Russian Chemical Bulletin, International Edition, Vol. 65, No. 3, pp. 779—783, March, 2016

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Nitric oxide donation by the binuclear tetranitrosyl iron complexes in the presence of erythrocytes N. I. Neshev, E. M. Sokolova, B. L. Psikha, T. N. Rudneva, and N. A. Sanina Institute of Problems of Chemical Physics, Russian Academy of Sciences, 1 prosp. Akad. Semenova, 142432 Chernogolovka, Moscow Region, Russian Federation. Fax: +7 (496) 522 3507. Email: [email protected] The kinetic regularities of nitric oxide donation in the presence of erythrocytes by represen tatives of a new class of synthetic nitric oxide donors, binuclear tetranitrosyl iron complexes (BTNIC) [Fe2(SR)2(NO)4] with thiolcontaining ligands, where R is pyrimidin2yl, 1methyl imidazol2yl, benzothiazol2yl, and penicillamine residue, were established. The NOdon ating ability of BTNIC was estimated from the apparent rate constants of the first order for the formation of intraerythrocyte methemoglobin with the variation of the initial concentration of the complex. During the standard experimental time (15—17 min), three of the four complexes released in the solution no more than a quarter of available NO groups. Their NOdonating ability turned out to be variable increasing with an increase in the initial concentration of the complex. At the same time, the complex bearing penicillamine residues as ligands donated almost all NO groups within approximately 1 min. Features of NO donation by the BTNIC in the presence of erythrocytes are related to the formation in the system of an additional equilib rium pool of the membraneassociated complex, which is characterized by a reduced rate of hydrolytic dissociation with NO releasing to the solution. The NOdonating ability of the BTNIC in the presence of erythrocytes is determined by the ratio of volumes of the free and membraneassociated pools of the complex. Key words: erythrocytes, methemoglobin, binuclear tetranitrosyl iron complexes, nitric oxide.

Nitric oxide performs the most important biological functions in the organism primarily related to the modu lation of the blood pressure level, inhibition of thrombo sis, transmission of nervous pulses, and nonspecific im mune protection.1 It is known that insufficiency of enzy matic synthesis of nitric oxide is a pathogenic factor of many cardiovascular diseases.2 Therefore, exogenic donors of nitric oxides can be used as efficient remedies for the pharmacological correction of the indicated disorders. Representatives of a new class of synthetic NO donors, binuclear tetranitrosyl iron complexes (BTNIC) with thiol containing ligands based on various azaheterocyclic thiols and aliphatic thioamines, are presently studied actively.3 An important property governing the prospect of prac tical use of BTNIC is their NOdonating ability. As it turned out that this property of BTNIC depends on both the chemical nature of the complex and the medium in which donation occurs. We have earlier shown that the level of NO donation in the presence of erythrocytes de creases with an increase in the hematocrit (volume per centage content of cells) of the suspension.4 Since this effect can directly affect the pharmacological activity of BTNIC in the circulatory system, it seems necessary to continue studies of specific features of nitric oxide dona

tion by BTNIC in the presence of erythrocytes in order to establish the respective mechanisms. In this work, we intended to determine how the initial BTNIC concentration affects the level of NO donation in a suspension of erythrocytes. Experimental Preparation of erythrocytes. Mice of the line C 57 Bl/6f (age 3 months, body weight 18—20 g) served as a blood source. Erythrocytes were isolated using a standard procedure.5 A sus pension of erythrocytes with the hematocrit value 1.6% in an isotonic solution of NaCl containing 5 mM sodium phosphate buffer (pH 7.4) was used in experiments. A suspension with hema tocrit 1.6% corresponded to the concentration of intraerythro cyte hemoglobin 2•10–4 mol L–1 based on heme evaluated from the absorbance of the hemolysate at the isosbestic point 525 nm using a molar absorption coefficient of 7.5 mmol–1 L cm–1.6 Determination of methemoglobin. The concentration of in traerythrocyte hemoglobin was determined spectrophotometri cally5 at 630 nm using molar absorption coefficients of 0.11 and 3.8 mmol–1 L cm–1 for oxy and methemoglobin, respectively.7 NO donors. The BTNIC [Fe2(SR)2(NO)4], where R is py rimidin2yl (1),8 1methylimidazol2yl (2),9 benzothiazol2 yl (3),10 and penicillamine residue (4),11 earlier synthesized at

Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 0779—0783, March, 2016. 10665285/16/65030779 © 2016 Springer Science+Business Media, Inc.

