The bioluminescent oxygen metabolite indicator protein pholasin was characterized with respect to the type and location of reactive oxygen metabolites detected in suspensions of stimulated human neutrophils. Whereas pholasin detected reactive oxygen metabolites from neutrophil suspensions stimulated with soluble agents, particulate stimulants were apparently not effective triggering agents for pholasin-dependent neutrophil chemiluminescence. Neutrophils stimulated with fMet-Leu-Phe (1 to 100 nmol/l) showed maximum pholasin-dependent chemiluminescence 45 to 60 s after stimulation. The time of maximum chemiluminescence was virtually independent of fMet-Leu-Phe concentration. In contrast, the time to reach maximum light emission increased from 60 s with 100 nmol/l phorbol ester to 295 s with 1 nmol/l phorbol ester. Significant inhibition of stimulated chemiluminescence was caused by both superoxide dismutase (20 micrograms/ml, 80% inhibition) and reduction of the oxygen concentration in the incubation medium to less than 0.5 mumol/l (95% inhibition). In contrast, the myeloperoxidase inhibitor sodium azide (0.1 mmol/l) afforded only 50% inhibition of the pholasin-dependent neutrophil chemiluminescence. Our results show that pholasin detects superoxide radicals released from cells stimulated by soluble stimulants but not intracellular oxidative activity elicited by particulate stimulants.

Azides↗

PubMedDate not supplied

The chromophore of pholasin: a highly luminescent protein.

Pholasin is the photoprotein extracted from the marine bivalve Pholas dactylus. It undergoes an oxidative chemiluminescent reaction to oxypholasin with superoxide anion, hypochlorite, peroxidases and other oxidants. Since the observed absorbance and chemiluminescent emission spectra of pholasin solutions cannot be brought about solely by the amino acids composing the protein, there has to be a chemiluminescent chromophore. However, little is known about the chemical nature of this molecule. This work seeks to identify the chemical structure of the luminescent prosthetic group of pholasin. Pholasin could not be reactivated using chromophores from the hydroid Obelia geniculata (coelenterazine) and from the ostracod shrimp Vargula (formerly Cypridina) hilgendorfi. Furthermore, the reaction product of the Vargula chromophore could not be detected in solutions containing oxypholasin. Fluorescence analysis of such a solution revealed a compound with an emission spectrum (lambda max 480 nm; excitation at 320 nm), resembling the emission spectrum of the chemiluminescent reaction. This fluorescent substance was separated by gel filtration. It exhibited an apparent molecular mass of less than 2000. Fluorescence measurements of extracts of partially purified pholasin suggested that a flavin moiety may be involved in pholasin luminescence.

Animals↗