Regeneration of the Photoprotein Aequorin.

摘要

The calcium-triggered blue luminescence of aequeous solutions of the photoprotein aequorin, extracted and purified from the coelenterate Aequorea, results in a blue-fluorescent protein product (BFP) that has a fluorescence emission spectrum exactly like that of the chemically excited luminescence. The light-emitting chromophore in BFP is 2(p-hydroxyphenylacetyl)amino-3-benzyl-5-(p-hydroxyphenyl)pyrazine. The same chromophore occurs in various coelenterates including some which have a separately identifiable luciferin-luciferase system, as in Renilla. When BFP (the spent photoprotein after reacting with Ca(2+) in the light-emitting process) is incubated with the synthetic chromophore in the presence (but not in the absence) of oxygen, the active photoprotein is slowly regenerated as attested by the major properties including the same sensitivity to Ca(2+) in a luminescent reaction, quantitatively the same specific light emitting activity of 1.55 x 10 to the 15th power photons per ml of solution having A280 = 1.0/cm, same kinetics of light-emission in presence or absence of air, same absorption spectrum and same chromatographic behavior on DEAE-cellulose and on Sephadex G100.