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the Institute of Problems of Chemical Physics (Russian Acade my of Sciences, Chernogolovka, Moscow Region) were used. The complexes were introduced into an erythrocyte suspension as solutions in isotonic sodium chloride (4) or as solutions in DMSO (1—3), which were prepared prior to experiments. The DMSO concentration in the samples did not exceed 3%.

Neshev et al.

[HbFe3+]•105/mol L–1

a

20

4

16 12

Results and Discussion The molecular structures of the BTNIC used in the work belong4 to two structural types: μS (A, complexes 1 and 4) and μS—C—N (B, complexes 2 and 3). In the complexes of the A type, the Fe atoms (the distance be tween them is ∼2.7 Å) are linked through bridges of the S atoms of the ligand similarly to the structure of the Fe2S2 clusters in the active centers of the nonheme enzymes. In the complexes of the B type, the Fe atoms (the distance between them is ∼4.0 Å) are bound through bridges of the —S—С—N— chain of the atoms of aromatic thiolyl, which causes their characteristic ESR signal with g ∼2.03 in so lutions similar to the ESR signal of the cellular dinitrosyl iron complexes.

3

8

2 4 1 2

4

6

8

kapp•103/s–1

10

t/min

b

20

15

1

The simplest model of the processes occurred in the course of BTNIC decomposition in an erythrocyte sus pension can be presented as follows: [Fe(SR2)(NO)4]

С•104/mol L–1

Fig. 1. Kinetic curves of methemoglobin formation (a) in sus pensions with the concentration of 1 0.6•10–4 (1), 0.8•10–4 (2), 1.2•10–4 (3), and 2.4•10–4 (4) mol L–1 (hemoglobin concentra tion 2•10–4 mol L–1) and the apparent rate constant (kapp) for methemoglobin formation in the presence of 1 (b).

4 NO + Decomposition (1) products of the complex.

Nitric oxide coming inside an erythrocyte via diffusion is rapidly oxidized in the reaction with oxyhemoglobin [HbFe2+]O2 + NO

2

HbFe3+ + NO3–.

(2)

Equation (1) describes the hydrolytic dissociation of BTNIC outside erythrocyte, whereas the oxidation of nitric oxide diffusing inside an erythrocyte is described by Eq. (2). We have previously developed a procedure for the evaluation of the NOdonating ability of BTNIC. The procedure is based on the study of the kinetics of intra erythrocyte methemoglobin formation.5 The apparent rate constant of the first order for methemoglobin formation (kapp) can serve as a quantitative characteristic of the NOdonating ability of BTNIC. The kinetic curves of intraerythrocyte methemoglobin formation under the action of various concentrations of complexes 1—3 are presented in Figs 1—3, a. It can be seen that not more than a quarter of available NO groups

is liberated from the complexes within the standard exper imental time (15—17 min) taking into account the initial concentrations of complexes 1—3 and stoichiometry of reactions (1) and (2) (the stoichiometric limit of the re lease of NO groups is equal to the quadruplicate con centration of the complex indicated in the captions to the figures). An analysis of the obtained data showed that the kin etics of methemoglobin formation in the presence of BTNIC is satisfactorily described, in the most cases, by the firstorder equation [HbFe3+] = [HbFe3+]∞•(1 – e–kappt),

(3)

where t is time, [HbFe3+] is the concentration of methe moglobin, kapp is the apparent rate constant of the first order reaction, and [HbFe3+]∞ is the limiting value of methemoglobin concentration at t → ∞. The apparent firstorder rate constant (kapp) was de termined for each kinetic curve. A relationship between

NOdonating ability of nitrosyl iron complexes

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a

[HbFe3+]•105/mol L–1

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a

20 25

4 3

15

20 3 15

10

10

2

2 5

5

1

1 2

4

6

8

10

12

kapp•103/s–1

14

16

2

t/min

b

20

4

6

8

10

12

14

kapp•103/s–1

16 t/min

b

6

16 12

4

8 2

4

1 0.5

1.0

1.5

С•104/mol L–1

Fig. 2. Kinetic curves of methemoglobin formation (a) in sus pensions with the concentration of 2 0.6•10–4 (1), 0.8•10–4 (2), 1.2•10–4 (3), and 2.4•10–4 (4) mol L–1 (hemoglobin concentra tion 2•10–4 mol L–1) and the apparent rate constant (kapp) for methemoglobin formation in the presence of 2 (b).

