Abstract
The interaction of L-cysteine with bismuth compounds bismuth(III) salicylate, bismuth(III) citrate, and bismuth(III) nitrate, was studied at pH 1.0 (0.100 M HNO3 and 0.100 M HCl) and pH 7.4 MOPS buffer by cyclic voltammetry at glassy carbon and boron-doped diamond electrodes. pH 1.0, at which bismuth (III) exists as the simple Bi3+ ion, was chosen to approximate the acid strength of stomach contents. pH 7.4, at which bismuth(III) exists as BiO, was used for its similarity to general physiological conditions. The amino acid L-cysteine was chosen because its sulfhydryl group undergoes intense interaction with many metal cations, serving as a model for cysteine-containing proteins in the digestive system. It was determined that Bi(III) and L-cysteine (Cys) form soluble complexes at both pH 1.0 and pH 7.4. UV–vis spectroscopic investigations support interaction of Bi(III) and L-cysteine to form a 1:2 Bi(III): Cys complex in pH 7.4 MOPS buffer. L-cysteine addition to solutions of the pharmaceutical bismuth(III) salicylate was found to alter the voltammetric behavior of the salicylate complex. These results, especially at pH 1.0, are relevant to understanding the interaction of various cysteine-containing proteins in the human digestive system with bismuth pharmaceuticals and may help guide future explorations of bismuth formulations.
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This paper is part of the JES Focus Issue on Organic and Inorganic Molecular Electrochemistry. This paper 2530 was accepted to be presented at the Montreal, QC, Meeting of the Society, May 10–14, 2020.
Due to their essential roles in the treatment of gastrointestinal disorders, colitis, and other various conditions, bismuth compounds have played an important role in the field of medicine.1–5 Bismuth drugs, such as bismuth subsalicylate, colloidal bismuth subcitrate, and ranitidine bismuth citrate, have been used to treat various gastrointestinal disorders.1,2,4 As such, many studies have been conducted to shed light on the interaction between bismuth and various proteins found in the digestive system.1,2,4 Recent research on bismuth compounds has encompassed many techniques which include inductively coupled plasma mass spectrometry (ICP-MS), fluorescence, X-ray diffraction, and NMR spectroscopy.1,2 Some of the effects of bismuth-based drugs are thought to be due to the intense interaction between bismuth(III) and certain amino acid side-chains in various proteins. L-Cysteine, with its sulfhydryl side-chain, figures prominently in these mechanisms.2,4 Analytical methods for bismuth pharmaceuticals have appeared, some of which are based on electrochemical techniques.6–8 To our knowledge, however, few if any studies have utilized electrochemistry as a method to study the interactions between bismuth and amino acids. Electrochemical methods provide an inexpensive yet powerful way to evaluate the details of metal—ligand interactions, primarily by means of potential shifts as ligands are added to the metal cations. In the present work, electrochemical methods are applied to the study of L-cysteine interactions with bismuth(III) ions. In particular, a change in the voltammetric behavior of bismuth(III) salicylate, a common bismuth-based pharmaceutical, upon L-cysteine addition is expected if there is a significant interaction between the two substances. In this way, a general pathway of such interactions can be established, which may also be extended to various cysteine-containing proteins in the digestive system. Electrochemical methods, then, represent a powerful, and in this case, a rather novel way to evaluate bismuth interactions with sulfur-containing substances such as L-cysteine and related proteins. By studying such interactions, the results of this work should be of considerable interest to the pharmaceutical community by establishing a general pathway by which bismuth-based pharmaceuticals interact with sulfur-based ligands in the digestive tract. Previous research from this laboratory has involved the electrochemical study of L-cysteine and other amino acids with side-chains capable of complexing metal cations.9–11 The results of electrochemical studies of the interaction of L-cysteine with trivalent bismuth compounds are presented in this work. It should be noted here that the structures of bismuth complexes with ligands such as salicylate and citrate are extremely complicated,1–3 and the structures given in this work are simple representations.
