Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 11/17/2014
Publication Date: N/A
Technical Abstract: Neutrophil elastase (NE) activity in urine, sputum and nasal mucous is used as an indicator of inflammation due to viral or bacterial infection. However, bovine nasal mucous neutrophils collected, lysed and stored in Dulbecco's minimal medium containing Phenol Red, showed no NE activity with methoxysuccinly-ala-ala-val-pro-7 aminofluorocoumarin (MeOsuc-AAPV-AFC) as substrate, whereas the Uristix@) strip assay was positive for NE activity. Titration of 7-aminofluorocoumarine (AFC), the fluorescent leaving group, with increasing concentrations of Phenol Red showed the Phenol Red did not quench fluorenscence. Dixon plots of elastase inhibition with Phenol Red using MeOsucAAPV-AFC as substrate revealed uncompetitive inhibition, suggesting that Phenol Red binds to the enzme-substrate complex. In contrast, a different substrate, N-(tosyl-alanyloxy)-3phenlpyrrole (the substrate inbedded in the Uristix@ strips), revealed that Phenol Red does not inhibit NE activity, a result that is identical to that using the Uristix@ strip test for NE. The inhibition constant of Phenol Red for human neutrophil elastase is 0.103 mg/ml (0.29 mM). Similarly, the inhibition constant with a known inhibitor of human neutrophils elastase, N-(methoxysuccinyl)-ala-ala-pro-val-chloromethyl ketone, is 0.27 mM. These results suggest that caution must be used when considering using Dulbecco's solution containing Phenol Red for collecting neutrophils that are to be used in subsequent enzyme assays.