Introduction

Cobalamins (vitamin B12; Cbls) are the ubiquitous cobalt corrin complexes involved in numerous enzymatic processes, i.e. methyl transfer reactions, deoxyadenosyl radical assisted processes, dehalogenation reactions [1] and processes utilizing S-adenosylmethionine [2]. Besides the role as essential nutrients, Cbls have been successfully examined as antioxidants [3,4,5]; aquacobalamin (H2OCbl) is the well-known cyanide antidote [6,7,8,9], as well as the other prospective medicinal applications have been reported [10]. Cobinamide (Cbi; Fig. 1) is a nucleotide-free analog of cobalamin. Cbi exhibits a more pronounced effect as cyanide antidote than H2OCbi [9, 11], that can be partially explained by higher Cbi affinity toward CN−, i.e. equilibrium constant for CN− coordination on H2OCbl is 1012 M−1 [12], and those for first and second CN− molecules in the case of the reaction with aquahydroxocobinamide ((H2O)(HO−)Cbi) are > 1014 and ca. 108 M−1 [12, 13], respectively. Moreover, nitrocobinamide ((H2O)(NO2−)Cbi), a complex of Cbi with nitrite, possesses excellent intramuscular adsorption, whereas H2OCbl can be administered only intravenously [14].

Fig. 1
figure 1

Structure of diaquacobinamide (A), lysine and ethylenediamine (B), histidine and imidazole (C), and arginine and guanidine (D)

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Cobinamides have been examined in other applications as well. (H2O)(HO−)Cbi readily reacts with hydrogen sulfide via series of coordination and redox steps [15], and its reactivity toward H2S is superior to that of H2OCbl [16]. These facts explain successful experiments utilizing Cbi as H2S antidote [17, 18], whereas H2OCbl was inefficient in this respect [19]. (H2O)(HO−)Cbi is efficient as a scavenger of nitric oxide [20]. Nitrosylcobinamide, a complex of one-electron reduced Cbi with nitric oxide, possesses pronounced hypotensive effect [21], and facilitates wound healing [22] and formation of bone tissue [23]. Cobinamides (e.g., (CN−)2Cbi and its derivatives) have been successfully used to activate soluble guanylyl cyclase [24, 25]. Cbi acts as a redox catalyst as has been shown for dehydroascorbic acid reduction to ascorbic acid by glutathione [26]. Cobinamides are shown to possess antioxidant properties [27].

Efficacy of corrinoids as cyanide antidotes can be affected by their side reactions with biomolecules. For example, H2OCbl binds thiocyanate to give thiocyanato-Cbl, which reacts with CN− more slowly than H2OCbl, whereas SCN− does not affect reactivity of (H2O)(HO−)Cbi toward CN− [28]. H2OCbl binds with bovine serum albumin (BSA), a structurally close analog of human serum albumin [29], to give an inert in redox and ligand exchange processes amino complex, which is poorly reactive toward CN− [30, 31]. Nevertheless, affinity of cobinamides toward serum albumin remains poorly evaluated: it is just reported that (H2O)(HO−)Cbi tightly binds with BSA [32]. Interaction of (H2O)(HO−)Cbi with extracellular macromolecules may cause its poor absorption upon intramuscular injection [14, 33]. Here, we report results of study on binding of (H2O)(HO−)Cbi, (H2O)(NO2−)Cbi, (H2O)(CN−)Cbi and (CN−)2Cbi with BSA and the reactivity of generated complexes toward cyanide.

Experimental

Cyanocobalamin (vitamin B12; ≥ 98%; Sigma-Aldrich), bovine serum albumin (Sigma; heat shock fraction, pH 5.2; ≥ 96%), sodium borohydride (≥ 98%; Sigma-Aldrich), imidazole (99%; Alfa Aesar), ethylene diamine (99%; Alfa Aesar), sodium nitrite (≥ 98%; Sigma-Aldrich), tyrosine (≥ 98%; Sigma-Aldrich), guanidine hydrochloride (≥ 98%; Sigma-Aldrich), sodium periodate (≥ 99.8%; Sigma), diethyl pyrocarbonate (99%; Sigma-Aldrich), zinc acetate dihydrate (≥ 98%; Sigma-Aldrich) were used as received. Other chemicals were of analytical reagent grade. Buffer solutions (phosphate and the mixture of phosphate and borate; 0.1 M) were used to maintain pH during the measurements.