Experimental
L-Cysteine, bismuth(III) subsalicylate, bismuth(III) citrate, bismuth subnitrate, and MOPS were obtained from Sigma-Aldrich and used as received. [It should be noted that the term "bismuth subsalicylate" is an older term referring to bismuth(III) salicylate, but the term is still used for bismuth pharmaceuticals.] Cyclic voltammetric experiments were carried out using a Gamry Interface 1000 Potentiostat, and potentials were measured vs a Ag/AgCl reference electrode. Glassy carbon electrodes (3 mm diameter) were obtained from Bioanalytical Systems, and the boron-doped diamond (BDD) electrode (3 mm diameter) was obtained from Windsor Scientific as purchased through eDAQ. The electrodes were manually polished using Buehler 0.05 μm alumina polishing suspension on a felt polishing pad, rinsed with deionized water, then placed in a Cole-Parmer 8890 sonicator for five minutes to remove alumina particles. The electrochemical cell was a 20 ml cylindrical cell fitted with a PTFE top and 1 mm diameter platinum wire counterelectrode. Bismuth compounds were added to appropriate electrolytes to prepare 10.0 ml of 1.0 mM bismuth solution. The contents in the cell were bubbled with N2 gas and mixed with a magnetic stirring bar for 10 min to remove oxygen from the solutions. The cell was cleaned by soaking the cell and all of its components in aqueous 0.100 M K2EDTA solution overnight in order to remove any bismuth ions adsorbed to the glass cell walls. All experiments were carried out at 22 ± 1 °C.
A Jasco V-670 spectrophotometer was utilized to obtain UV–vis spectra. 2.0 ml of 0.10 M pH 7.4 MOPS buffer was pipetted into a quartz cuvette (1.00 cm path length). Bismuth(III) nitrate was added to the MOPS buffer solution into the cuvette to prepare a 1.0 mM Bi(NO3)3 solution. The cell was de-aerated with a nitrogen stream, then the top of the cell was sealed with Parafilm® in order to exclude oxygen from the cell. Following acquisition of Bi(NO3)3 UV–vis spectra, 0.100 M L-cysteine solution was added in increments into the cuvette through the Parafilm® covering using a microsyringe. In this way, spectra of the various stages of Bi(III) complexation by L-cysteine were obtained.
Results and Discussion
These studies were carried out at pH 1.0 and pH 7.4; therefore, it is helpful to specify the forms in which L-cysteine and bismuth(III) species exist under these conditions. According to the Pourbaix diagram,12 bismuth exists as the simple Bi3+ ion at pH 1.0 and as BiO+ at pH 7.4 For L-cysteine, Fig. 1 presents the pKa values for the three functional groups on this amino acid.13 At pH 1.0, all three functional groups are in the protonated forms, and at pH 7.4 only the carboxylic acid moiety is fully de-protonated (zwitterionic form). The chemical forms of both bismuth and cysteine are important because they affect the stability of complexes formed between them. The 1.0 mM concentration level for bismuth(III) was chosen in order to correspond to the rather low levels of L-cysteine (<1 mM) found in most physiological fluids.14,15 Typical levels of bismuth pharmaceuticals in bodily fluids are likely slightly higher than 1 mM. A dose of 500 mg bismuth subsalicylate (362 g mol−1) in 1 liter of stomach contents would produce a nominal 1.4 mM bismuth(III) concentration. The use of 1.0 mM bismuth compounds seems, then, appropriate to match typical pharmaceutical bismuth(III) concentrations as well as the L-cysteine concentrations found in the body.
Figure 1. pH distribution for L-Cysteine species. The structure of L-cysteine is at right.
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Standard image High-resolution imageStudies at pH 1.0
At pH 1.0 (0.10 M HNO3), bismuth(III) nitrate produces only a small reduction process at −0.37 V vs Ag/AgCl at glassy carbon due to poor solubility, with a broad bismuth stripping peak at + 0.07 V. The high hydrogen overpotential at the glassy carbon working electrode allowed voltammetric experiments in this highly acidic medium, whereas the use of platinum or gold would have produced large current responses for proton reduction. The stripping peak potential corresponds rather closely with the value given for the Bi/Bi3+ couple (+0.317 V vs SHE,16 or +0.120 V vs Ag/AgCl). Upon addition of L-cysteine, the current for the reduction process was found to greatly increase, indicating that Bi3+ ion was now forming a soluble complex with L-cysteine. This interaction greatly increases the solubility of the bismuth ion. In addition, the peak potential for the reduction process is shifted from −0.37 V to −0.27 V, reflecting the difference in reduction potentials for the complex compared to the Bi3+ ion. The stripping peak current on the return sweep is also much greater, due to the higher complex concentration in solution, even with a shorter negative potential excursion. Further L-cysteine additions produced greater reduction, and stripping peak, currents, suggesting successive higher complexation steps and possibly faster electron-transfer kinetics.
