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.