A new probe for detecting zinc-bound carbonic anhydrase in cell lysates and cells

Abstract

We report the synthesis and application of a small molecule probe for carbonic anhydrase (CA) to track holo-CA in cell lysates and live-cell models of zinc dyshomeostasis. The probe displays a 12-fold increase in fluorescence upon binding to bovine CA and also responds to human CA isoforms.

Pull‐Down of Metalloproteins in Their Native States by Using Desthiobiotin‐Based Probes

“…Probe 4 was then used to pull down CA from a red blood cell (RBC) lysate (Figure B). The RBC lysate was prepared by using a mixture of chloroform and ethanol according to literature procedures; this process significantly reduces the Hb concentration in these samples . Under these conditions, the aqueous supernatant lysate should contain bCA, Hb, oxidases, and a small amount of catalase.…”

Visualizing the Dynamic Metalation State of New Delhi Metallo-β-lactamase-1 in Bacteria Using a Reversible Fluorescent Probe

“…To make a fluorescent probe specific for dizinc NDM-1, we linked the environment-sensitive fluorophore 4- N,N -dimethylaminopthalimide ( 4-DMAP ) with thiol derivatives predicted to bind NDM-1 with micromolar affinity via direct interactions with both Zn 2+ ions in the enzyme active site (Figure ). These small-molecule fluorescent probes for NDM-1 (Figure B) were synthesized in multiple steps from N , N -dimethylaminophthalic anhydride ( DMAP ). , Here, DMAP was coupled with cysteamine to generate probe 1 . Probe 2 , using glycine as a precursor, was generated to compare interactions with a non-thiol-based metal binding group.…”

“…Previously, we studied the effects of cellular metal homeostasis perturbations on carbonic anhydrase (CA) in mammalian HeLa cells using a CA-binding fluorescence probe. 24 Treatments with 10 μM TPEN and 1 μM zinc pyrithione (ZnPT) to decrease and increase intracellular zinc availability, respectively, did not affect observed fluorescence associated with the fluorescent probe-CA complex. This result was consistent with in vitro studies where TPEN failed to affect the fluorescence of the probe-CA complex.…”

“…Previously, we studied models of zinc dyshomeostasis in HeLa cells by designing small molecule fluorescent probes that report on the metallation status of intracellular carbonic anhydrase. 24 Some existing NDM-1 targeted fluorophores consist of fluorogenic substrates [25][26] or covalent modifiers, [27][28] and represent irreversible "switch on" probes. However, these probes do not necessarily report on metallation and lack the ability to monitor dynamic reversible changes.…”

Detection of a multi‐disease biomarker in saliva with graphene field effect transistors

“…Carbonic anhydrases (CAs) are a family of enzymes that catalyse the reversible reaction of carbon dioxide and water into bicarbonate and protons (Krishnamurthy et al, 2008). Several studies have pointed to the importance of the CAs and their inhibitors as an indicator for various diseases (Finkelmeier et al, 2018; Koutnik et al, 2017; Li et al, 2019; Mehta et al, 2018; Zamanova et al, 2019). Of particular importance is the human isozyme CA1, which has been identified as a potential marker of various cancers (breast, oral, colorectal, lung, prostate), ankylosing spondylitis, diabetes, Sjogren's syndrome and pancreatitis (Bánová Vulić et al, 2019; Chang et al, 2010, 2012; Frulloni et al, 2000; Giusti et al, 2007; Hu et al, 2007; Inagaki et al, 1991; Kardoush et al, 2017; Lee et al, 2015; Liu et al, 2012; Peng et al, 2012; Ryu et al, 2006; Torella et al, 2014; Wang et al, 2016; Zheng et al, 2015).…”