Hydrogen Sulfide and Carbonyl Sulfide Delivery for Biological Applications

项目摘要 R 01资助的长期目标是促进活性硫的开发和使用 物种（RSS）捐助者调查RSS在生物学中的多方面作用，特别是组织 再生这项工作的重点是开发和使用供体分子，这些分子被激活， 不同的刺激释放羰基硫（COS），它被碳酸酐酶（CA）迅速转化为H2S， 内切酶R 01母基金资助的目标是扩大COS/H2S捐助者的平台， CA介导COS转化为H2S的同种型特异性，并将开发的供体构建体应用于模型 骨骼再生的过程。母基金的每个目标目前都受到直接COS检测挑战的限制 并且将直接受益于所要求的硫优化的气相色谱（GC）仪器。到 完成父母补助金中概述的具体目标，我们要求补充工具，以支持 购买带有脉冲火焰光度检测器（PFPD）和自动顶空的GC系统 具有固相微萃取（SPME）功能的自动进样器。这台仪器将使我们能够检测和 定量COS，H2S和其他气态RSS，无论是独立或同时，在水溶液样品。的 要求这一工具的理由是，GC将能够完成所述的目标，在资助的 赠款，将有助于克服方法中的挑战，并将大大增加每一个 研究。我们选择仪器设置的动机是标准GC检测器的低灵敏度， 含硫分子，以及在该领域中使用PFPD和SPME进行GC检测的先前工作 提取以量化挑战性水性基质中的RSS。所要求的文书将有助于完成 R 01的目标是：（1）直接测量COS/H2S供体的COS和H2S 化合物;（2）在CA酶活性测量中COS的定量;（3）在CA酶活性测量中COS的检测和测量。 从非生物条件下产生COS。此外，该工具还将通过以下方式支持研究活动： 提供：（1）与复杂样品环境如水凝胶和细胞培养基更好的相容性;（2） 与我们现有的方法相比，显著提高了对不同含硫分析物的灵敏度;（3） 在单个分析工作流程中同时监测来自一个样品的多个RSS;以及（4）增强 与本项目相关的机理和定量研究及数据的严谨性和可重复性。

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.