Synthesis of (H2O)(CN−)Cbi was performed according to published procedure [34]. Decyanation of (H2O)(CN−)Cbi included its reduction to Cbi(II) by equal amount of sodium borohydride under anaerobic conditions, acidification of solution by acetic acid to pH 3…4, addition zinc acetate (tenfold excess over (H2O)(CN−)Cbi) to bind cyanide with Zn2+, and oxidation of Cbi(II) to (H2O)2Cbi by equal amount of sodium periodate (our earlier experiments indicated that periodate does not modify corrin macrocycle [35]). (H2O)2Cbi was further purified using column chromatography on silica gel (Sigma-Aldrich; average pore size 60 Å (52–73 Å), 70–230 mesh, 63–200 μm) using 10% aqueous acetic acid as eluent. Identity of product to (H2O)2Cbi was carried out using ultraviolet–visible (UV–vis spectroscopy; λmax: 349, 496 and 519 nm at pH 7.4 for (H2O)(HO−)Cbi [28]) and Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-MS; m/z: 990.5 for [Cbi + H]+ ion). Concentrations of corrinoids were determined spectrophotometrically via their conversion to dicyano-species (ε367 = 30,400 M−1 cm−1 [36]). (H2O)(NO2−)Cbi and (CN−)2Cbi were prepared in situ by mixing (H2O)2Cbi with a two-fold excess of sodium nitrite and potassium cyanide, respectively.

Ultraviolet–visible (UV–Vis) spectra were recorded on a cryothermostated (± 0.1 °C) Cary 50 or Shimadzu UV-1800 UV–vis spectrophotometer in quartz cells. Kinetics of rapid reactions was studied on a thermostated (± 0.1 °C) RX2000 (Applied Photophysics, UK) rapid mixing stopped-flow accessory connected to Cary 50 spectrophotometer.

Equilibrium constants were calculated using function A = f([BSA]) derived from Eq. 1 [28]:

$$\frac{A-{A}_{0}}{{A}_{\infty }-A}=K\left(\left[\mathrm{BSA}\right]-\frac{A-{A}_{0}}{{A}_{\infty }-{A}_{0}}\left[\mathrm{Compl}.\right]\right),$$
(1)

Here [BSA] is the total (free + bound) BSA concentration in solution, M; [Compl.] is the total concentration of complex, M; A, A0, A∞ are absorbances at the monitoring wavelength for the complex at a particular BSA concentration, for the starting complex, and for the final complex, respectively; K is the equilibrium constant, M−1.

Fluorescence emission spectra were recorded on a Shimadzu RF-6000 spectrofluorophotometer in non-fluorescent cells under aerobic conditions at room temperature (23 ± 2 °C). The excitation wavelength was 280 nm, the excitation and emission bandwidths were 1.5 and 20.0 nm, respectively. The fluorescence intensities were corrected with respect to the inner filter effect [37].

Ultrafiltration experiments were performed using spin-filters with a molecular weight cut-off of 30 kDa (Amicon). Before experiments, spin-filters were first hydrated with 3 mL water by one cycle of centrifugation and then washed by 3 mL of 0.1 M phosphate buffer at 6000 rpm, 10 min in EBA 20 centrifuge (Hettich).

MALDI-MS measurements were performed on Shimadzu AXIMA Confidence mass-spectrometer with 2,5-dihydroxybenzoic acid as the matrix.

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).

