indistinct, but its quality is clearer than that obtained with the use of 488 nm radiation) provides more details about additional interaction between Fe3+ and the axial ligand, such as H2O, NO2 or NO. Similar to the case of human metHb–H2O, and in the case of Hb–NO2 adducts, the crystallographic structures suggest the uncommon interaction with Fe3+ via oxygen (O–nitrite ion mode) for human Hb.41 The similarity of the O–nitrito binding mode in the case Fe3+‒O‒N=O and O interactions in the case of Fe3+‒OH242 may have an impact on the existence of the bands originating from some of the iron–ligand modes in a similar wavenumber region. Moreover, it is suspected that axial-ligand stretches originating from Fe3+‒ONO vibrations will not be easy to differentiate with the use of 14N/15N labeling due to interaction via oxygen. Those issues as well as a quite noisy Raman signal in the low spectral region (due to low power applied to minimize the impact on functional RBCs) make assignments of the ferric–axial ligand quite difficult. The previous RRS obtained from human Hb standard and its derivatives show various bands assigned to Fe3+‒O grouping modes. The band at 514 cm–1 has been previously assigned to


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the Fe–O stretching mode in hematin.47 The

3+ HSFe ‒OH

stretching, typical for metHb, was

defined by Asher and Schuster48 at 495 cm–l and later studies supported this finding.49 The

closest band to the one defined by Asher, which increases with NO2– addition but is not shifted due to isotope labeling is observed in the region of 490–510 cm–1 with the strongest signal at 502 cm–1. We believe that this broad band could be assigned to the stretching ν(Fe3+‒ O) modes typical for the mixture of the Fe3+‒OH2 and Fe3+‒ONO species. The enhancement of this band supports the increase formation of adducts with ligand bonded to Fe3+ core via oxygen (metHb-OH2 and/or Fe3+‒ONO) with the increase of nitrite concentration. The band at 502 cm–1 was previously reported as H/D sensitive and assigned to the Fe–O[Tyr] stretching mode in the case of Tyr63 mutation.50 We may also suggest that isotope

15

N-labeling insensitive band at

around 459 cm–1, could be assigned with high probability to the vibrations of Fe3+‒O-type interaction species. Moreover, the doublet at around 471/478 cm-1, which shows some isotope 15

N-labeling sensitivity, could be rather assigned to the vibrations of Fe3+‒N-type interaction.

Some other weak bands in the region of 475‒611 cm–1 for 633 nm and 471‒530 cm–1 for 488 nm excitation show some slight changes to the isotope labeling at the level of signal noise. The biggest variation between Hb14NO2 and Hb15NO2 is observed for 633 nm excitation in the region of 560‒611 cm–1. This may suggest the existence of another adduct with Raman spectra which are more sensitive to

14

N/15N labeling. The existence of the isotope labeling sensitive bands at

around 550‒570 cm–1 was previously reported for the ferric heme–nitro species of some cytochromes and assigned to the ν(Fe3+‒NO2).46 However, as such interaction for Hb via nitrogen was previously excluded,41 we suggest that this band could originate from the vibrations of the ν(Fe3+‒NO) in the Hb–NO adduct. The ν(Fe2+–NO) stretching mode was defined by Tsubaki51 at 551 cm–1 and later assigned rather to an Fe2+–N–O bending mode37. Benko and Yu


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assigned the ν(Fe3+–NO) mode for human HbA at 594 cm–1, which is at a higher frequency than Fe2+‒NO and agrees with the spectral region that is sensitive to

14

N/15N labeling presented in

Figure 4. It was previously reported that protonation of the O-bonded nitrito ligand will generate the ferric–hydroxo species as well as NO molecule through a homolysis reaction.43 It was also postulated that oxyHb–nitrite ion reaction is essential for NO release.1 Moreover, it is known that the ligation process in the case of the aquometHb–H2O, which is preceded by the dissociation of the coordinated water molecule from Fe3+, is more favorable for NO than for NO2–.21 Therefore, it is not surprising to observe also the existence of the Hb–NO adduct with Fe3+–NO core in such conditions inside RBCs. Moreover, this suggests the possibility of the predominance of such adducts in the case of low nitrite ion concentrations, which will be proven below. Hb Adducts Observed at Low Nitrite Ion Concentrations: metHb–H2O and Fe3+–NO and/or Fe3+–O–nitrito species of Hb. The exact interpretation of Raman spectra for such low concentrations is most difficult due to very subtle changes in the Raman spectra. However, it is important to stress that even though the exact determination of the type of adducts is impossible at such low concentration, it is certainly clear that species additional to metHb–H2O are formed also for such low nitrite ion concentrations. The main difference between low and high concentration of nitrite ion interacting with oxyHb inside RBCs is the lack of nitration of vinyl groups of heme in the case of nitrite ion concentrations up to 2 mM. This is supported by the absence of bands at 1322, 761, 442 and 410 cm–1 for Raman spectra obtained with the use of 488 nm excitation from RBCs treated with 2 mM and 600 µM NO2–. The modes assigned to nitrovinyl species are also invisible in the case of Raman spectra obtained with the use of 633 nm excitation from RBCs treated with 2 mM NO2–.


