Ruđer Bošković Institute Research Data Repository
| Title (english) | Interaction between Cu and Thiols of Biological and Environmental Importance: Case Study Using Combined Spectrophotometric/Bathocuproine Sulfonate Disodium Salt Hydrate (BCS) Assay |
| Author | Elvira Bura Nakić Ruđer Bošković Institute |
| Author | Dora Crmarić Ruđer Bošković Institute |
| Scientific / art field, discipline and subdiscipline | NATURAL SCIENCES Interdisciplinary Natural Sciences Marine Science |
| Abstract (english) | Considering the biological and ecological importance of Cu–thiol interactions and the discrepancies in previous research, this study focuses on Cu interactions with biologically and ecologically relevant thiols: glutathione (GSH), L-cysteine (L-cys), 3-mercaptopropionic acid (MPA), and thioacetic acid (TAA) in aqueous solution. The addition of Cu(II) to a thiol-containing solution led to a rapid reduction of Cu(II) and the formation of a Cu(I)–thiol complex. The mechanism of Cu(II) reduction and Cu(I) complex formation as well as the kinetics of Cu(I) oxidation strongly depend on the structural properties of the individual thiols investigated. The reducing power of the investigated thiols can be summarized as follows: L-cys ≅ GSH > MPA > TAA. The reaction order, with respect to Cu(I) oxidation, also changes over the time of the reaction course. The deviation of the reaction kinetics from the first order with respect to Cu(I) in the later stages of the reaction course can be attributed to a Fenton-like reaction occurring under low thiol concentration conditions. At high Cu:thiol ratios, in the case of GSH, L-cys, and MPA, the early stage of the reaction course is characterized by high Cu(I) stability, most likely as a result of Cu(I) complexation by the thiols present in excess in the reaction mixture. |
| Methods (english) | All solutions were prepared with deionized water from the Milli-Q (MQ) system (18.2 MΩ, Millipore, Burlington, MA, USA), and all chemicals were of analytical grade. The Cu(I) standard solution used for the calibration curve of the Cu(I)–bathocuproine complex (see Section 3.2) was prepared by dissolving copper(I) chloride (CuCl; Thermo Fisher Scientific, Waltham, MA, USA) in a solution containing 1 M sodium chloride (NaCl; Grammol, North Salt Lake, UT, USA) and 0.1 M hydrochloric acid (HCl; Roth, Newport Beach, CA, USA), which was previously purged with high purity nitrogen to remove oxygen [66]. The bathocuproine sulfonate disodium salt hydrate (BCS; Thermo Fisher Scientific), was prepared by dissolving BCS in MQ water to a concentration of 1000 µM. The Cu(II) standard solution was prepared by dissolving copper(II) sulfate (CuSO4; VWR BDH Prolabo Chemicals, Radnor, PA, USA) in MQ water to a final concentration of 0.01 M Cu(II). Thiol solutions of L-cysteine (L-cys), reduced glutathione (GSH), thioacetic acid (TAA), and 3-mercaptopropionic acid (MPA) were purchased from Thermo Fisher Scientific and prepared fresh daily by dissolving the thiols in MQ water to a final concentration of 0.01 M. A constant ionic strength and a pH = 8.4, relevant to the environmental and physiological conditions, in the model solutions containing Cu and thiol were achieved using 0.1 M borate buffer. The borate buffer was prepared from ortho-boric acid (VWR BDH Prolabo Chemicals), and its pH was adjusted to pH = 8.4 with sodium hydroxide (NaOH; Lach-ner Chemicals, Neratovice, Czech Republic). Copper reduction by individual thiol species was investigated by measuring the Cu(I) concentration with a UV–Vis spectrophotometer (Analytik Jena, Jena, Germany) in a 1 cm quartz cuvette. The interaction of Cu with thiols was studied by adding aliquots of a Cu(II) stock solution to a solution containing thiol (L-cysteine, glutathione, 3-mercaptopropionic acid, or thioacetic acid) buffered to pH = 8.4 with 0.1 M borate buffer. The Cu concentration of 100 µM was the same in all experiments, while the thiol concentrations ranged from 100 to 1000 µM. The kinetics of the reduction of Cu(II) and the oxidation of Cu(I) were studied by adding aliquots of the Cu–thiol solution to the mixture of BCS and EDTA (BCS assay). Previous studies have shown that BCS effectively binds Cu(I) in an orange complex with an absorption maximum (Amax) of 484 nm, while a masking ligand is necessary to avoid Cu(II) interference [66,67]. In this study, EDTA was used as the masking ligand for Cu(II) with a fivefold excess of EDTA over BCS, which has been found to be optimal for ensuring Cu(II) complexation while avoiding Cu(I) oxidation [66,67]. A volume of 1 mL of the Cu–thiol model solution was added to the mixture containing 3 mL of BCS (1000 µM) and 0.15 mL of EDTA (0.1 M), resulting in a dilution factor (DF) of 4.15 in the Cu–thiol model solution and final concentrations of 723 µM BCS and 0.00361 M EDTA. The addition of the Cu–thiol solution to the mixture of BCS and EDTA resulted in the formation of an orange-colored solution with an absorption maximum (Amax) of 484 nm, indicating the presence of Cu(I) in the solution. Cu(I) concentrations were determined by measuring the A484 solution, calculating the Cu(I) concentration from the Cu(I)–BCS calibration curve, and correcting the Cu(I) concentration by a dilution factor of 4.15. For the Cu(I)–BCS calibration curve, one blank and five standard additions of Cu(I) were prepared. The blank solution contained 3 mL of 1000 µM BCS, 0.15 mL of 0.1 M EDTA, 0.1 mL of 1 M borate buffer, and MQ water with a final volume of 4.15 mL. The final concentrations of 723 µM BCS and 0.00361 M EDTA were the same as those used to measure the absorbance of Cu–thiol solutions. The Cu(I) standard solutions were prepared in the same way as the blank solution, adding five different Cu(I) aliquots that resulted in a linear absorbance over the concentration range studied, from 1.1 to 35.2 µM. The Cu(I) standard solution was freshly prepared before the experiment, following the procedure described in Section 3.1. To better understand the reaction mechanism, in addition to monitoring the Cu(I) concentration, UV–Vis spectra of solutions containing Cu and L-cys were recorded at the same reaction time as was the Cu(I) determination. |
| Keywords (english) | |
| Language | english |
| URN:NBN | urn:nbn:hr:241:001650 |
| Publication | 2023-05-30 |
| Geolocation | Croatia |
| Project | Number: IPCH-2020-10-4965 Title (croatian): Razumijevanje pojavnosti kemijskih vrsta bakra i redoks transformacija u morskoj vodi Title (english): Understanding copper speciation and redox transformations in seawater Acronym: SeCuTrans Leader: Elvira Bura-Nakić Jurisdiction: Croatia |
| Publisher | Institut Ruđer Bošković Ruđer Bošković Institute |
| Access conditions | Open access |
| Terms of use |  |
| Created on | 2025-03-12 11:59:30 |
