PROPERTIES OF BIOLUMINESCENT PROTEIN AEQUORIN

A protein, aequorin, having no more than the usual, slight fluorescence of proteins in general, gives a bright, blue, light-emitting reaction with Ca2+, leading irreversibly to a strongly blue fluorescent protein product; the reaction mechanism presumably involves an unusual intramolecular chemical change catalyzed by Ca2+. A functional moiety in this bioluminescence reaction can be separated from the protein in the form of either of two blue fluorescent compounds designated as “AF-350” and “AF-400.” The former has an absorption maximum at 350 mμ, and molecular weight of 277 by mass spectrometry; its separation from aequorin is by treatment with urea and mercaptoethanol at alkaline pH, leaving the protein moiety as “apoaequorin-SH.” The latter fluorescent compound (AF-400) has an absorption maximum at 400 mμ; its separation from aequorin is by treatment with NaHSO3, leaving apoaequorin-SO. Critically determined properties of aequorin include: E1cm1% at 280 mμ = 27.0; s20,w = 2.90 × 10-13 sec; D20,w = 8.70 × 10-7 cm2/sec; MW by Sephadex G-75 gel filtration = 23,000; MWsD = 30,000 (using partial specific volume = 0.729); MW by amino acid analysis = 32,000; and luminescence activity = 4.5 × 1015 photons per mg dry weight at 25°. The chemical composition of aequorin includes, in addition to the above-mentioned functional moiety, 18 different amino acids, an unidentified other amino compound, glucose, no phosphate, and no acetylneuraminic acid. © 1969, American Chemical Society. All rights reserved.