BACKGROUNDNitric oxide (NO) is a signaling molecule that plays many roles during infection, inflammation, and wound healing processes. Due to the role of NO in wound repair, a novel NO generation system was developed based on copper–chitosan complexes that can be used for the topical generation of NO. Chitosan, a biocompatible polymer, chelates copper ions. Copper in the +1 state can reduce nitrite (NO2–) and convert it into NO. With glucose, a reducing sugar, present in the system, Cu+2 can be returned to Cu+1 to complete the catalytic cycle.RESULTSCopper–chitosan milli‐ and micro‐sized particles were produced using microfluidic techniques. Copper–chitosan milli‐particles (Cu‐chito) did produce nitric oxide (NO). The maximum rates of NO production were ∼ 1.40 nmol min‐1 g‐1 (Cu‐chito) and 1.08 nmol min‐1 g‐1 (Cu‐chito + glucose). The milli‐particles were tested with ARPE‐19 cell lines in cell proliferation assays. Cu‐chito particle treatments with nitrite showed 130% more growth in comparison with chitosan milli‐particles not containing copper. Furthermore, Cu‐chito treatments of nitrite + glucose showed 152% more growth in comparison with control groups, and 118% in comparison with Cu‐chito with nitrite alone. The activity of intracellular NO target, matrix metalloproteinases (MMP‐2 and ‐9), were shown to increase by 60% after 48 h of Cu‐chito ± glucose treatments.CONCLUSIONNO‐releasing copper–chitosan derivatives were produced, with proof of concept for nitric oxide release and positive effects on a cell culture model of wound healing. © 2018 Society of Chemical Industry
Current research has identified S-nitrosoglutathione reductase (GSNOR) as the central enzyme for regulating protein S-nitrosylation. In addition, the dysregulation of GSNOR expression is implicated in several organ system pathologies including respiratory, cardiovascular, hematologic, and neurologic, making GSNOR a primary target for pharmacological intervention. This study demonstrates the kinetic activation of GSNOR by its substrate S-nitrosoglutathione (GSNO). GSNOR kinetic analysis data resulted in nonhyperbolic behavior that was successfully accommodated by the Hill–Langmuir equation with a Hill coefficient of +1.75, indicating that the substrate, GSNO, was acting as a positive allosteric affector. Docking and molecular dynamics simulations were used to predict the location of the GSNO allosteric domain comprising the residues Asn185, Lys188, Gly321, and Lys323 in the vicinity of the structural Zn2+-binding site. GSNO binding to Lys188, Gly321, and Lys323 was further supported by hydrogen–deuterium exchange mass spectroscopy (HDXMS), as deuterium exchange significantly decreased at these residues in the presence of GSNO. The site-directed mutagenesis of Lys188Ala and Lys323Ala resulted in the loss of allosteric behavior. Ultimately, this work unambiguously demonstrates that GSNO at large concentrations activates GSNOR by binding to an allosteric site comprised of the residues Asn185, Lys188, Gly321, and Lys323. The identification of an allosteric GSNO-binding domain on GSNOR is significant, as it provides a platform for pharmacological intervention to modulate the activity of this essential enzyme.
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