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Addition of nitrite ions at 600 µM concentration to oxyHb suggests formation of an Fe3+ Hb (ν4 at 1372 cm‒1) species different from oxyHb as the band at 572 cm‒1 ν(Fe–O2) is not present. The RRS of RBCs in such conditions is highly dominated by the population of the HS state, which suggests that metHb–H2O is the major component (the band at 1582 cm‒1 is relatively weak, while bands at around 1551 cm‒1 are relatively strong). Moreover, it was reported that upon Soret excitation (406.7 nm) Hb–NO adducts may be additionally characterized by the band at around 1620 cm–1 originating from ν(N–O).51 Here, in Figure 5 we observe a weak isotope-labeling sensitive shoulder at 1623 cm–1 of the band at 1608 cm–1 assigned to the ν19 mode. The addition of NO2– up to 2 mM decreases the HS population, which suggests an increase of the binding in the sixth coordination site. The appearance of additional bands at around 450 and 530 cm–1 observed in the case of 488 nm laser excitation, as shown in Figure 2, supports such ligation. The bands at similar frequencies were previously assigned to the pyrrole swiveling modes.52 It was postulated for the H–NOX family, which act as sensors for NO,53,54 that their hemes are highly distorted from their normal planar geometry, which is reflected by enhancement of the pyrrole rings swiveling and twisting. The presence of such out-of-plane modes may be an indicator of heme distortion caused by NO ligand binding. The fact that those bands, observe at 450 and 530 cm–1, are the strongest for 2 mM concentration suggests that, in this condition, the majority of additional to metHb–H2O adducts are those, that accounted from the smallest population of higher concentration of nitrite ions. This indicates that, in contrast to high nitrite ion concentration, the biggest population besides Fe3+–OH2 adducts belongs rather to Fe3+–NO than to Fe3+–ONO. Such a statement is additionally supported by the analysis of the comparison presented in Figure 5, which shows the existence of the isotope-labeling sensitive bands for 2 mM concentration and therefore is a proof


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for Fe3+–N interaction rather than Fe3+–O mode. It is however worth to mention that such effect is very weak as Figure 5 presents only the slight shifts of some bands due to isotope labelling, while the values for band maxima are not changed (as presented in Table 3).

Figure 5. The average Raman spectra measured from isolated RBCs with oxyHb content treated with 2 mM concentrations of Na14NO2 and Na14NO2 (cumulative addition) with the difference spectrum. Each average spectrum is obtained from around 1200 single spectra recorded from different RBCs with the use of 633 nm laser excitation and with the laser intensity in the focus spot approximately equal to 0.5 mW.


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Table 2. Observed Wavenumber Values (cm–1) for the Most Prominent Bands of the Average Raman Spectra Obtained with the Use of 488 nm Excitation and from 1200 Single RBCs (one single spectrum from one individual RBC) with Assignments and Symmetry Terms Assuming an Idealized D4h Symmetry for Observed Hb species.11,17,25,31,52,55,56 Bands are assigned for spectra obtained from functional RBCs with deoxyHb, oxyHb, as well as oxyHb treated with 100 mM Na14NO2 and Na15NO2 and 2 mM Na14NO2. Wavenumber/cm–1

Band assignment/Type of interaction

Local coordinatesa

deoxyHb

oxyHb

Na14NO2 (100 mM)

Na15NO2 (100 mM)

Na14NO2 (2 mM)

Ca=Cb–NO2

δ(4-vinyl)

–

–

410(vw)

–

–

Ca=Cb–NO2

δ(2-vinyl)

–

–

442(w)

442(w)

–

δ (pyr swivel)

3+

Fe –NO

–

–

–

–

450(w)

3+

–

–

459(vw)

459(vw)

–

3+

–

–

471/478(vw)

476/485(vw)

–

3+

–

–

502(m)

502(m)

508(w)

–

–

518/530(w,sh)

518(w,sh)

530(w)

Fe3+–O2

ν/δ(Fe –O–) ν/δ(Fe3+–N–)/ δ (pyr swivel) ν(Fe–O2)

–

572(w)

–

–

–

ν7

δ(pyr def)sym

674(m)

678(m)

680(m)

680(m)

682(m)

ν15

ν(pyr br)

757(m)

757(m)

755 (m)

753(m)

759(m)

Ca=Cb–NO2

δ(NO2)

–

–

761(sh)

761(sh)

–

Ca=Cb–NO2

νsym(NO2)

