Measuring intracellular free zinc using carbonic anhydrase as a fluorescent biosensor

Abstract

Zinc is a metal ion of great importance in the biological world. It is a cofactor in over 300 enzymes and is necessary for proper function, growth, and development of the human body. Zinc is also a vital component in the immune and reproductive systems, and in zinc fingers of transcription factors. Zinc also has been implicated in apoptosis where there is still debate over whether it is protective or pro-apoptotic. Recent papers also have focused on zinc and its relation to oxidative stress as well as mitochondrial function and membrane potential disruption. It has been proposed that oxidative stress causes release of zinc from binding sites such as metallothionein; where upon it induces apoptosis by inhibiting electron transport chain enzymes and/or other mitochondrial enzymes. However, an increase in the amount of intracellular zinc could also initiate these events in the absence of oxidative stress. A necessary starting point in understanding the many roles of zinc is a quantitative measurement of readily exchangeable zinc. Due to the modest amounts of zinc and the presence of other divalent cations zinc measurements require high selectivity and sensitivity. To meet these requirements we have employed fluorescently labeled carbonic anhydrase in conjunction with raiometric fluorescence detection to quantitated zinc in cells. CA is an ideal sensor transducer for zinc binding due to its picomolar affinity for zinc. Aryl sulfonamides are known inhibitors of carbonic anhydrase (CA) as their binding is zinc-dependent. When zinc binds to fluorescently tagged CA, resonance energy transfer occurs from a zinc-bound aryl sulfonamide to the fluorescent label on carbonic anhydrase. Zinc levels are then measured by taking the ratio of fluorescence intensities at two different excitation wavelengths. Using this system we have successfully measured the level of labile zinc to be 5pM in resting PC-12 cells. To monitor zinc levels in mitochondria, an expressible system was developed with the red fluorescent protein DsRed2 fused to carbonic anhydrase that contains a mitochondrial target sequence at its 5' end thus creating an expressible biosensor to measure free zinc in mitochondria. The expressible sensor was used to measure 0.5-5nM free zinc in mitochondria.