Results and discussion

Reaction between (H2O)(HO−)Cbi with bovine serum albumin

Addition of BSA to solution of (H2O)(HO−)Cbi results in changes in ultraviolet–visible (UV–vis) spectrum shown in Fig. 2 i.e. maxima at 357 and 538 nm are emerged. There was no sharp isosbestic points indicating a presence of several consecutive steps in the reaction mechanism. UV–vis spectrum similar to the spectrum of the product of the reaction between (H2O)(HO−)Cbi and BSA can be generated via mixing (H2O)(HO−)Cbi with nitrogenous ligands, i.e. mixing (H2O)(HO−)Cbi with high excess of ethylene diamine (en) provides maxima at 356 and 536 nm due to the formation of amino complex (Fig. S1), whereas binding of one and two imidazole (ImH) molecules on Co(III)-ion produces species with maxima at 354 and 534 nm and 357 and 541 nm (Fig. S2), respectively. No changes in UV–vis spectrum were observed upon mixing with (H2O)(HO−)Cbi with tyrosine and guanidine (Figs. S3 and S4). In these experiments, en, ImH and guanidine mimic motifs of lysine, histidine and arginine side chains, respectively (Fig. 1). Therefore, UV–vis spectra provided by Fig. 2 can be attributed to complexation between (H2O)(HO−)Cbi and nitrogenous group of BSA. It is known that nitrogen atom of histidine can be efficiently ethoxylated by diethyl pyrocarbonate (DEPC) [38]. We modified BSA by tenfold excess of DEPC and compared reactions of (H2O)(HO−)Cbi with native and ethoxylated BSA. Using DEPC modified BSA, the final product exhibits maximum in UV–vis spectrum at 538 nm slightly less intense than in the case of the reaction with native BSA (Figs. S5 and S6). This results assumes that predominant coordination of Co(III) occurs via lysine amino groups, whereas histidine binding occurs at a lesser extent.

Fig. 2
figure 2

UV–vis spectra of reaction between (H2O)(HO−)Cbi (5.0 × 10–5 M) and BSA (1.0 × 10–4 M) at pH 7.4, 25.0 °C

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Further, we determined stoichiometry of the reaction by mixing (H2O)(HO−)Cbi with different quantities of BSA and allowing mixtures to react for 6 h. The results are provided by Fig. 3 indicating complete transformation of (H2O)(HO−)Cbi to the final product in almost equimolar mixture.

Fig. 3
figure 3

UV–vis spectra recorded in the course of titration of (H2O)(HO−)Cbi (5.0 × 10–5 M) by BSA at pH 7.4, 25.0 °C. Inset: a plot of absorbance at 540 nm versus [BSA]

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Next, we studied the kinetics of the reaction using an excess of BSA over (H2O)(HO−)Cbi. The typical kinetic curve of the reaction is shown in Fig. 4. It is described by a two-exponential equation, which can be explained by the presence of two consecutive steps in the system. The dependence of observed rate constants (kobs.) on [BSA] for the faster step is linear and exhibits a positive Y-intercept (Fig. 5), that is typical for ligand exchange process. Dividing the value of the slope by the value of the intercept gives value of the equilibrium constant of 7.5·103 M−1, which is far from equimolar binding between reactants. Observed rate constants for the second step of the reaction were poorly reproducible, probably, due to the low contribution of this step in overall UV–vis spectral changes of the reaction in comparison with the first step. Apparently, the second step involves rearrangement of BSA-Cbi complex and increases overall equilibrium constant of the reaction.

Fig. 4
figure 4

Kinetic curve of reaction between (H2O)(HO−)Cbi (5.0 × 10–5 M) and BSA (5.0 × 10–4 M) at pH 7.4, 25.0 °C

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Fig. 5
figure 5

Plot of kobs. versus [BSA] for the reaction between (H2O)(HO−)Cbi and BSA at pH 7.4, 25.0 °C

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The plot of slopes of concentration dependencies of the first step (k′) versus pH is shown in Fig. 6. It exhibits sigmoid profile at pH ca. 6.5 and increase at pH > 8. The sigmoid profile can be explained by the protonation of (H2O)(HO−)Cbi to (H2O)2Cbi (pKa = 5.9 at 25 °C [39]), as well as by contribution of histidine binding to Co(III) and its protonation in acidic medium (pKa ca. 6.6 at 25 °C in proteins [40]). Acceleration of the reaction upon alkalinization is characteristic to the amino group coordination, which is deprotonated in alkaline medium (pKa ca. 10.5 at 25 °C in proteins [40]). The conversion of (H2O)(HO−)Cbi to (HO−)2Cbi is characterized by pKa = 10.3 at 25 °C [39].

