A practical guide to the preparation and use of metal ion‐buffered systems for physiological research

Abstract

Recent recognition that mobile pools of Zn and Cu are involved in the regulation of neuronal, endocrine and other cells has stimulated the development of tools to visualize and quantify the level of free trace metal ions. Most of the methods used to measure or control loosely bound metals require reference media that contain exactly defined free concentrations of the target ions. Despite the central importance of proper metal ion buffering, there is still a lack of international standards and beginners in the field may have difficulties finding a coherent description of how to prepare trace metal ion buffers, especially when experiments are to be performed in multimetal systems. To close this gap, we provide a guide for the design, preparation and use of metal ion-buffered systems that facilitate immediate application under physiologically relevant ionic conditions. Thermodynamic and kinetic concepts of chemical speciation as well as general protocols and specific examples are outlined for the accurate preparation of single- and dual-metal ion buffers. In addition, experiments have been performed with FluoZin-3 to illustrate that metal ion-buffered systems are required for reliable preparation of nanomolar Zn solutions and that dual-metal ion buffers can be used to calibrate suitable fluorescent Zn sensors in the presence of millimolar Ca concentrations. Together, the information provided should sensitize readers to the many potential pitfalls and uncertainties that exist when working with physiologically relevant concentrations of trace metal ions and enable them to formulate their own metal ion buffers for most in vitro applications.

Metal Chelation Therapy and Parkinson’s Disease: A Critical Review on the Thermodynamics of Complex Formation between Relevant Metal Ions and Promising or Established Drugs

“…Equations to correct K d values, by taking into account the effect of a competing ligand (e.g., the buffer) and of the different pH, have been proposed [316]. Standardized conditions to measure the K d of metal-protein complexes are also being proposed [317][318][319], and this should lead to more reproducible results, thus eventually allowing a more reliable comparison among K d numbers. Table S2 (Supplementary Materials) reports the metal-ligand speciation available in the literature for the ligands listed in Table 2 (rows) and the relevant metal ions, i.e., Cu(II), Cu(I), Fe(III), Fe(II), Mn(II), and Zn(II) (columns).…”

Zn2+-induced changes in Cav2.3 channel function: An electrophysiological and modeling study

“…To avoid trace metal contamination and reduce uncertainties with regard to free metal ion concentrations in our recording solutions, we employed suitable labware and sample handling procedures, which have been outlined in detail in a previous publication ( Neumaier et al, 2017 ). Briefly, all solutions were prepared using type 1 ultrapure water (Purelab Flex 2 by ELGA Labwater), metal-free pipette tips, and reagents of the highest purity available (see previous section).…”

Multiparametric analysis of the effectiveness of cisplatin on cutaneous squamous carcinoma cells using two different types of adjuvants

“…This AuNP concentration was chosen considering already published data elsewhere [11]. For tricine, a millimolar scale concentration was chosen as described in the literature [9,12,13].…”