kapp and the initial concentrations of the corresponding BTNIC is shown in Figs 1—3, b. The lines drawn are linear approximations of the corresponding points in the Origin program. It is seen that the plots are characterized by a positive slope ratio in all cases, indicating an increase in kapp with an increase in the initial concentration of the complex. On the contrary, the kapp values decreased with an increase in the hematocrit in the previous series of experi ments with variable hematocrit at a constant concentra tion of the complex.4 Thus, the overall results of two ex perimental series reveal the characteristic "drift" of kapp in opposite directions. Correspondingly, the NOdonating ability of complexes 1—3 also changes in opposite direc tions. This fact needs to be explained, because it does not follow from the presented above scheme of reactions (Eqs (1) and (2)). The behavior of complex 4 with a substantially higher NOdonating ability turned out to be basically different

1.0

С•104/mol L–1

Fig. 3. Kinetic curves of methemoglobin formation in suspen sions with the concentration of 3 0.6•10–4 (1), 0.8•10–4 (2), 1.2•10–4 (3), and 2.4•10–4 (4) mol L–1 (hemoglobin concentra tion 2•10–4 mol L–1) and the apparent rate constant (kapp) for methemoglobin formation in the presence of 3 (b).

(Fig. 4). Its concentrations in experiments were decreased sixfold compared to other complexes to avoid the com plete oxidation of oxyhemoglobin. It is seen that the sto ichiometric limits of NO evolution according to Eqs (1) and (2) (shown in the figure by dotted lines) are achieved within 0.5—1 min. This result is very important in two aspects. First, the fast and stoichiometrically complete evolution of nitric oxide by complex 4 distinguishes it from other BTNIC, indicating good prospects of using com plex 4 as both pharmacological agent and nitric oxide source in biochemical research. Second, this result is a direct experimental substantiation of the efficiency of using an erythrocyte suspension as a nitric oxide trap for the quantitative estimation of the NOdonating ability of exogenic NO donors. As already said above, the regular change in the NO donating ability of complexes 1—3 with a change in the hematocrit of the suspension and initial concentration of the complex cannot be explained in terms of the simple chemical model (reactions (1) and (2)). Therefore, not

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[HbFe3+]•105/mol L–1

The equation describing a relationship between the equilibrium ratio of volumes of the free and membrane bound pools of the complex and the concentration of free binding sites directly follows from Eq. (7)

16 4 12

[X]/[PX] = 1/(Ka[P]).

3 8 2 4 1 2

4

6

8

t/min

Fig. 4. Kinetic curves of methemoglobin formation in suspen sions with the concentration of complex 4 1•10–5 (1), 2•10–5 (2), 3•10–5 (3), and 4•10–5 (4) mol L–1 (hemoglobin concentration 2•10–4 mol L–1).

only purely chemical but also possible physicochemical interactions between the components of the studied sys tem should be taken into account. Earlier we already as sumed4 that the binding of a portion of BTNIC mole cules with the erythrocyte surface can result in a decrease in the hydrolytic dissociation rate because of the restricted contact with the aqueous medium. The assumption about binding of some molecules of the complex with the cell surface implies that two equilibrium pools of the complex, free and membranebound, differing in NOdonating abil ity, appear in the system. This problem can be analyzed on the basis of the as sumption about the equilibrium character of binding of the complex with an array of onetype and independent binding sites on the erythrocyte membrane [P] + [X]

[PX],

X0 = [X] + [PX],

(5)

P0 = [P] + [PX],

(6)