Fig. 6
figure 6

Plot of k’ versus pH for the reaction between (H2O)(HO−)Cbi and BSA at 25.0 °C

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Next, we examined complexation between (H2O)(HO−)Cbi and BSA using fluorescence spectroscopy. Addition of (H2O)(HO−)Cbi to BSA results in a low quenching of tryptophan fluorescence, whereas prolonged incubation of reactants leads to a deep fluorescence quenching (Fig. S7) that agrees with slow complexation between reactants and formation of weakly fluorescent BSA-Cbi complex.

Strengths of complexation between (H2O)(HO−)Cbi and BSA was examined by ultrafiltration, since retention of Cbi species by BSA may involve hydrogen bonding, which can be poorly supported by UV–vis and fluorescence data. Although (H2O)(HO−)Cbi is slightly adsorbed on membrane upon filtration (ca. 10%), its contribution cannot explain significant decrease in cobinamide concentration in permeate in comparison with the initial solution (Fig. S8): i.e. permeate contains ca. 5% ([(H2O)(HO−)Cbi]0:[BSA]0 = 1: 8), ca. 10% ([(H2O)(HO−)Cbi]0:[BSA]0 = 1: 1) % of Cbi in comparison with initial solutions. Thus, ultrafiltration supports relatively tight complexation between (H2O)(HO−)Cbi and BSA, though minor fraction of (H2O)(HO−)Cbi is weakly bound by BSA and is washed through the membrane in the course of ultrafiltration.

In several experiments, cobinamides are administered as cyanide antidotes prior to the toxin introduction [41], thus, we tested the influence of (H2O)(HO−)Cbi binding with BSA on its reactivity toward cyanide. Addition of CN− to (H2O)(HO−)Cbi results in the rapid formation of (CN−)2Cbi exhibiting maxima at 367, 540 and 580 nm (Fig. S9). In the case of complex between BSA and Cbi, generation of (CN−)2Cbi occurs significantly slower: although the minor fraction of (CN−)2Cbi is formed within 1 min after addition of CN−, complete transformation complex between Cbi and BSA to (CN−)2Cbi is not observed for 2 h of the reaction (Fig. 7).

Fig. 7
figure 7

UV–vis spectra collected after mixing complex between (H2O)(HO−)Cbi ([(H2O)(HO−)Cbi]0 = 5.0 × 10–5 M) and BSA ([BSA]0 = 4.0 × 10–4 M) with CN− (1.0 × 10–4) at pH 7.4, 25.0 °C. Inset: a time-course curve of the reaction

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Despite the high reactivity of (H2O)(HO−)Cbi toward CN−, it is rarely used in experiments as antidote due to certain toxicity [20]. This drawback can be eliminated by using (H2O)(HO−)Cbi derivatives (e.g. nitrocobinamide; (H2O)(NO2−)Cbi). Thus, we examined (H2O)(NO2−)Cbi reaction with BSA as well. Changes in UV–vis spectrum of the reaction between (H2O)(NO2−)Cbi and BSA are shown in Fig. S10, i.e. the same product is formed as in the case of reaction of (H2O)(HO−)Cbi with BSA. Thus, nitrite molecule in (H2O)(NO2−)Cbi is substituted by one of BSA amino groups. Binding of (H2O)(NO2−)Cbi to BSA is accompanied by quenching of tryptophan fluorescence (Fig. S11) as in the case of the reaction involving (H2O)(HO−)Cbi. Data shown in Fig. S12 indicate that significant fraction of (H2O)(NO2−)Cbi is retained by BSA upon ultrafiltration, i.e. permeate contains ca. 5% ([(NO2−)Cbi]0:[BSA]0 = 1: 8), ca. 15% ([(NO2−)Cbi]0:[BSA]0 = 1: 1) % of Cbi in comparison with initial solutions, whereas membrane adsorbs ca. 12% of (H2O)(NO2−)Cbi. Binding of (H2O)(NO2−)Cbi to BSA decreases its reactivity toward CN− as well: complex cannot be transformed completely to (CN−)2Cbi for 2 h, although the major fraction of CN− binds to cobinamide within ca. 2 min (i.e. faster than in the case of the reaction involving the complex between (H2O)(HO−)Cbi and BSA).

