Pholasin--a bioluminescent indicator for detecting activation of single neutrophils.
Pholasin is the protein-bound luciferin from the bivalve mollusc Pholas dactylus which reacts with its luciferase and molecular oxygen to produce light. Pholasin was purified 226-fold with a yield of 58% from P. dactylus to give a preparation free from luciferase contamination. The ratio (k) of endogenous pholasin chemiluminescence to that when maximally stimulated by luciferase was 8.12 X 10(-6) +/- 0.87 X 10(-6) (mean +/- SD, n = 6), equivalent to a t 1/2 of 23.7 h at pH 9. Pholasin could detect reactive oxygen metabolite production from neutrophils stimulated by the chemotactic peptide N-formyl-Met-Leu-Phe, in the presence and absence of 2-chloroadenosine or cytochalasin B, and by latex beads in the presence and absence of cytochalasin B. Pholasin was also able to detect a longer-lived oxidative activity distinct from myeloperoxidase, and released from neutrophils activated by latex beads or chemotactic peptide; luminol could not. Under optimal conditions pholasin produced a signal some 50-100 times that of luminol in the presence of activated neutrophils. This enabled activation of a single neutrophil by chemotactic peptide (1 microM) to be detected, giving a signal of 194 +/- 21 chemiluminescent counts per second, some six times that of the background signal (mean +/- SD, n = 2). Pholasin thus provides an indicator sufficiently sensitive to establish whether neutrophil activation occurs through thresholds in individual cells.
D000818:Animals; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003571:Cytochalasin B / Q000494:pharmacology; D049454:Firefly Luciferin / Q000302:isolation & purification*; D008156:Luciferases; D008163:Luminescent Measurements; D008974:Mollusca; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D009195:Peroxidase / Q000097:blood
D003571:Cytochalasin B; D009240:N-Formylmethionine Leucyl-Phenylalanine; D049454:Firefly Luciferin; C052157:pholasin; D009195:Peroxidase; D008156:Luciferases