Equation (8) is a convenient mathematical model for the analysis of the whole array of the obtained experimen tal results. Since the free and membranebound pools of the com plex have different NOdonating ability, they will be char acterized by different individual rate constants of methe moglobin formation. In this case, the apparent rate con stant kapp measured in experiment will be a combination of these individual constants. The kapp value will change depending on the ratio of concentrations of the indicated pools. The hyperbolic dependence of the [X]/[PX] ratio on the concentration of free binding sites [P] specified by Eq. (8) is shown in Fig. 5. Correspondingly, kapp will also depend functionally on the value of [P]. An increase in the hematocrit of the suspension at an unchanged concentra tion of the complex means an increase in the concentra tion of free binding sites, which should result in a decrease in kapp as it was observed.4 On the contrary, the experi mental conditions of this work (increasing concentration of the complex at an unchanged hematocrit) provide a decrease in the number of free binding sites. This should result in an increase in kapp, which is really observed (see Figs 1—3). Thus, the experimentally obtained regularities of a change in the NOdonating ability of BTNIC in the presence of erythrocytes can be explained using the pro posed model of equilibrium binding of BTNIC with the cellular fraction. It is important to note that the dependence of NOdo nating ability of BTNIC on the incubation medium can [X]/[PX]

Increases

where X0 is the initial concentration of the complex, and P0 is the total concentration of binding sites. The condition of detailed equilibrium in the system can be presented as follows:

where Кa is the binding constant.

(8)

(4)

where X is a molecule of the complex, and P is the binding site of the complex on the cell surface. The equilibrium concentrations of the complex and the concentration of the binding sites in the suspension bulk can be presented, as usual, by material balance equations

[PX] = Ka[P][X],

Neshev et al.

(7)

kapp Decreases

[P] Fig. 5. Theoretical dependence of the ratio of concentrations of the free and membranebound pools of the complex on the con centration of the free binding sites (see Eq. (8)).

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be considered a unique feature of this class of NO donors, which is directly related to the mechanism of NO dona tion based on the hydrolytic dissociation of the complex. The NOdonating ability of BTNIC depends on both the presence of binding sites for the complex and the value of the affinity of BTNIC to these sites. The latter, in turn, depends on the level of lipophilicity of the Sligands. This provides possibilities for the purposeful optimization of the basic structure of the NO donor based on BTNIC taking into account a specific pharmacological target. References 1. P. Pacher, J. S. Beckman, L. Liaudet, Physiol. Rev., 2007, 87, 315. 2. L. J. Ignarro, C. Napoli, J. Loscalzo, Circ. Res., 2002, 90, 21.aaa 3. N. A. Sanina, S. M. Aldoshin, Russ. Chem. Bull. (Int. Ed.), 2011, 60, 1223 [Izv. Akad. Nauk, Ser. Khim., 2011, 1199]. 4. N. I. Neshev, E. M. Sokolova, B. L. Psikha, N. A. Sanina, T. N. Rudneva, Russ. Chem. Bull. (Int. Ed.), 2014, 63, 2020 [Izv. Akad. Nauk, Ser. Khim., 2014, 2020].

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5. N. I. Neshev, B. L. Psikha, E. M. Sokolova, N. A. Sanina, T. N. Rudneva, S. V. Blokhina, Russ. Chem. Bull. (Int. Ed.), 2010, 59, 2215 [Izv. Akad. Nauk, Ser. Khim., 2010, 2160]. 6. R. Lemberg, J. W. Legge, Hematin Compounds and Bile Pig ments; their Constitution, Metabolism, and Function, Inter science Publishers, New York, 1949, 748 pp. 7. W. G. Zijlstra, A. Buursma, W. P. Meeuwsenvan der Roest, Clin. Chem., 1991, 37, 1633. 8. N. A. Sanina, G. V. Shilov, S. M. Aldoshin, A. F. Shestakov, L. A. Syrtsova, N. S. Оvanesyan, E. S. Chudinova, N. I. Shkondina, N. S. Emel´yanova, A. I. Kotel´nikov, Russ. Chem. Bull. (Int. Ed.), 2009, 58, 572 [Izv. Akad. Nauk, Ser. Khim., 2009, 560]. 9. N. A. Sanina, T. N. Rudneva, S. M. Aldoshin, G. V. Shilov, D. V. Korchagin, Yu. M. Shul´ga, V. M. Martinenko, N. S. Ovanesyan, Inorg. Chim. Acta, 2006, 359, 570. 10. Pat. RF No. 2441872; Byul. Izobr. [Invention´s Bulletin], 2012, No. 4 (in Russian). 11. Pat. RF No. 2460531; Byul. Izobr. [Invention´s Bulletin], 2012, No. 25 (in Russian).

Received July 1, 2015; in revised form October 22, 2015