Reaction between (H2O)(CN−)Cbi with bovine serum albumin

Mixing of (H2O)(CN−)Cbi with BSA produces complex with absorption maxima at 360, 520 and 549 nm (Fig. 8). It proceeds faster than complexation of (H2O)(HO−)Cbi with BSA, i.e. for several seconds and hundreds of seconds in the case of ((H2O)(CN−)Cbi and ((H2O)(HO−)Cbi, respectively (pH 7.4, 25.0 °C). The final product of the reaction between (H2O)(CN−)Cbi and BSA exhibits spectrum, which is close to those of (en)(CN−)Cbi (λmax: 359, 519 and 547 nm; Fig. S14) and (ImH)(CN−)Cbi (λmax: 360, 519 and 552 nm; Fig. S15). Tyrosine and guanidine do not react with (H2O)(CN−)Cbi (Figs. S16 and S17). Modification of BSA by DEPC slightly decreases intensity of peaks at 520 and 549 nm in UV–vis spectrum of reaction product (Fig. S18) indicating formation of minor fraction of BSA(histidine)–(CN−)Cbi complex and predominance of BSA(lysine)–(CN−)Cbi form.

Fig. 8
figure 8

UV–vis spectra recorded before (1) and after (2) mixing of (H2O)(CN−)Cbi (4.0 × 10–5 M) and BSA (3.3 × 10–4 M) at pH 7.4, 25.0 °C

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In contrast to binding of (H2O)(HO−)Cbi, the complexation between (H2O)(CN−)Cbi and BSA occurs more weakly and requires ca. tenfold excess of BSA over (H2O)(CN−)Cbi to completely convert it to bound state (Fig. 9). Fitting the plot of absorbance at 555 nm versus [BSA] to Eq. 1 gives value of equilibrium constant K = (6.9 ± 0.6) × 103 M−1 (pH 7.4, 25.0 °C).

Fig. 9
figure 9

UV–vis spectra recorded in the course of titration of (H2O)(CN−)Cbi (3.9 × 10–5 M) by BSA at pH 7.4, 25.0 °C. Inset: a plot of absorbance at 555 nm versus [BSA] fitted to Eq. 1

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Typical kinetic curve of the reaction between (H2O)(CN−)Cbi and an excess of BSA is shown in Fig. 10. It is described by exponential equation indicating the first order with respect to cobinamide. The plot of observed rate constants versus [BSA] is linear and shows a positive Y-intercept (Fig. 11) that is typical to the reversible complexation between reactants. Dividing the value of the slope by the value of the intercept gives value of the equilibrium constant K = 5.9·103 M−1, which agrees with the value obtained by the titration (6.9·103 M−1).

Fig. 10
figure 10

Kinetic curve of reaction between (H2O)(CN−)Cbi (4.0 × 10–5 M) and BSA (4.0 × 10–4 M) at pH 7.4, 25.0 °C

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Fig. 11
figure 11

Plot of kobs. versus [BSA] for the reaction between (H2O)(CN−)Cbi and BSA at pH 7.4, 25.0 °C

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Dependence of the rate constant of the forward reaction between (H2O)(CN−)Cbi and BSA on pH (Fig. 12) is similar to that involving (H2O)(HO−)Cbi (Fig. 6) with the exception of the absence of sigmoid profile in a near neutral region. Conversion of (H2O)(CN−)Cbi to (HO−)(CN−)Cbi is characterized by pKa = 11.0 (25 °C) [42]. Thus, increase of rate upon alkalinization corresponds to deprotonation of lysine residues to the more reactive NH2-form.

