EFFECT OF BOVINE SERUM ALBUMIN ON REDOX AND LIGAND EXCHANGE REACTIONS INVOLVING AQUACOBALAMIN Текст научной статьи по специальности «Химические науки»

Porphyrins Порфирины

Макрогэтзроцмклы

http://macroheterocycles.isuct.ru

Paper Статья

DOI: 10.6060/mhc200498d

Effect of Bovine Serum Albumin on Redox and Ligand Exchange Reactions Involving Aquacobalamin

Ilia A. Dereven'kov,@ Sergei V. Makarov, and Pavel A. Molodtsov

This paper is dedicated to prof. Rudi van Eldik on the occasion of his 75th birthday. His works on vitamin B12 have been a source of inspiration for us

Institute of Macroheterocyclic Compounds, Ivanovo State University of Chemistry and Technology, 153000 Ivanovo, Russia @Corresponding author E-mail: derevenkov@gmail.com

Bovine serum albumin (BSA) is capable of binding aquacobalamin (H2OCbl) by two diffe ent modes, viz. (i) via hydrogen bonding or n-n interactions without substitution of water molecule, or (ii) via water substitution to give amino BSA-Cbl(III) complex. In this work, we showed that the first type of complex exhibits the same reactivity toward ascorbic acid and thiocyanate as H2OCbl. The amino BSA-Cbl(III) complex is substantially less reactive toward ascorbic acid, thiocyanate and sulfite than H2OCbl. Using sulfite, we showed that ligand exchange in this complex proceeds via two pathways, i.e. via (i) a direct substitution of BSA by sulfite species or (ii) a slow dissociation of BSA from the complex followed by rapid sulfite binding by Cbl(III).

Keywords: Cobalamin, serum albumin, ascorbic acid, redox reactions, ligand exchange.

Влияние бычьего сывороточного альбумина на редокс реакции и лигандный обмен с участием аквакобаламина

И. А. Деревеньков,@ С. В. Макаров, П. А. Молодцов

Институт макрогетероциклических соединений, Ивановский государственный химико-технологический университет, 153000Иваново, Россия @Е-таИ: derevenkov@gmail.com

Бычий сывороточный альбумин (БСА) способен связывать аквакобаламин (Н20СЬ1) двумя способами: за счет образования водородных связей или п-п взаимодействия без замещения молекулы воды или (и) в результате замещения воды и образования аминокомплекса. В этой работе показано, что комплекс первого типа обладает такой же реакционной способностью по отношению к аскорбиновой кислоте и тиоцианату, как и Н20СЬ1. Аминокомплекс БСА-СЫ(Ш) является намного менее реакционноспособным по отношению к аскорбиновой кислоте, тиоцианату и сульфиту, чем Н20СЬ1. С использованием сульфита показано, что лигандный обмен в этом комплексе протекает по двум маршрутам: через (0 прямое замещение БСА сульфитом или (И) медленную диссоциацию БСА из комплекса, сопровождающуюся быстрым связыванием сульфита кобаламином(Ш).

Ключевые слова: Кобаламины, сывороточный альбумин, аскорбиновая кислота, редокс реакции, лигандный обмен.

Introduction

Human serum albumin (HSA) is the most abundant protein in blood plasma.[1,2] Bovine serum albumin (BSA) is frequently used as a model of HSA due to close structural resemblance.[3] Serum albumins bind and transport numerous endo- and exogenous molecules.[4,5] Serum albumins are capable of binding at several specific sites[6] and transferring fatty acids,[7-9] steroids,[10,11] bilirubin,!12,131 carotenoids,[14] porphyrins,[15-17] and other compounds. They are also capable ofbinding drugs and drug-like molecules.181

Serum albumins are capable of reacting with cobalamins (Cbls; Figure 1).[19-25] Cyano- (CNCbl) and aqua- (H2OCbl) Cbls act as quenchers of HSA and BSA fluorescence, i.e. in the case of CNCbl and HSA the quenching occurs via the dynamic mechanism,[23] CNCbl quenches BSA fluorescence via the static or combined mechanism,[24-25] whereas the static mechanism was reported for H2OCbl and BSA.[22] It is reported that complexation of BSA with CNCbl leads to structural changes in protein molecule.[25] HSA tightly binds several hydrophobic Cbl derivatives to give hybrid materials capable of catalyzing carbon-skeleton rearrangement upon light irradiation.[26,27]

Figure 1. Structure of aquacobalamin.

Earlier, we showed that the reaction between H2OCbl and native BSA includes two steps.[22] The first step proceeds rapidly and generates a complex via hydrogen bonding or interactions without substitution of water molecule (hereafter, 'the labile complex'). In the course of the second step, relatively slow substitution of water molecule occurs and the amino complex between Co(III) and one of BSA lysine residues is formed. The formation of amino BSA-Cbl(III) complex does not proceed stoichiometrically, and

its fraction co-exists in solution with the labile complex. The interaction of H2OCbl and BSA does not affect the formation of methemealbumin, a complex between Fe(III)-heme and BSA, indicating involvement of different binding sites ofBSA in reactions withthese tetrapyrroles. Reduction of BSA changes the reaction mechanism: BSA forms with Co(III) thiolate complex, which is further decomposed to Co(II)-species.