Figure 2. Cyclic voltammograms for 1.0 mM Bi(NO3)3 at glassy carbon in 0.100 M HNO3, 100 mV s−1, showing the effects of L-cysteine additions. (a) no added L-cysteine blue square dotted line. (b) 1:1 Bi3+: L-Cysteine green round dotted line. (c) 1:2 Bi3+: L-Cysteine red dashed line. (d) 1:3 Bi3+: L-Cysteine black solid line. (e) 1.0 mM L-Cysteine orange thin line.
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Standard image High-resolution imageSimilar studies using bismuth subsalicylate, a commonly used medication for stomach ailments,1–3 revealed that bismuth subsalicylate is soluble to at least 1.0 mM in 0.10 M HNO3, producing a clear, colorless solution. The cyclic voltammetric response (Fig. 3) shows a clearly defined reduction peak at −0.30 V, and a bismuth stripping peak at +0.07 V vs Ag/AgCl. This behavior is similar to that seen in Fig. 2 after complex formation due to the addition of L-cysteine. It is clear that bismuth subsalicylate does not form the uncomplexed Bi3+ ion by dissociation in 0.10 M HNO3; otherwise, a voltammetric response similar to Fig. 2a would have been observed. Upon addition of L-cysteine to bismuth subsalicylate, small changes in the voltammetric behavior were observed. At the 1:1 Bi(sal):L-cysteine point, the reduction potential shifted to −0.194 V in a broad process, with bismuth stripping at +0.116 V. At the 1:2 point, the reduction potential shifted back to −0.27 V in a narrower process, with bismuth stripping at the same potential as for the 1:1 point. Finally, further L-cysteine addition to the 1:3 point produces only a slight further negative reduction potential shift. These results are generally consistent with the replacement of the salicylate ligand with L-cysteine. It is not possible to fully characterize the resulting complexation behavior with these results; however, the 1:1 point may be due to a mixed salicylate/L-cysteine complex, with complete replacement of the salicylate by L-cysteine at the 1:2 point. The further slight negative shift at the 1:3 point is probably due to excess L-cysteine in the solution after formation of Bi(Cys)2. This small shift is typical of metal complexes in the presence of excess ligand.17 These results show that L-cysteine is capable of displacing the salicylate ligand from Bi(III), implying that bismuth(III) salicylate may interact in a similar fashion with cysteine-containing proteins under the low pH conditions found in stomach contents.
Figure 3. Cyclic voltammograms for 1.0 mM Bi(III) salicylate at glassy carbon in 0.100 M HNO3, 100 mV s−1, showing the effects of L-cysteine additions. (a) no added L-cysteine blue square dotted line. (b) 1:1 Bi(III) salicylate: L-Cysteine green round dotted line. (c) 1:2 Bi(III) salicylate: L-Cysteine red dashed line. (d) 1:4 Bi(III) salicylate: L-Cysteine black solid line. (e) 1.0 mM L-Cysteine orange thin line.
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Standard image High-resolution imageA brief study was also carried out for bismuth(III) citrate, another commonly used pharmaceutical, at pH 1.00 using both 0.100 M HNO3 and 0.100 M HCl, at a boron-doped diamond electrode (Fig. 4). The HCl solution was intended to simulate the chemical environment of typical stomach contents. As shown in the figure, voltammetry in 0.100 M HNO3 produced a lower current response than did a scan in 0.100 HCl. The response in 0.100 M HNO3 is very similar to that observed in Fig. 2a, indicating that the citrate complex dissociates in 0.100 M HNO3, leaving a slightly soluble Bi3+ ion with its relatively low reduction current. For the 0.100 M HNO3 scan, the reduction potential for Bi3+ is also very similar to that in Fig. 2a, further supporting the dissociation hypothesis. The higher currents, and different potential values, found in the 0.100 M HCl study suggest that formation of soluble bismuth chloride complexes is probably involved in HCl solution.16 Although citrate is a multidentate ligand, the relatively high concentration of chloride in 0.100 M HCl apparently allows the formation of such complexes as the citrate complex dissociates in the acidic solution.
