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Peptide-based, two-fluorophore, ratiometric probe for quantifying mobile zinc in biological solutions.

Zhang DY, Azrad M, Demark-Wahnefried W, Frederickson CJ, Lippard SJ, Radford RJ - ACS Chem. Biol. (2014)

Bottom Line: Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology.Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications.A plate reader-based assay using HcZ9 was developed, the accuracy of which is comparable to that of atomic absorption spectroscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.

ABSTRACT
Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology. Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications. Using solid-phase peptide synthesis, we conjugated a zinc-sensitive Zinpyr-1 derivative and a zinc-insensitive 7-hydroxycoumarin derivative onto opposite ends of a rigid P9K peptide scaffold to create HcZ9, a ratiometric fluorescent probe for mobile zinc. A plate reader-based assay using HcZ9 was developed, the accuracy of which is comparable to that of atomic absorption spectroscopy. We investigated zinc accumulation in prostatic cells and zinc levels in human seminal fluid. When normal and tumorigenic cells are bathed in zinc-enriched media, cellular mobile zinc is buffered and changes slightly, but total zinc levels increase significantly. Quantification of mobile and total zinc levels in human seminal plasma revealed that the two are positively correlated with a Pearson's coefficient of 0.73.

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Mobile and total zinc measurements ofcell lysates. (a) The mobilezinc concentration of cell lysate was measured before and after theaddition of 250 nM (0.5 equiv) of ZnCl2. The measured increasein mobile zinc is shown in nM and compared to the expected increase(% recovery). (b) RWPE-1 and RWPE-2 cells were bathed for 30 min ineither unmodified KSFM (gray bars) or KSFM supplemented with 10 μMzinc pyrithione (ZnPT) (red bars). (c) Mobile and (d) total zinc concentrationsin RWPE-1 and RWPE-2 cells were measured after cells were bathed for24 h in either unmodified KSFM (gray bars) or in KSFM supplementedwith 50 μM ZnCl2 (blue bars). The measured mobilezinc concentration is calculated relative to protein content. Datafor total zinc in the RWPE cells were adapted from ref (28). Data are means ±SE; n.s. is not significant; *p < 0.05; **p ≪ 0.01.
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fig3: Mobile and total zinc measurements ofcell lysates. (a) The mobilezinc concentration of cell lysate was measured before and after theaddition of 250 nM (0.5 equiv) of ZnCl2. The measured increasein mobile zinc is shown in nM and compared to the expected increase(% recovery). (b) RWPE-1 and RWPE-2 cells were bathed for 30 min ineither unmodified KSFM (gray bars) or KSFM supplemented with 10 μMzinc pyrithione (ZnPT) (red bars). (c) Mobile and (d) total zinc concentrationsin RWPE-1 and RWPE-2 cells were measured after cells were bathed for24 h in either unmodified KSFM (gray bars) or in KSFM supplementedwith 50 μM ZnCl2 (blue bars). The measured mobilezinc concentration is calculated relative to protein content. Datafor total zinc in the RWPE cells were adapted from ref (28). Data are means ±SE; n.s. is not significant; *p < 0.05; **p ≪ 0.01.

Mentions: After confirmation that HcZ9 accurately measuresmobile zinc concentrationsunder controlled conditions, we investigated its performance in celllysates. For cell lines, we chose RWPE-1 and -2, PC-3, and HeLa. Spikeand recovery experiments were performed to assess the accuracy ofthe method. In a typical experiment, the mobile zinc levels are measuredbefore and after an addition of 250 nM (0.5 equiv) ZnCl2. In all cell lines, the amount of spiked zinc measured was within10% of the expected value (Figure 3a), suggestingthat the activity of HcZ9 is not significantly altered by variousbiomolecules in these biological samples and validating the abilityof HcZ9 to quantify mobile zinc in cell lysates (Supplemental Figure S11).


Peptide-based, two-fluorophore, ratiometric probe for quantifying mobile zinc in biological solutions.

Zhang DY, Azrad M, Demark-Wahnefried W, Frederickson CJ, Lippard SJ, Radford RJ - ACS Chem. Biol. (2014)

Mobile and total zinc measurements ofcell lysates. (a) The mobilezinc concentration of cell lysate was measured before and after theaddition of 250 nM (0.5 equiv) of ZnCl2. The measured increasein mobile zinc is shown in nM and compared to the expected increase(% recovery). (b) RWPE-1 and RWPE-2 cells were bathed for 30 min ineither unmodified KSFM (gray bars) or KSFM supplemented with 10 μMzinc pyrithione (ZnPT) (red bars). (c) Mobile and (d) total zinc concentrationsin RWPE-1 and RWPE-2 cells were measured after cells were bathed for24 h in either unmodified KSFM (gray bars) or in KSFM supplementedwith 50 μM ZnCl2 (blue bars). The measured mobilezinc concentration is calculated relative to protein content. Datafor total zinc in the RWPE cells were adapted from ref (28). Data are means ±SE; n.s. is not significant; *p < 0.05; **p ≪ 0.01.
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fig3: Mobile and total zinc measurements ofcell lysates. (a) The mobilezinc concentration of cell lysate was measured before and after theaddition of 250 nM (0.5 equiv) of ZnCl2. The measured increasein mobile zinc is shown in nM and compared to the expected increase(% recovery). (b) RWPE-1 and RWPE-2 cells were bathed for 30 min ineither unmodified KSFM (gray bars) or KSFM supplemented with 10 μMzinc pyrithione (ZnPT) (red bars). (c) Mobile and (d) total zinc concentrationsin RWPE-1 and RWPE-2 cells were measured after cells were bathed for24 h in either unmodified KSFM (gray bars) or in KSFM supplementedwith 50 μM ZnCl2 (blue bars). The measured mobilezinc concentration is calculated relative to protein content. Datafor total zinc in the RWPE cells were adapted from ref (28). Data are means ±SE; n.s. is not significant; *p < 0.05; **p ≪ 0.01.
Mentions: After confirmation that HcZ9 accurately measuresmobile zinc concentrationsunder controlled conditions, we investigated its performance in celllysates. For cell lines, we chose RWPE-1 and -2, PC-3, and HeLa. Spikeand recovery experiments were performed to assess the accuracy ofthe method. In a typical experiment, the mobile zinc levels are measuredbefore and after an addition of 250 nM (0.5 equiv) ZnCl2. In all cell lines, the amount of spiked zinc measured was within10% of the expected value (Figure 3a), suggestingthat the activity of HcZ9 is not significantly altered by variousbiomolecules in these biological samples and validating the abilityof HcZ9 to quantify mobile zinc in cell lysates (Supplemental Figure S11).

Bottom Line: Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology.Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications.A plate reader-based assay using HcZ9 was developed, the accuracy of which is comparable to that of atomic absorption spectroscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.

ABSTRACT
Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology. Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications. Using solid-phase peptide synthesis, we conjugated a zinc-sensitive Zinpyr-1 derivative and a zinc-insensitive 7-hydroxycoumarin derivative onto opposite ends of a rigid P9K peptide scaffold to create HcZ9, a ratiometric fluorescent probe for mobile zinc. A plate reader-based assay using HcZ9 was developed, the accuracy of which is comparable to that of atomic absorption spectroscopy. We investigated zinc accumulation in prostatic cells and zinc levels in human seminal fluid. When normal and tumorigenic cells are bathed in zinc-enriched media, cellular mobile zinc is buffered and changes slightly, but total zinc levels increase significantly. Quantification of mobile and total zinc levels in human seminal plasma revealed that the two are positively correlated with a Pearson's coefficient of 0.73.

Show MeSH
Related in: MedlinePlus