–

–

1322(s)

1292/1309(s)

–

ν4

ν(pyr hr)sym

1357(s)

1379(s)

1376(s)

1376(s)

1376(s)

ν3

ν(CαCm)sym

1470(m)

1507(m)

1507(vw)

1507(vw)

–

Fe –ONO

ν(N=O)

–

–

1479(vw)

–

–

ν11

ν(CβCβ) ν(CβCβ)

1551(s)

–

–

–

1548(s)

1564(s)

–

1563(m,sh)

1568(m,sh)

1564(m)

ν(CαCm)as ν(CαCm)as

1585(m)

1586(s)

1587(s)

1588(s)

1586(m)

1604(s)

1604(m)

–

–

–

ν(Ca=Cb) ν(CαCm)as

–

–

1616(m)

1615(m)

1612(m)

3+

Fe –ONO/OH2 3+

Fe –NO 3+

Fe –ONO/OH2 Fe3+–NO

3+

ν2 ν37 ν19

ν/δ(Fe –O–) ν/δ(Fe –N–)

ν10 – 1640(s) 1642(s) 1642(s) 1640(s) The mode notation is based on that proposed by Abe et al. [55] and Hu et al. [52]. a ν – stretching, δ – scissoring, def– deformation, br – breathing, hr – half-ring, qr – quarter-ring, as – asymmetric, sym – symmetric, pyr – pyrrole, swivel – swiveling, sh-shoulder, vw – very weak, w – weak, m- medium, s – strong.


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Table 3. Observed Wavenumber Values (cm–1) for the Most Prominent Bands of the Average Raman Spectra Obtained with the Use of 633 nm Excitation and from 1200 Single RBCs (one single spectrum from one individual RBC) with Assignments and Symmetry Terms Assuming an Idealized D4h Symmetry for Observed Hb Species.11,17,25,31,52,55,56 Bands are assigned for spectra obtained from functional RBCs with oxyHb treated with 2 and 100 mM Na14NO2 and Na15NO2. Band assignment/Type of interaction

Local coordinatesa

ν9/ν52

δ(CβC1)sym

CaF2

Na14NO2 (2 mM) 270(w)

Wavenumber/cm–1 Na15NO2 Na14NO2 (2 mM) (100 mM) 270(w) 257/270(w)

Na15NO2 (100 mM) 257/270(w)

324(w)

324(w)

324(w)

324(w)

ν8

ν(Fe–N)

342(w)

342(w)

342(w)

342(w)

Ca=Cb–NO2

δ(4-vinyl)

–

–

410(w)

406/416(w)

Ca=Cb–NO2

δ(2-vinyl)

–

–

436(w)

438(w)

Fe3+–NO

δ (pyr swivel)

425(w)

425(w)

–

–

508(w)

508(w)

506(w)

508(w)

536(vw)

542(vw)

536(vw)

542(vw)

570(vw,sh)

561(vw)

570(vw,sh)

561(vw)

3+

Fe –ONO/OH2 Fe3+–NO Fe3+–NO 3+

3+

ν/δ(Fe –O–) ν/δ(Fe3+–N–)/ δ (pyr swivel) ν/δ(Fe3+–N–) 3+

Fe –NO

ν/δ(Fe –N–)

611(vw)

607(vw)

611(vw)

582(vw)

ν7

δ(pyr def)sym

675(m)

675(m)

675(m)

675(m)

ν15

ν(pyr br)

757(s)

757(s)

757(s)

757(s)

–

–

944(m)

954(m)

1121(s)

1121(s)

1121(s)

1121(s)

ν5/ν13/ν18/ν42

ν(N–O) ν(pyr half– ring)asym δ(CmH)

1214/1229/1244(s,sh)

1214/1229/1244(s,sh)

1230/1258(s)

1230/1258(s)

Ca=Cb–NO2

νsym(NO2)

–

–

1318(m)

1287/1305(s)

ν11

ν(CβCβ)

1546(s)

1546(s)

1546(s)

1546(s)

ν2

ν(CβCβ)

1564(sh)

1564(sh)

1558(sh)

1564(sh)

ν19

ν(CαCm)asym

1608(s)

1608(s)

1608(s)

1608(s)

3+

Fe –ONO ν22

ν10 ν(CαCm)asym 1639(sh) 1639(sh) 1639(sh) 1639(sh) The mode notation is based on that proposed by Abe et al. [55] and Hu et al. [52]. a ν – stretching, δ – scissoring, def– deformation, br – breathing, hr – half-ring, qr – quarter-ring, as – asymmetric, sym – symmetric, pyr – pyrrole, swivel – swiveling, sh-shoulder, vw – very weak, w – weak, mmedium, s – strong.


