Testing protease activity in the presence of a nitric oxide-generating foam

The purpose of this study was to test the effects of a two-component nitric oxide-generating foam on a variety of proteases. A fluorescent substrate 96-well assay was used to measure the protease activity of five human and one bacterial enzymes. The enzymes were tested at levels associated with extremely high proteolytic activity in human hard-to-heal (chronic) wounds (10µg/ml). The test product comes as a two-part unmixed pair of solutions (Solution A and Solution B) which are mixed 1:1 prior to use. In all, three test groups were used, consisting of one each of the separate solutions and a mixture of the two solutions. Dilutions of each solution were also tested. The reaction was monitored for fluorescence generation with 485nm excitation and 528nm emission, and one reading per minute for 10 minutes. The relative fluorescence unit (RFU) per minute (min) was calculated for each sample and the residual activity was reported as the RFU/min observed divided by the RFU/min of the non-treated control (the enzyme not exposed to any test solution). The undiluted test solutions reduced or eliminated detectable protease activity (multiple p-values, all p≤1.69×10^-8). Dilution had no apparent effect on activity reduction for Solution A or Solution A+B, and there was a dose-dependent loss of inhibition with Solution B. Two inhibitory mechanisms remain possible, but untested: the low pH of both Solution A and Solution A+B; or the formation of nitric oxide protein adducts. Additional testing in wound fluids or wound fluid surrogates is necessary to determine if the effects will be diminished in the presence of additional biomolecules.