Abstract
Ruthenium-based CO-releasing molecules (CO-RMs), CORM-2 and CORM-3, have been widely used as surrogates of CO for studying its biological effects in vitro and in vivo with much success. However, several previous solution-phase and in vitro studies have revealed the ability of such CO-RMs to chemically modify proteins and reduce aromatic nitro groups due to their intrinsic chemical reactivity under certain conditions. In our own work of studying the cytoprotective effects of CO donors, we were in need of assessing chemical factors that could impact the interpretation of results from CO donors including CORM-2,3 in various in vitro assays. For this, we examined the effects of CORM-2,3 toward representative reagents commonly used in various bioassays including resazurin, tetrazolium salts, nitrites, and azide-based H2S probes. We have also examined the effect of CORM-2,3 on glutathione disulfide (GSSG), which is a very important redox regulator. Our studies show the ability of these CO-RMs to induce a number of chemical and/or spectroscopic changes for several commonly used biological reagents under near-physiological conditions. These reactions/spectroscopic changes cannot be duplicated with CO-deleted CO-RMs (iCORMs), which are often used as negative controls. Furthermore, both CORM-2 and -3 are capable of consuming and reducing GSSG in solution. We hope that the results described will help in the future design of control experiments using Ru-based CO-RMs.