Date of Award

August 2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

David H. Petering

Committee Members

Nicholas Silvaggi, Arsenio Pacheco, Guilherme Indig, Graham Moran

Keywords

Fluorescence, Page, Zinc, Zinquin

Abstract

Fluorescent sensors have been a main microscopic tools used to understand Zn2+ physiology on a cellular level. The use of the fluorescent Zn2+ sensor Zinquin (ZQ) and its analogues have revealed that transient Zn2+ is a chief component in a variety of biochemical pathways. Yet, little work has been performed to validate the exact targets of Zinquin in a cellular environment. The goals of this investigation are to determine the types of Zinquin reactions that take place in the cell as well as the identities of its cellular targets.

It has been hypothesized that Zinquin reacts with free Zn2+ within cells at nanomolar concentrations. Instead, by using robust analytical methods such as fluorescence spectrophotometry, column chromatography, and ICP-MS, this report shows a consistent pattern across seven cell and tissue types that Zinquin reacts in micromolar concentrations with Zn2+ bound to proteins. The main source of evidence for this reaction is a unique emission spectrum for a ZQ-Zn-protein adduct compared to Zn(ZQ)2. Zn(ZQ)2 can be generated by Zinquin sequestering Zn2+ from members of the Zn-proteome and can undergo additional ligand substitution reactions with apo-Zn-binding sites within the proteome to form new ZQ-Zn-protein adducts.

With these new insights in mind, previously published investigations were revisited to reevaluate the conclusions drawn using Zinquin as a Zn-reporter molecule. A parallel investigation was also performed using a Zinquin analogue, TSQ, which revealed that the reactions and fluorescence observed were dependent on the chemical properties of the sensor used, not on the physiology being evaluated.

Lastly, the development of a novel affinity chromatography method led to a partial isolation of the intracellular targets of Zinquin. In addition, a high resolution native polyacrylamide gel electrophoresis method was developed to sufficiently separate protein mixtures while still retaining their metal cofactors. These gels can then be analyzed using laser ablation-inductively coupled plasma-mass spectrometry to assess the metal content of the protein bands. The development of these two techniques enhances the ability to isolate and identify members of the Zn-proteome, and thus are great tools for advancing the field of metallomics.

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