The pH dependence of the PHM-catalyzed monooxygenation of dansyl-YVG was studied in two different buffer systems in the pH range of 4-10. The pH-activity profile measured in a sulfonic acid buffer exhibited a maximum at pH 5.8 and became inactive at pH >9. The data could be fit to a model that assumed a protonated unreactive species A, a major reactive species B, and a less reactive species C. B formed in a deprotonation step with pK(a) of 4.6, while C formed and decayed with pK(a)s of 6.8 and 8.2, respectively. The pH dependence was found to be dominated by k(cat), with K(m)(dansyl-YVG) remaining pH-independent over the pH range of 5-8. Acetate-containing buffers shifted the pH maximum to 7.0, and the activity-pH profile could be simulated by formation and decay of a single active species with pK(a)s of 5.8 and 8.3, respectively. The pH-dependent changes in activity could be correlated with a change in the Debye-Waller factor for the Cu-S(met) (M314) component of the X-ray absorption spectrum which underwent a transition from a tightly bound inactive "met-on" form to a conformationally mobile active "met-off" form with a pK(a) which tracked the formation of the active species in both sulfonic acid and acetate-containing buffer systems. The data suggested that the conformational mobility of the bound substrate relative to the copper-superoxo active species is critical to catalysis and further suggested the presence of an accessible vibrational mode coupling Cu-S motion to the H tunneling probability along the Cu-O...H...C coordinate.
The basic cellular internalization processes of endocytosis and phagocytosis are important to many areas of cell biology including receptor internalization, pathogen response, and apoptotic cell clearance. However, the ability to study these processes has historically been limited by the lack of quality tools to directly monitor the internalization and acidification of cargo. Here we present an assortment of tools to enable research into cellular internalization pathways with particular relevance to immunology. Two fluorogenic, pH-sensitive probes, one green and one red, are presented. These dyes are non-fluorescent at the neutral pH typically found in the cytosol and extracellular environment, but become brightly fluorescent in acidic cellular compartments, permitting direct monitoring of internalization and acidification processes. We present experimental data in which these dyes were used to label microorganisms including yeast and gram-positive and -negative bacteria. Live cell imaging and flow cytometry were then used to monitor the phagocytosis and acidification of these labeled particles by cultured murine macrophages over time. Additionally, we investigated the utility of these dyes to study phagocytic clearance of apoptotic cells by primary human monocyte-derived macrophages. We anticipate that these tools will enable additional research into the fields of receptor trafficking, drug delivery, host-pathogen response, and clearance of apoptotic cells.
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