Extraction, partial purification and properties of obelin, the calcium-activated luminescent protein from the hydroid Obelia geniculata.
1. The Ca(2+)-activated luminescent protein obelin was extracted from the hydroid Obelia geniculata. 2. After the addition of a large excess of calcium (greater than 5mm) a peak in the rate of luminescence occurred within 100ms, followed by an exponential decay (k=2.8s(-1)). The obelin activity (light emitted) was measured by the peak height or by the total number of counts recorded on a scalar in the first 10s after addition of Ca(2+). 3. After an overnight extraction in 40mm-EDTA-200mm-Tris-HCl, pH7.0, 7.2x10(11) counts were obtained from 186g of wet hydroids. 4. The stability of the crude extracts was dependent on pH, being optimal at pH7.0. 5. Obelin could be purified threefold with a yield of 69% by selecting the protein precipitated between 60%- and 100%-saturated (NH(4))(2)SO(4). The precipitate could be stored for at least 6 months as a suspension in 40mm-EDTA+saturated (NH(4))(2)SO(4), pH7.0, frozen at -70 degrees C with a recovery of 95-100%. 6. Luminescence was also stimulated by Sr(2+). However, obelin appeared to have a lower affinity for Sr(2+) than for Ca(2+). Mg(2+) inhibited Ca(2+)-activated luminescence. 7. Obelin could be used to assay as little as 50pmol of Ca(2+) in a final volume of 1ml. 8. At pH7.0 in Ca(2+)-EGTA [ethanedioxybis(ethylamine)tetra-acetate] buffers the rate of obelin luminescence was proportional to the square of the free Ca(2+) concentration in the presence and absence of 1 and 10mm-Mg(2+). Over the range 0.1-10mum-Ca(2+) less than 0.03% of the obelin was consumed/s. 9. In order to use obelin to study free ionized Ca(2+) concentrations similar to those found inside cells in the presence of 10mm-Mg(2+) a minimum of 10(8) counts were required. A total of 10(12) counts can be readily extracted from about 200g of wet hydroids. Thus a sufficient quantity of an aequorin-like calcium-activated luminescent protein should now be available to workers in the United Kingdom in order to carry out physiological experiments.
D000331:Aequorin; D000645:Ammonium Sulfate; D002118:Calcium; D011232:Chemical Precipitation; D003063:Cnidaria / Q000032:analysis*; D004739:England; D006863:Hydrogen-Ion Concentration; D008163:Luminescent Measurements; D008274:Magnesium; D011506:Proteins / Q000302:isolation & purification*; D013324:Strontium
D011506:Proteins; D000331:Aequorin; D008274:Magnesium; D000645:Ammonium Sulfate; D002118:Calcium; D013324:Strontium