H2OCbl is a relatively reactive complex, i.e. it can be involved in numerous ligand exchange[28-30] and redox reactions.[301 However, it remains unclear how binding to serum albumins affects its reactivity. This work provides data on reactions of H2OCbl in the absence and in the presence of BSA.

Experimental

Hydroxocobalamin hydrochloride (Sigma-Aldrich; HOCbl; >96 %), bovine serum albumin (Sigma-Aldrich; heat shock fraction, pH 5.2; >96 %), ascorbic acid (Sigma-Aldrich; AA; >99 %), sodium sulfite (Sigma-Aldrich; >98 %), potassium thiocyanate (Sigma-Aldrich; 99 %) were used without additional purification. Oxygen-free argon was used to deoxygenate reagent solutions.

Phosphate buffer solutions (0.1 M) were used to maintain pH during the measurements. The pH values of solutions were determined using Multitest IPL-103 pH-meter (SEMICO) equipped with ESK-10601/7 electrode (Izmeritelnaya tekhnika) filled by 3.0 M KCl solution. The electrode was preliminarily calibrated using standard buffer solutions (pH 1.65-12.45).

Concentrations of Cbl stock solutions were determined using UV-Vis spectroscopy via a conversion of Cbl to its dicyano-form (extinction coefficient is 30400 M-Lcm-1 at 368 nm[31]).

Ultraviolet-visible (UV-Vis) spectra were recorded on a cryothermostated (± 0.1 °C) Cary 50 UV-Vis spectrophotometer in quartz cells.

Equilibrium constants (K) were calculated using Eq. (1).[32]

A + K [L]

A =-

1 + K [L]

(1)

where [L] is the ligand concentration in solution, M; A is absor-bance at the monitoring wavelength for the complex at a particular ligand concentration; A0isthe absorbance forthe starting complex; Arn is the absorbance for the final complex.

Experimental data were analyzed using Origin 7.5 software.

Results and Discussion

Effect of BS on the Reaction between H2OCbl and Ascorbic Acid

It is well-known that H2OCbl can be efficiently reduced to Co(II)-form (Cbl(II)) by AA (Figure 2A).[33-35] We found that preliminary incubation of H2OCbl with BSA substantially decreases the yield of Cbl(II) in the reaction with AA, i.e. the UV-Vis spectrum of the product includes peaks at 358 and 537 nm (Figure 2B), which are not typical to Cbl(II) and H2OCbl. Addition ofBSA to Cbl(II) does not change its UV-Vis spectrum.

The yield of Cbl(II) strongly depends on incubation time of H2OCbl with BSA (Figure 3). The yield of Cbl(II) and kinetic curve profile coincide for experiments in the

Figure 2. UV-Vis spectra of the reaction of H2OCbl (7.640-5 M) with AA (7.540-4 M) in the absence (A) and in the presence (B) of BSA (4.040-4 M) at pH 7.0, 25.0 °C. The mixture of H2OCbl with BSA was incubated for 1 h prior adding AA.

Figure 3. Time-course curves of the reaction between H2OCbl (7.640-5 M) and AA (7.540-4 M) in the absence (1) and in the presence of BSA(4.0^10"4 M) atpH 7.0, 25.0 °C. Incubation time of H2OCbl with BSA is 0 (2), 15 (3), 30 (4) and 60 (5) min.

Figure 4. UV-Vis spectra of the second step of the reaction between H2OCbl (7.6-10-5 M) incubated with BSA (4.0-10-4 M) for 1 handAA(4.5-10-3 M) atpH7.0, 25.0 °C. Insert: a time-course curve of the reaction.

absence and in the presence of BSA, where BSA and AA are simultaneously added to Cbl(II), whereas the yield of Cbl(II) becomes lower upon increasing incubation time.

These observations can be explained by the slow formation of the amino complex between Cbl(III) and BSA,[22] which is relatively inert toward reduction by AA, whereas the labile complex between BSA and H2OCbl formed within mixing time has virtually the same reactivity as free H2OCbl. Despite the interaction between BSA and AA occurs via static mechanism (viz, via complexation with equilibrium constant K = 2.3-104 M-1 at 22.9 °C),[36] this process cannot account for the significant decrease in reaction rate between H2OCbl and AA in the presence of BSA, because a major AA fraction exists in unbound state under experimental conditions.

Further, we examined whether the amino complex between BSA and Cbl(III) can be reduced by higher

quantities of AA. The slow decay of absorbance at 535 nm indicating the reduction of the BSA-Cbl(III) complex is observed, although the reaction does not complete after 3 hours (Figure 4).

