Metal Ion Buffers
Introduction: Why use metal ion buffers?
Metal ion buffers are solutions of metal ions that are similar to, and share many advantages of, pH buffers. Like pH buffers with protons, they “resist” changes in free metal ion concentration that would occur if, for instance, additional metal ion from another source somehow contaminated the sample under study. Particularly when working with free metal ion concentrations below micromolar, the presence of contaminating metal ions in common buffer salts and water makes the use of metal ion buffers almost essential; please see our discussion of free vs. total metal ion concentration under Resources. For instance, ACS Reagent Grade sodium chloride is specified as having up to 5 ppm by weight heavy metals as contaminants (determined as Pb). Thus making a physiological saline solution with 100 mM NaCl means it can contain roughly 150 nanomolar heavy metals, which are likely to interfere if one is trying to measure free zinc at the 100 picomolar level. An analogous problem arises when buffer components or impurities bind the metal ion of interest: for instance, phosphate, citrate and Tris (tris(hydroxymethyl)aminomethane) all bind zinc with significant affinity. Thus adding one micromolar total zinc ion to a 20 millimolar pH 7.5 Tris buffer only leaves 1.3 picomolar free and bioavailable, a very large error.
While the details of formulating metal ion buffers are beyond our scope here, the principle is actually straightforward: the metal ion buffer incorporates a fairly strong ligand together with a predetermined amount of the metal ion in question, such that a fixed proportion (preferably 99+%) of the metal is bound to the ligand. Thus addition or subtraction of metal ion causes a much smaller change in the free metal ion concentration than it would in the absence of the metal ion buffer. For example, if we have a 20 mM pH 7.5 Bicine buffer with 0.62 picomolar free zinc ion present (1 uM total zinc ion added), addition of 100 nanomolar additional zinc ion only results in a free zinc ion concentration of 0.69 picomolar, a much smaller change than in the absence of the buffer.
Use of Metal Ion Buffers to Calibrate Molecular Sensors
One of the best and most important uses of metal ion buffers is to calibrate fluorescence (and other) sensors. The first reason this is important is that the response of the instrument one is using with the sensor varies a lot depending on the individual microscope, plate reader or fluorometer; i.e., even with a ratiometric sensor the actual numerical value of the ratio will vary from machine to machine. The value of buffers for calibration will be evident if we consider the following: for a high affinity (KD ≈ 0.1 nM) fluorescence-based molecular zinc sensor, suppose we want to determine how big a fluorescence change corresponds to a particular value of free zinc concentration. We might try to put 0.1 nanomolar (KD ) of ZnCl2 in a 4 ml cuvette (presuming there's no contaminating zinc ion, other metals, or things that bind zinc, which is probably not true), but then we face another problem: if we then put (say) 1 nM of our sensor in the solution, it will largely scavenge up all the free zinc ( ≈ total zinc in this case) since the sensor concentration is 10x the free zinc concentration and also above KD. In this case, the sensor fluorescence change will correspond to only 10% fractional occupancy (because that's all the zinc there is) rather than the 50% corresponding to KD: obviously a big error. The problem of course, is that the free zinc concentration declines as it gets bound by the sensor: this is not so much the case in natural waters, and manifestly not the case in growth media, serum, or cell cytoplasm, which have abundant zinc-binding components which act as metal ion buffers. In addition, lowering the sensor concentration is seldom an option since the fluorescence signal drops correspondingly, and interfering fluorescence and electronic noise in the measurement grow in importance. The metal ion buffer largely solves these problems for calibration because binding of the free zinc doesn't consume all the available zinc since 99+% is still bound by the buffer and the equilibrium merely shifts slightly as it is bound by the sensor. Moreover, enough sensor can be used to provide adequate signal to noise, with no concern about scavenging all available zinc. Pokegama MetalloBuffers are designed to make such calibrations easy.
Pokegama Technologies MetalloBuffer products
Pokegama Technologies MetalloBuffersTM are formulated to provide reliable free zinc and copper concentrations for calibrating Pokegama and other metal ion sensors. We offer three different sets of metal ion buffers:
1) Basic MetalloBuffers: These metal ion buffer sets are the simplest MetalloBuffer sets we offer. They are designed for calibration of fluorescent or bioluminescent molecular zinc sensors (Pokegama’s and others on the market) in your microscope, fluorometer, or plate reader. Since the lamp output, monochromator/filter throughput, and detector efficiency of any of these instruments varies with wavelength and from instrument to instrument, it is essential to calibrate the sensor in situ with known concentrations of free zinc. This is especially true for non-ratiometric sensors such as TSQ, Zn-AF2, or
2) Physiological Salts MetalloBuffers (in development): We have formulated MetalloBuffer sets in widely used physiological balanced salt solutions such as Earle’s Balanced Salts and Henke’s . These buffers are intended for use with cells of all types to provide physiologically compatible media having known free zinc concentrations.
3) MetalloBuffer Medium Supplements (in development): Pokegama has formulated a series of zinc buffer supplements designed to be used together with common growth media such as DMEM to achieve preselected free zinc concentrations for use with mammalian cell cultures. When used as directed, this permits the investigator to reliably carry out experiments with known, fixed free zinc concentrations to elucidate zinc effects, or calibrate sensors in situ. Please note that although the supplements may be used with media that include fetal bovine serum, the variable amounts of zinc in serum make the derived values of free zinc in such media less accurate.