Chemical Mechanism of Dinoflagellate Bioluminescence

Luciferin binding protein (LBP) binds luciferin at pH 8 but not at pH 6 (Fogel and Hastings, 1971) thus, LBP inhibits the luciferin-luciferase reaction at pH 8 but not at pH 6. Luciferin bound to LBP is stable, differing from the free form of luciferin that is extremely unstable. The molecular size of the Gonyaulax LBP was considered 

Purified LBP is obtained from the crude LBP separated in the gel filtration of the 35 kDa luciferase on Sephadex G-100 (see Fig. 8.2). The fractions of crude LBP are combined and the protein is precipitated with ammonium sulfate (75% saturation). The precipitate is dissolved in a small volume of lOmM Tris-HCl/5 mM 2-mercaptoethanol, pH 8, and a small amount of luciferin is added as a tracer. Then, the crude LBP is purified on a column of Sephadex G-200 (Hastings and Dunlap, 1986). The fractions of LBP are identified by luminescence produced by the addition of luciferase at pH 6.3 the luminescence due to the tracer luciferin is proportional to the amount of LBP in each fraction. 

To assay the amount of LBP, first an excess amount of luciferin is added to the sample at pH 8 to saturate the binding site of LBP, and then the excess luciferin is removed by gel filtration using a small column of Sephadex G-25 (about 1 ml volume) also at pH 8. The luciferin-bound LBP is eluted at the void volume. To measure the amount of LBP, the following assay buffer is added to a small portion of the elu-ate 0.2 M phosphate, pH 6.3, containing 0.25 mM EDTA, 0.1 mg/ml of BSA, and luciferase (Morse and Mittag, 2000). The total light obtained represents a relative amount of LBP the absolute amount (the weight or the number of molecules) cannot be obtained because the quantum yield of the luminescence reaction is not known. 

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