Search

Two coumarin derived alpha-dicarbonyls, HOCGO and DMACGO, are presented as pH, polarity and quencher sensitive fluorescent reporters for proteins that can be targeted at reactive arginines. Both inactivate a number of enzymes that feature functionally critical and chemically susceptible arginyls. Both are chemoselective in responding towards Arg side chain but not towards Cys or Lys side chains under suitably dilute conditions. With pK(app) approximate to 6.7, HOCGO can serve as a pH sensor, while with pK(app) much less than 4.0, DMACGO is better suited as a polarity sensor. A contrasting set of changes are manifest in the CGOs upon protein interaction that are either attributable to Arg modification or to the noncovalent probe associations with hydrophobic protein domains. DMACGO probes a single hydrophobic site on ovalbumin while HOCGO is largely unresponsive to this protein. Three to five arginyls are modified in HSA and BSA by HOCGO as well as DMACGO, while the latter also probes two hydrophobic sites on both these proteins. HOCGO modifies a single arginine in LDH active site, while its adducts with H-4 and M(4)LDH isozymes titrate to the apparent pK(a) of 7.8. Other proteins labeled with HOCGO or DMACGO reveal a number of variations that can furnish information about the microenvironment in the sites probed. The CGOs are thus potentially useful reporters of protein domains that feature reactive arginines. Suitable experimental condition are defined based on mechanistic considerations that may be used in applying the CGOs as Arg modifiers and as fluorescent probes.