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Table 4. Description of the Oxidation Number (+2, +3), Spin State (low-spin state–LS, highspin state–HS)46 as Well as the Molecule Present in the Sixth Coordination Site for the Iron Ion of Hb Species Used in the Manuscript type of Hb iron ion DeoxyHb OxyHb metHb–H2O metHb–NO2– metHb–H2O/NO

oxidation state

spin state

+2 +3 +3 +3 +3

HS LS LS/~90% HS LS/~40% HS LS/HS

molecule in the sixth coordination site absent O2 H 2O NO2– H2O/NO

4. CONCLUSIONS This work describes the interaction between nitrite ions and functional RBCs in vitro with the use of RRS supported with UV‒vis spectrophotometry. Our studies proof the possibility of in situ detection and differentiation of some products of the reaction between oxyHb and NaNO2 inside functional RBCs. The detailed UV–vis analyses, as well as vibrational analysis of the obtained Raman spectra, based on the previous reports connected with model compounds, suggest the presence of metHb–H2O with additional Hb adducts. Their formation was dependent on sodium nitrite concentration, on the method of its addition to the solution (a single administration or cumulative addition) as well as on the measurement time from the moment of nitrite ion addition. For RBCs treated with NaNO2 concentration higher than 15 mM (up to 100 mM, cumulative addition) the metHb–H2O as well as various Hb3+–NO2 adducts and Hb3+–NO were studied in situ. UV–vis profile without the QIV band and with more intense Qv than Q0 band support the hypothesis of formation of metHb–NO2 adducts in such conditions.3 RRS allowed detection and differentiation of the O–nitrito bound to Fe3+ core Hb species (Fe3+–O–N=O), nitrovinyl adducts with 2-vinyl nitration favored over 4-vinyl nitration as well as Fe3+–NO


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adducts. The isotope-labeling sensitive band originating from symmetric νsym(NO2) of the nitrovinyl group of the heme at 1322 cm–1 confirms the presence of the nitrovinyl adducts for RBCs treated with 100 mM NO2– as well as allows for in vitro visualization of the Hb–NO2 species inside functional RBCs. Raman maps confirm that this band, connected with the presence of the nitrovinyl group, is observed only for RBCs and not for surrounding buffer. Vibrational analysis of bands assigned to the 2-vinyl and 4-vinyl bending of the Cβ–Ca=Cb in RRS, obtained with the use of 488 and 633 nm excitations, allowed for confirmation of the regiospecificity of such processes in vitro (2-vinyl nitration is favored over 4-vinyl nitration). Vibrational analysis of the spin and oxidation state marker bands showed the increase of the LS character with the increase of the NO2– concentration, which suggests the binding of the NO2– ligand to the Fe3+ core. The HS state of the Fe3+ dominates for aquometHb–H2O while for NO2– derivatives of metHb, such population changes to a majority of LS state of the Fe3+. The –O– N=O unit bound to the Fe3+ via oxygen was suggested in this manuscript based on detailed vibrational analysis and previous reports. However, some bands that were particularly sensitive to 14N/15N labeling revealed also the possibility of additional ligation to the Fe3+ via nitrogen. As interaction for Hb with NO2 via nitrogen was previously excluded,41 we found that these bands could originate from the vibrations of the ν(Fe3+‒NO) in the Hb–NO adduct. At lower NaNO2 concentration (below 2 mM) the exact determination of the type of adducts is impossible due to very subtle changes in the Raman spectra. However, it is certainly clear that species additional to metHb–H2O are formed also for such low nitrite ion concentrations. UV–vis profile similar to oxyHb with the Q bands slightly shifted toward lower wavelengths, observed for isolated RBCs after treatment with low concentration of NaNO2, supports such a hypothesis. Detailed analysis of the RRS results suggested that the presence of


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Fe3+–NO beside metHb–H2O is the most plausible, however the presence of Fe3+–O–N=O in such condition cannot be excluded. Results presented here prove that RRS could be a useful tool for in vitro detection and differentiation of various stable Hb3+NO2 and Hb3+NO adducts inside functional human RBCs.

AUTHOR INFORMATION Corresponding Author: *Katarzyna M. Marzec [email protected]

ACKNOWLEDGMENT This work was supported by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Program (grant coordinated by JCET–UJ, No. POIG.01.01.02–00–069/09) and National Centre of Science (DEC– 2013/08/A/ST4/00308). K.M.M. acknowledges financial support from a Go8 Fellowship, enabling a research stay at Monash University.


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TOC IMAGE


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Figure 1 208x161mm (300 x 300 DPI)


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Figure 2 79x100mm (300 x 300 DPI)



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Figure 3 97x112mm (300 x 300 DPI)


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Figure 4 51x40mm (300 x 300 DPI)



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Figure 5 39x27mm (300 x 300 DPI)


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TOC 40x20mm (300 x 300 DPI)


Resonance Raman in Vitro Detection and Differentiation of the Nitrite

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