Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea

Parts used

Key findings

• Extraction of marginal-strip tissue from nearly 10,000 specimens of the hydromedusan Aequorea, followed by ammonium sulfate precipitation, EDTA-2Na extraction of the resulting filter cake, and repeated DEAE column chromatography with NaCl gradient elution, yielded approximately 5 mg (5.3 mg dry weight) of a purified light-emitting protein named aequorin. Aequorin was the first bioluminescent system described as a single organic component with the properties of a protein, rather than the two-component luciferin-luciferase systems — a substrate oxidized by an enzyme — that characterized every other bioluminescent organism extracted up to that time.
• Purified aequorin emits light only upon addition of Ca++; among thirteen other cations tested — magnesium, barium, potassium, ammonium, zinc, cobalt, manganese, ferric iron, ferrous iron, copper, lead, cadmium, and strontium — none activates the reaction, with the slight activation seen for cadmium attributable to impurities and the weak strontium effect inconclusive.
• The velocity of aequorin's Ca++-triggered luminescent reaction depends on the molar ratio of Ca++ to EDTA present in solution: when EDTA is in molar excess over Ca++, no luminescent reaction occurs; at equimolar Ca++ and EDTA, the reaction proceeds at about half its maximal velocity; and a slight molar excess of Ca++ over EDTA drives the reaction to near-maximal velocity. In every case, the light-emitting reaction follows first-order kinetics.
• Aequorin's Ca++-triggered light-emitting reaction requires no dissolved oxygen: solutions equilibrated with pure hydrogen, air, or pure oxygen produce the same rate and total light output, within about 10 percent, distinguishing this reaction from every other bioluminescent system extracted up to that time, all of which depend on free oxygen or added peroxide.
• Aequorin does not act catalytically in its own light-emitting reaction, unlike the luciferase enzyme of the Cypridina bioluminescent system: a solution in which the aequorin luminescent reaction has already run to completion produces no acceleration of a fresh reaction when mixed with new aequorin, whereas spent Cypridina luciferase solution measurably speeds a fresh Cypridina luciferin reaction assayed under the same conditions.
• Aequorin has the general chemical properties of a protein — positive Biuret and Ninhydrin reactions, precipitation by ammonium sulfate, rapid denaturation below pH 4 even at 0°C, and an isoelectric point below pH 5.5 by paper electrophoresis — and behaves as a single substance, since no separate light-emitting component can be resolved from it by dialysis, DEAE chromatography, ammonium sulfate precipitation, or paper electrophoresis. Its molecular weight was calculated at approximately 35,000 from the ultraviolet absorption changes that accompany the luminescent reaction, using an assumed extinction coefficient for a reduced pyridinium-type chromophore analogous to protein-bound diphosphopyridine nucleotide (DPN) complexes.
• Aequorin's thermal stability depends on the solvent it is dissolved in: half of its luminescent activity is lost after a one-minute exposure to 58°C in EDTA-2Na solution, but the same one-minute half-inactivation temperature rises to approximately 83°C when the aequorin solution is instead saturated with ammonium sulfate.
• Total light output from the aequorin-Ca++ reaction is independent of pH between 5.1 and 8.3, declining only outside that range, while the rate of light emission behaves differently — showing a near-plateau between pH 6.5 and 7.5, decreasing at more acidic pH, and increasing at more alkaline pH — indicating that more than one pH-sensitive step underlies the reaction's kinetics even though total light yield is comparatively pH-insensitive.
• The bulge in aequorin's ultraviolet absorption spectrum at 310 mμ disappears upon the Ca++-triggered luminescent reaction and is replaced by a new absorption peak at 333 mμ; this spectral shift, together with the inhibitory effect of diphosphopyridine nucleotide (DPN) and N1-methylnicotinamide chloride on the reaction, is consistent with aequorin containing a reduced pyridinium-type functional group similar to that found in dehydrogenase-DPN protein complexes.
• Among the many substances tested against aequorin, only EDTA, malonic acid, and a homologous series of aliphatic aldehydes (propionaldehyde, isobutylaldehyde, caprylaldehyde, capraldehyde, and caproaldehyde) reversibly inhibit the rate of the Ca++-triggered reaction without substantially reducing the total light eventually produced, and the degree of aldehyde inhibition increases with the length of the aliphatic chain. Aromatic aldehydes such as benzaldehyde and p-dimethylaminobenzaldehyde instead reduce total light output (quenching) without measurably slowing the reaction's rate.
• Aliphatic alcohols — n-butyl, n-amyl, iso-amyl, n-hexyl, n-heptyl, and cyclohexanol — increase the total light produced by aequorin's Ca++-triggered reaction, with the increase tending to grow with the length of the alcohol's carbon chain (up to a 30 percent increase for saturated cyclohexanol), while the aromatic alcohol benzyl alcohol instead decreases total light output by about 10 percent.
• The luminescent reaction of purified aequorin produces bluish light, differing in color from the greenish luminescence of intact Aequorea tissue or freshly prepared tissue squeezates. A separate protein, distinct from aequorin, has also been isolated from Aequorea squeezates that appears greenish in sunlight and yellowish under tungsten light and shows a very bright green fluorescence under ultraviolet light, but exhibits no light-emitting reaction of its own; this protein occurs in the photogenic tissue together with aequorin and may account for the greenish luminescence color of the intact organism through a light-filtering or fluorescence effect.
• Aequorin's luminescence efficiency, calculated from its estimated molecular weight of 35,000 and a measured potency of 42,500 light units per milligram, is about half the luminescence efficiency of Cypridina luciferin per molecule under comparable assay conditions.