Effect of BS on the Reaction of H2OCbl with Thiocyanate and Sulfit

Next, we checked whether BSA can affect binding properties of H2OCbl toward thiocyanate and sulfite. H2OCbl is capable of reacting with SCN- to give mixture of N- and S-bound complexes[37-38] (K = 1.1-103,[39] 1.4-103[40] M-1, 25.0 °C) and almost stoichiometrically forms S-bound sulfitocobalamin.[41,42]

For binding SCN- by H2OCbl, K = (1.4 ± 0.1)-103 M-1 (25.0 °C) was obtained that coincides with literature data. [40] In the case of H2OCbl incubated in the presence of BSA

Wavelength, nm

Wavelength, nm

Figure 5. UV-Vis spectra collected in the course of H2OCbl (5.0-10 5 M) titration by SCN- in the absence (A) and in the presence (B) ofBSA (4.0-10-4 M) at рЯ 7.0, 25.0 °C. The mixture of H2OCbl with BSA was incubated for 1 h prior adding SCN". Inserts: plots of absorbance at 560 nm versus [SCN-] fitted to Eq. (1)

for 1 h, K = (1.1 ± 0.1)-103 M-1 (25.0 °C) was determined that is close to equilibrium constant for free H2OCbl, although in the presence of BSA UV-Vis spectral changes were less pronounced than for free H2OCbl (Figure 5). Formation of thiocyanato-Cbl(III) in these reactions proceeds rapidly, whereas prolonged incubation ofH2OCbl/BSA mixture with higher [SCN-] is accompanied by further changes in UV-Vis spectrum, i.e. a slight decay of absorbance at 358 nm is observed (Figure 6). However, we were unable to study this reaction due to its low rate. Probably, in the course of the rapid step, the labile complex of H2OCbl with BSA reacts with SCN- similarly to free H2OCbl. Further, the fraction of amino BSA-Cbl(III) complex is slowly transformed to thiocyanato-Cbl(III).

To study reactivity of amino BSA-Cbl(III) complex in ligand-exchange reactions, we used sulfite, which possesses

Figure 6. UV-Vis spectra of the second step of the reaction between H2OCbl (5.0-10-5 M) incubated with BSA (4.0-10-4 M) for 1 h and SCN" (8.0-10-2 m) atpH 7.0, 25.0 °C. Insert: a time-course curve of the reaction.

higher affinity toward Cbl(III) than thiocyanate. UV-Vis spectra of the reaction between an excess of sulfite and amino BSA-Cbl(III) complex is provided in Figure 7A, i.e. decrease in absorbance at 358 nm is observed. The spectrum of the product coincides with that of sulfito-Cbl(III).[43] A typical kinetic curve of the reaction (Figure 7A) is well described by exponential equation indicating first order with respect to amino BSA-Cbl(III) complex. Dependence of observed rate constant (kobs) versus sulfi e concentration is linear indicating first order with respect to sulfite and exhibits a positive intercept (Figure 7B), which can be explained by the parallel pathway of sulfito-Cbl(III) formation, i.e. sulfito-Cbl(III) canbe formed via substitution ofBSA in amino BSA-Cbl(III) complex by sulfite (which is ascribed to the slope of concentration dependence in Figure 7B) or via slow BSA dissociation from BSA-Cbl(III) and further rapid sulfite binding by Cbl(III) (which is ascribed to intercept of concentration dependence in Figure 7B; Scheme 1).

Conclusions

This study showed that two types of complexes of bovine serum albumin with aquacobalamin exhibit different reactivity in redox and ligand exchange reactions. A complex of H2OCbl and BSA rapidly formed without substitution of water molecule reacts with ascorbic acid and thiocyanate similarly to free H2OCbl. The slowly generated amino BSA-Cbl(III) complex can be reduced to Cbl(II) by ascorbic acid at substantially lower rate than H2OCbl. Ligand substitution reactions involving amino BSA-Cbl(III) proceed slowly as well. Using sulfite, we showed that substitution of BSA in this complex proceeds via two pathways, i.e. via a direct substitution of BSA by sulfite species or a slow dissociation of BSA from the complex followed by rapid sulfite binding by Cbl(III).

Figure 7. UV-Vis spectra of the second step of the reaction between H2OCbl (5.0-10-5 M) incubated with BSA (4.0-10-4 M)

for 1 h and SO32- (8.0-10-2 M) at pH 7.0, 25.0 °C (A; the insert is a time-course curve of the reaction), and plot of kobs versus [SO32-]

for the reaction (B).

slow

so;

slow

+ SO?- fast

Scheme 1. Mechanism of reaction between amino BSA-Cbl(III) complex and sulfite

Acknowledgements. This work was supported by the Russian Science Foundation (project no. 19-73-00147) to IAD.

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Received 01.05.2020 Accepted 13.05.2020