The Luciferase of Gonyaulax polyedra

The active luciferase in the soluble luminescence system occurs in two molecular sizes, 130 kDa and 35kDa. The 130 kDa luciferase is the native form and occurs in extracts made at pH 8, and if luciferase is extracted with a pH 6 buffer, 130 kDa luciferase is converted into 35 kDa luciferase by the action of a protease (Krieger and Hastings, 1968 Fogel and Hastings, 1971 Krieger et al., 1974). The 130 kDa species is considered the naturally occurring form. 

The solution is dialyzed against the same buffer using a hollow fiber assembly, and then added onto a column of Affi-Gel Blue (50-100 mesh, 2 x 15 cm, Bio-Rad) prepared with the same buffer. The column is washed with the same buffer. Then luciferase is eluted with 50 mM Tris-HCl, pH 8.5, containing 5mM EDTA, 3 mM DTT, and 0.5 M NaCl (Hastings and Dunlap, 1986, state that it may be preferable to omit the Affi-Gel step because of difficulties encountered). 

The eluted luciferase is precipitated with ammonium sulfate. The precipitate is dissolved in 1 mM Tris-HCl, pH 8, containing 0.1 mM EDTA, 3 mM DTT and 0.1 M NaCl, and chromatographed on a column of Sephacryl S-300 (2.6 x 97 cm) using the same buffer. Luciferase is eluted in two peaks, corresponding to the molecular weights of about 420,000 (an aggregate) and 130,000, in a ratio of about 8 1. The fractions of these two peaks are pooled separately the Mr 420,000 luciferase is concentrated by either ultrafiltration or precipitation with ammonium sulfate. 

The concentrated luciferase solution is dialyzed overnight against 4 liters of 1 mM Tris-HCl buffer, pH 8.5, containing, 0.1 mM EDTA and 3 mM DTT. Then luciferase is further purified on a column of DEAE-BioGel A (1 x 25 cm, Bio-Rad) by elution with a linear increase of NaCl from 0 to 100 mM in the same buffer as that used in dialysis. The purified luciferase had a specific activity (based on initial maximum intensity) of approximately 8.5 x 1014 quanta sec 1mD1Aj810. 

Assay of the activities of luciferin and luciferase. Small volumes (5-50 xl) of luciferase and luciferin (Section 8.2.4) are mixed in 2 ml of 0.2 M citrate buffer, pH 6.3, or 0.2 M phosphate buffer, pH 8. The measurement is made in terms of the total light emission or the initial maximum light intensity that is reached within a few seconds. The 

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