Figure 4. Cyclic voltammograms for 1.0 mM Bi(III) citrate at boron-doped diamond, 100 mV s−1, in the solutions indicated below. (a) 0.100 M HNO3 black solid line. (b) 0.100 M HCl red dotted line.
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Standard image High-resolution imageStudies at pH 7.4
An investigation of bismuth(III) nitrate at pH 7.4 was also carried out in order to assess the behavior of bismuth in more usual physiological conditions. MOPS buffer was used because it generally exhibits minimal complexation of metal ions.18 PBS buffer was also considered; however, MOPS also avoids the likely interaction of bismuth(III) with the phosphate ions in the PBS buffer. Figure 5 illustrates the incremental additions of L-cysteine to bismuth(III) nitrate at pH 7.4 using a glassy carbon electrode. With no added L-cysteine, there are no redox processes. This is due to the fact that bismuth(III) nitrate did not dissolve in the solution, which had a cloudy appearance. Upon addition of L-cysteine at a 1:1 Bi3+:Cys ratio, the solution became clear, resulting in a voltammogram having one reduction peak and two stripping peaks. This behavior indicates that L-cysteine forms a soluble 1:1 complex with Bi3+ ion. At a 1:2 Bi3+:Cys ratio, the reduction peak current increases and a slight potential shift to more negative value occurs. This behavior is possibly due to an increase in the electron-transfer rate for the 1:2 complex reduction process, or perhaps due to complete solubility of this complex. At a 1:3 ratio, the reduction peak potential shifts to the more negative values due to excess L-cysteine in the solution.17 This behavior has already been noted for the bismuth subsalicylate/L-cysteine case, implying that two L-cysteine ligands are involved in this complex. A brief explanation of the two stripping peaks is necessary at this point. As can be observed in Fig. 5, there are two stripping peaks, one at −0.20 V and the other at 0.00 V. The relative peak current for the stripping peaks changes as L-cysteine is added, the current for the process at −0.20 V increasing relative to that for the process at 0.00 V. A plausible explanation for this behavior relies on the fact that electrodeposition of bismuth metal from the Bi(Cys)2 complex leaves the solution immediately next to the electrode surface depleted of bismuth ions, with L-cysteine remaining in solution at concentrations similar to the bulk concentration values. As bismuth is stripped from the electrode surface, the bismuth ion concentration next to the surface momentarily increases well beyond bulk Bi3+ values, so that L-cysteine is in relatively short supply as it complexes the newly formed Bi3+. At the lowest L-cysteine concentration, the relatively large bismuth ion concentration during stripping partially reforms the bismuth:L-cysteine complex; however, the relatively low L-cysteine concentration means that most of the bismuth(III) ions are not complexed. This behavior accounts for the observation of the stripping process in curve (a) predominantly at 0.00 V, close to the standard potential for Bi/Bi3+ noted above. As the L-cysteine concentration is increased, more bismuth ions are complexed with L-cysteine in solution as stripping proceeds, resulting in a more facile stripping process at less positive potentials due to this interaction. This is the process observed at −0.20 V vs Ag/AgCl. This process now has a peak current equal to that of the stripping process at 0.00 V.
Figure 5. Cyclic voltammograms for 1.0 mM Bi(NO3)3 at glassy carbon in pH 7.4 MOPS buffer, 100 mV s−1, showing the effects of L-cysteine additions. (a) no added L-cysteine blue square dotted line. (b) 1:1 Bi3+: L-Cysteine green round dotted line. (c) 1:2 Bi3+: L-Cysteine red dashed line. (d) 1:3 Bi3+: L-Cysteine black solid line. (e) 1.0 mM L-Cysteine orange thin line.
