Extraction, partial purification and properties of obelin, the calcium-activated luminescent protein from the hydroid Obelia geniculata

Abstract

1. The Ca2+-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 Ca2+. 3. After an overnight extraction in 40mm-EDTA–200mm-Tris–HCl, pH7.0, 7.2×1011 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 (NH4)2SO4. The precipitate could be stored for at least 6 months as a suspension in 40mm-EDTA+saturated (NH4)2SO4, pH7.0, frozen at −70°C with a recovery of 95–100%. 6. Luminescence was also stimulated by Sr2+. However, obelin appeared to have a lower affinity for Sr2+ than for Ca2+. Mg2+ inhibited Ca2+-activated luminescence. 7. Obelin could be used to assay as little as 50pmol of Ca2+ in a final volume of 1ml. 8. At pH7.0 in Ca2+–EGTA [ethanedioxybis(ethylamine)tetra-acetate] buffers the rate of obelin luminescence was proportional to the square of the free Ca2+ concentration in the presence and absence of 1 and 10mm-Mg2+. Over the range 0.1–10μm-Ca2+ less than 0.03% of the obelin was consumed/s. 9. In order to use obelin to study free ionized Ca2+ concentrations similar to those found inside cells in the presence of 10mm-Mg2+ a minimum of 108 counts were required. A total of 1012 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.