Zinc is one of the most widespread metal ions found in biological systems. Of the expected 3000 zinc proteins in the human proteome, most contain zinc in structural sites. Among these structures, the most important are zinc fingers, which are well suited to facilitate interactions with DNA, RNA, proteins and lipid molecules. Knowledge regarding their stability is a critical issue in understanding the function of zinc fingers and their reactivity under fluxing cellular Zn(II) availability and different redox states. Zinc stability constants that have been determined using a variety of methods demonstrate wide diversity. Recent studies on the stability of consensus zinc fingers have demonstrated that the known metal-ion affinities for zinc fingers may have been underestimated by as much as three or more orders of magnitude. Here, using four natural ββα zinc fingers, we compare in detail several different methods that have been used for the determination of zinc finger stability constants, such as common reverse-titration, potentiometry, competition with metal chelators, and a new approach based on a three-step spectrophotometric titration. We discuss why the stabilities of zinc fingers that are determined spectrophotometrically are frequently underestimated due to the lack of effective equilibrium competition, which leads to large errors during the processing of the titration data. The literature stability constants of many natural zinc fingers have been underestimated, and they are significantly lower when compared with the consensus peptides. Our data show that in the cell, some naturally occurring zinc fingers may potentially be unoccupied and are instead loaded transiently with Zn(II). Large variations in stability within the same class of zinc fingers have demonstrated that the thermodynamic effects hidden in the sequence and structure are the key elements responsible for the differentiation of the stability of the zinc finger metallome.

Ratiometric chemical probes and genetically encoded sensors are of high interest for both analytical chemists and molecular biologists. Their high sensitivity toward the target ligand and ability to obtain quantitative results without a known sensor concentration have made them a very useful tool in both in vitro and in vivo assays. Although ratiometric sensors are widely used in many applications, their successful and accurate usage depends on how they are characterized in terms of sensing target molecules. The most important feature of probes and sensors besides their optical parameters is an affinity constant toward analyzed molecules. The literature shows that different analytical approaches are used to determine the stability constants, with the ratio approach being most popular. However, oversimplification and lack of attention to detail results in inaccurate determination of stability constants, which in turn affects the results obtained using these sensors. Here, we present a new method where ratio signal is calibrated for borderline values of intensities of both wavelengths, instead of borderline ratio values that generate errors in many studies. At the same time, the equation takes into account the cooperativity factor or fluorescence artifacts and therefore can be used to characterize systems with various stoichiometries and experimental conditions. Accurate determination of stability constants is demonstrated utilizing four known optical ratiometric probes and sensors, together with a discussion regarding other, currently used methods.