Article Appropriate Buffers for Studying the Bioinorganic y Chemistry of Silver(I) Lucille Babel * , Soledad Bonnet-Gómez and Katharina M. Fromm * Chemistry Department, University of Fribourg, Chemin du Musée 9, 1700 Fribourg, Switzerland; [email protected] * Correspondence: [email protected] (L.B.); [email protected] (K.M.F.); Tel.: +41-2630-087-32 (K.M.F.) Dedicated to the radical chemist Prof. Bernd Giese on behalf of his 80th birthday. y Received: 21 February 2020; Accepted: 18 March 2020; Published: 22 March 2020 Abstract: Silver(I) is being largely studied for its antimicrobial properties. In parallel to that growing interest, some researchers are investigating the effect of this ion on eukaryotes and the mechanism of silver resistance of certain bacteria. For these studies, and more generally in biology, it is necessary to work in buffer systems that are most suitable, i.e., that interact least with silver cations. Selected buffers such as 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) were therefore investigated for their use in the presence of silver nitrate. Potentiometric titrations allowed to determine stability constants for the formation of (Ag(Buffer)) complexes. The obtained values were adapted to extract the apparent binding constants at physiological pH. The percentage of metal ions bound to the buffer was calculated at this pH for given concentrations of buffer and silver to realize at which extent silver was interacting with the buffer. We found that in the micromolar range, HEPES buffer is sufficiently coordinating to silver to have a non-negligible effect on the thermodynamic parameters determined for an analyte. Morpholinic buffers were more suitable as they turned out to be weaker complexing agents. We thus recommend the use of MOPS for studies of physiological pH. Keywords: silver; buffer; association constant; HEPES 1. Introduction A well-known list of buffers was published between 1966 and 1980, called Good’s buffers, for their use in biological systems [1]. This list contains essentially sterically hindered amines that aim to replace common buffers used in biology such as imidazole, sodium phosphate and sodium citrate. Indeed, these previously employed buffers are inadequate for certain experiments because of their reactivity towards small molecules (ATP), metal ions, or because of their toxicity for the cells [2–8]. For example, a phosphate buffer leads to precipitates with many cations and is known to inhibit or enhance certain reactions of a cellular system [2,3]. Imidazole is a very good complexing ligand for many metal cations and, due to its similar structure, could replace histidine residues in metal binding proteins [9–11]. Good’s buffers on the contrary were believed to be largely inactive towards the cell metabolism and thus should not interact with any biological molecule and/or metal ions. Nevertheless, since this list was established, many studies have proved that most of these sterically hindered tertiary amine-based buffers are able to coordinate slightly some metal ions [12,13]. Therefore, binding constants determined for other ligands could be affected by the presence of these buffers, which are usually in large excess compared to the ligand to ensure a stable pH, hence it is a necessity to know these values. A correction can then be applied to the thermodynamic model to take into consideration the effect of the buffer. To limit the effect of this correction, careful consideration of the metal ions in solution and the concentration of the buffer is necessary prior to use. 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Silver or in is studies used investigatingin in vitro studiestoxicity to investigate and/or antimicrobial e.g., the silver properties resistance of silver mechanism agents, yetof some appropriate bacteria bu orff iners studies for this investigating kind of studies toxicityare lacking and/or in antimicrobial the literature. properties We have recently of silver been agents, studying yet appropriate peptide models buffers inspired for this by thekind protein of studiesSilE, aare protein lacking of thein the silver literature. efflux pump We have in Gram recent negatively been bacteria,studying which peptide is able models to bind inspired a large by amount the proteinof silver(I) SilE, a [19 protein,20]. In of this the case, silver phosphate efflux pump buffer in could Gram not negative be used