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Standard image High-resolution imageUV–vis spectroscopy of bismuth/L-cysteine complex at pH 7.4
Although voltammetric information can give a good indication of the number of ligands involved in complexation, a study of the UV–vis behavior of the complex system can support these findings. In the present case, it was found that bismuth(III) ion in the form of bismuth(III) nitrate absorbs at 223 nm, and that L-cysteine absorbs at 237 nm, close to the 233 nm value previously reported.19 Starting with a solution of 1.0 mM Bi(NO3)3 in pH 7.4 MOPS solution, additions of 0.100 M L-cysteine by microsyringe produced a great increase in the absorbance of the band around 237 nm as well as the appearance of a new band at 325 nm due to the Bi/Cys complex. It is interesting to note that a UV–vis band at 350 nm has been observed upon addition of bismuth(III) to yeast alcohol dehydrogenase,1 as well as for bismuth(III) with cysteine-containing peptides,20 an effect ascribed to the interaction of Bi3+ with the protein thiolate groups.1,20 Beyond the 1:2 Bi:Cys point, the absorbance at 237 nm increases greatly and produces considerable background absorbance at 325 nm. The JASCO background correction software was used to subtract the contribution from the 237 nm band, and the results are shown in Fig. 6. It is apparent that the absorbance of the 325 nm process remains constant beyond the 1:2 Bi:Cys point, as illustrated in Fig. 7. These results suggest that Bi(NO3)3 and L-cysteine form a Bi(Cys)2 complex in pH 7.4 MOPS buffer. The unfortunate proximity of the Bi:Cys absorbance band at 325 nm to the intense 237 nm band contributes to the absorbance of the former band and therefore introduces some degree of uncertainty to the ligand value. It should be noted that a range of compositions has been reported for the interaction of Bi(III) with L-cysteine in aqueous media, undoubtedly due to the effect of other solution components and the solution pH. A Bi(Cys)3 complex has been reported,8 as well as Bi2Cys3.21 Both Bi(Cys) and Bi(Cys)2 have been observed by electrospray ionization mass spectrometry (ESI MS).22
Figure 6. UV–vis spectroscopy of 1.0 mM Bi(NO3)3 in pH 7.4 MOPS buffer, with incremental additions of L-cysteine, after subtraction of 237 nm background. Path length: 1.00 cm. (a) Bi(NO3)3 1.0 mM blue round dotted line. (b) L-Cysteine (separate run) green square dotted line. (c) 1:0.5 Bi3+: L-Cysteine brown dashed-dotted line. (d) 1:1.0 Bi3+: L-Cysteine red dashed line. (e) 1:1.5 Bi3+: L-Cysteine orange dash dotted line. (f) 1:2.0 Bi3+: L-Cysteine black solid line. (g) 1:2.5 Bi3+: L-Cysteine violet long dashed line.
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Figure 7. UV–vis spectroscopy, workup of data from Fig. 6 using a fourth order polynomial fit. A 20 μl addition of L-cysteine corresponds to an equimolar amount of Bi3+.
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Conclusions
Cyclic voltammetric experiments involving bismuth ion reduction have shown that L-cysteine and Bi3+ form soluble complexes in both pH 1.0 HNO3 and pH 7.4 MOPS buffer at the 1 mM level. The pH 1.0 level was chosen as representative of the acidity levels of stomach contents, whereas pH 7.4 is typical of physiological conditions in most of the rest of the human body. Following the reduction of the bismuth-cysteine complexes to bismuth metal, subsequent bismuth stripping is observed on the return sweep. UV–vis spectroscopy suggested that bismuth(III) and L-cysteine form a 1:2 complex in pH 7.4 MOPS buffer. These results support the intense interaction between the bismuth(III) oxidation state and L-cysteine in both pH 1.0 and pH 7.4 environments. The findings at pH 1.0 are particularly interesting because they deal with typical acidity levels found in human stomach contents. In 0.100 M HNO3, cyclic voltammetry shows that the bismuth(III) citrate complex dissociates to form Bi3+, whereas bismuth(III) salicylate does not exhibit this behavior. In addition, the bismuth(III) salicylate complex at pH 1.0 was found to show significant changes in its voltammetric behavior upon addition of L-cysteine, implying that L-cysteine is a stronger ligand toward Bi(III) than is salicylate. The present studies show, in all cases considered, a significant interaction between Bi(III) and L-cysteine, mostly likely due to the sulfhydryl side-chain on L-cysteine. L-cysteine can be regarded as a model for cysteine-containing proteins, so the present electrochemical investigations represent a novel way to study the interactions of bismuth(III) with both L-cysteine and proteins comprised of it. The very intense interactions between L-cysteine and Bi(III) species shown in this work may provide information helpful to understanding such issues as tolerance of the body to bismuth as well as the details of bismuth speciation.
Acknowledgments
The support of the United States Naval Academy Chemistry Department is gratefully acknowledged.