Hydrogen Sulfide and Carbonyl Sulfide Delivery for Biological Applications

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Project Summary The long-term goal of the funded parent R01 grant is to advance the development and use of reactive sulfur species (RSS) donors to investigate the multifaceted roles of RSS in biology, and in particular tissue regeneration. This work is centered on the development and use of donor molecules that are activated by different stimuli to release carbonyl sulfide (COS), which is rapidly converted to H2S by carbonic anhydrase (CA) enzymes. The goals of the funded parent R01 grant are to expand the platform of COS/H2S donors, to investigate isoform specificity of CA mediated conversion of COS to H2S, and to apply developed donor construct to models of bone regeneration. Each Aim of the parent grant is currently limited by challenges with direct COS detection and would benefit directly from the requested sulfur-optimized gas chromatography (GC) instrument. To complete the Specific Aims outlined in the parent grant, we request an instrument supplement to support the purchase of a GC system with a Pulsed Flame Photometric Detector (PFPD) and automated headspace autosampler with solid phase microextraction (SPME) capabilities. This instrument will allow us to detect and quantify COS, H2S, and other gaseous RSS, either independently or simultaneously, in aqueous samples. The rationale for requesting this instrument is that the GC will enable completion of the stated Aims in the funded grant, will facilitate overcoming challenges in the approach, and will significantly increase the impact of each of the studies. Our choice of instrumental setup is motivated by the low sensitivity of standard GC detectors toward sulfur-containing molecules, and on prior work in the field that has used GC detection by PFPD and SPME extraction to quantify RSS in challenging aqueous matrixes. The requested instrument will facilitate completion of the R01 Aims by allowing for: (1) Direct measurement of COS and H2S simultaneously for COS/H2S donor compounds; (2) Quantification of COS in CA enzyme activity measurements; (3) Detection and measurement of COS generation from abiotic conditions. In addition, this instrument would also support the research activities by providing: (1) Better compatibility with complex sample environments like hydrogels and cell culture media; (2) Significantly enhanced sensitivity for different sulfur-containing analytes over our current methods; (3) Simultaneous monitoring of multiple RSS from one sample in a single analytical workflow; and (4) Enhanced rigor and reproducibility of mechanistic and quantification studies and data related to this project.