bacteria, because ofwhich the immediate is able to formationbind a largeof theamount poorly of soluble silver(I) silver [19,20]. phosphate In this salt.case, phosphate buffer could not be used because of the immediateHEPES formation contains of the N-donors poorly andsoluble is not silver innocent phosphate with salt. respect to silver(I) as shown by a crystal structureHEPES ofcontains a HEPES-silver(I) N-donors and complex is not [innocent21]. Two wi nitrogenth respect atoms to silver(I) from the as piperazineshown by moietiesa crystal of structureHEPES of molecules a HEPES-silver(I) as well as twocomplex oxygen [21]. atoms Two from nitrogen the alcohol atoms and from sulfonate the piperazine functions coordinatemoieties of the HEPESsilver molecules ion in a distorted as well as tetrahedral two oxygen geometry. atoms from However, the alcohol the binding and sulfonat affinitye was functions not quantified. coordinate the silverHerein, ion inwe a distorted determined tetrahedral the affinity geometry. of HEPES However, for silver the ions binding in order affinity to quantize was not the quantified. buffer eff ect. InHerein, comparison, we determined we also studied the affinity the of eff HEPESect of other for silver buff ersions that in order were to expected quantize to the possess buffer effect. the least In interactioncomparison, with we silver also ionsstudied (Scheme the effect1) to find of other out which buffers one that would were be expected ideal for studiesto possess with the silver(I) least in interactionbiological with media. silver ions (Scheme 1) to find out which one would be ideal for studies with silver(I) in biological media. OH SO3H N N N N SO3H SO3H NH2 HEPES PIPES HO OH OH HO S SO3H 3 Tris N N O O MES MOPS Scheme 1. StructuresScheme of 1. buffersStructures investigated of buffers for investigated their affinities for theirwith asilverffinities ions. with silver ions. 2. Materials2. Materials and and Methods Methods Silver nitrate AgNO was purchased from Carlo Erba reagents (RPE, Analytical 99+%). Silver nitrate AgNO3 was3 purchased from Carlo Erba reagents (RPE, Analytical 99+%). 4-(2- hydroxyethyl)-1-piperazineethane4-(2-hydroxyethyl)-1-piperazineethane sulfonic sulfonic acid (HEPES), acid (HEPES), 3-(N-morpholino)propan 3-(N-morpholino)propanesulfonicesulfonic acid acid (MOPS), tris(hydroxymethyl)aminomethane (Tris) (Roche), sodium nitrate NaNO (MOPS), tris(hydroxymethyl)aminomethane (Tris) (Roche), sodium nitrate NaNO3 and potassium 3 hydrogenand potassium phthalate hydrogen(KHP) (Merck) phthalate were (KHP)purchased (Merck) from wereSigma-Aldrich. purchased Piperazine-1,4-bis(2- from Sigma-Aldrich. ethanesulfonicPiperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) and 2-(N acid)-morpholino)ethanesulfonic (PIPES) and 2-(N-morpholino)ethanesulfonic acid (MES) were purchased acid (MES) from were purchased from Roth. Nitric acid was purchased from Fluka and NaOH pellets from Acros. HNO Roth. Nitric acid was purchased from Fluka and NaOH pellets from Acros. HNO3 0.1 M stock 3 0.1 M stock solution in 0.1 M NaNO was standardized towards KHP (0.4 g) where the equivalence solution in 0.1 M NaNO3 was standardized3 towards KHP (0.4 g) where the equivalence point is point is followed with the help of phenolphthalein indicator. NaOH 0.2 M stock solutions in 0.1 M followed with the help of phenolphthalein indicator. NaOH 0.2 M stock solutions in 0.1 M NaNO3 NaNO were standardized with stock solution of HNO 0.1 M and used within two weeks to avoid were standardized3 with stock solution of HNO3 0.1 M and used3 within two weeks to avoid carbonate carbonate formation. Buffers and silver nitrate were dissolved at a concentration of 0.05 M in 0.1 M formation. Buffers and silver nitrate were dissolved at a concentration of 0.05 M in 0.1 M NaNO3. PIPESNaNO was3. insoluble PIPES was in insolublewater, and in NaOH water, had and to NaOH be added had up to to be a added 0.069 M up concentration to a 0.069 M concentration(1.4 eq.) (1.4 eq.). Buffers were titrated manually in presence of 0.1 M NaNO3 at 296 K over the pH range of 2-11 (HNO3 was addedBuff toers obtain were the titrated starting manually pH of 2) in with presence NaOH of 0.2 0.1 M M as NaNO titrant.3 at Changes 296 K over in pH the were pH rangemonitored of 2–11 with(HNO a glass3 was electrode added to obtain(Primatrode the starting with pHNTC of 2)Methrom, with NaOH combined 0.2 M as glass-Ag/AgCl titrant.