Fig. 12
figure 12

Plot of k′ versus pH for the reaction between (H2O)(CN−)Cbi and BSA at 25.0 °C

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Complexation between (H2O)(CN−)Cbi and BSA results in a weak tryptophan fluorescence quenching (Fig. S19), that is distinct from substantially deeper quenching effects of (H2O)(HO−)Cbi (Fig. S7) and (NO2−)2Cbi (Fig. S11). These observations can be explained by different binding sites of (H2O)(CN−)Cbi and (H2O)(HO−)Cbi or (NO2−)2Cbi in BSA molecule. In the case of (H2O)(HO−)Cbi and (NO2−)2Cbi, the binding site is located closer to BSA tryptophan residue than for (H2O)(CN−)Cbi.

Using ultrafiltration experiments (Fig. S20), we showed that (H2O)(CN−)Cbi retention by BSA is substantial in the case of a eightfold excess of BSA over (H2O)(CN−)Cbi (i.e. permeate contains ca. 8% of initial Cbi concentration) and becomes lower in the equimolar mixture (i.e. Cbi concentration in permeate is ca. 35% of the initial concentration). Binding of free (H2O)(CN−)Cbi on the membrane is ca. 8%.

We found that binding of (H2O)(CN−)Cbi by BSA slightly decreases rate of the reaction with CN− (Fig. S21), however, the reaction is substantially faster than processes involving complexes of BSA with (H2O)(HO−)Cbi (Fig. 7) or (NO2−)2Cbi (Fig. S13).

(CN−)2Cbi reacts with BSA as well (Fig. 13). The reaction is accompanied by slow gradual decrease of absorbance at 580 nm due to its partial decyanation. The reaction results in a slight tryptophan fluorescence quenching (Fig. S22), which is comparable with that observed for (H2O)(CN−)Cbi (Fig. S19).

Fig. 13
figure 13

UV–vis spectra collected for the reaction between (CN−)2Cbi ([(CN−)2Cbi]0 = 3.0 × 10–5 M) and BSA ([BSA]0 = 4.0 × 10–4 M) at pH 7.4, 25.0 °C. Time interval between spectra is 1 min, the final spectrum was recorded after 24 h of incubation. Inset: a time-course curve of the reaction

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Ultrafiltration experiments indicate that (CN−)2Cbi concentration in permeate is ca. 25% of the initial in the case of an eightfold excess of BSA and ca. 35% of the initial in the case of the equimolar mixture (Fig. S23). (CN−)2Cbi binding by membrane is ca. 15%. Therefore, introduction of tightly bound ligands to cobinamide decreases its retention by BSA. However, ultrafiltration data shows that weak complexation between (CN−)2Cbi and BSA occurs and is contributed probably by hydrogen bonding.

Conclusions

This work showed for the first time that cobinamide species are reactive toward proteins. Thus, bovine serum albumin binds (H2O)(HO−)Cbi, (H2O)(NO2−)Cbi and (H2O)(CN−)Cbi predominantly via aminogroups of lysine residues, as well as the minor fraction exists as the histidine complex. In the case of (H2O)(HO−)Cbi, binding occurs in equimolar ratio. Upon binding with BSA, the reactivity of (H2O)(HO−)Cbi and (H2O)(NO2−)Cbi toward cyanide is substantially decreased, although rate of CN− binding is higher in the case of nitro-Cbi-BSA complex that may partially explain why (H2O)(NO2−)Cbi is used more frequently as CN− antidote than (H2O)(HO−)Cbi. These results suggest that introduction of Cbi might be more efficient after cyanide poisoning than prior this event to prevent the formation of inert complex between Cbi and BSA. The binding of (H2O)(CN−)Cbi is much weaker than of (H2O)(HO−)Cbi, and sustains relatively high reactivity toward CN−. Moreover, binding of (H2O)(CN−)Cbi and (H2O)(HO−)Cbi occurs at different sites of BSA as was indicated by spectrofluorimetric titration. (CN−)2Cbi is the least reactive toward BSA Cbi species studied in this work, and is poorly retained by BSA.