Luciferin Obtained by Kuwabara and Wassink

According to the Kuwabara-Wassink paper, the purified luciferin in aqueous neutral buffer solution showed an absorption maximum at 320 nm, and a fluorescence emission peak at 490 nm. The luminescence emission maximum measured with Airth s fungal luciferase system was 524 nm at pH 6.5, whereas the chemiluminescence emission maximum of the luciferin with H2O2 plus a droplet of strong NaOH plus ferrous sulfate was 542 nm. No information was reported on the chemical nature of the luciferin. 

Assay methods. Luciferin is measured by mixing 5-20 pi of a sample, 1-2 mg of CTAB, 1-2 mg of NaHC03, 3 ml of 50 mM Tris-HCl (pH 8), 10 pi of 10% H2O2, and 15 pi of 50 mM FeSCTj, in this order. The addition of FeSC 4 triggers the luminescence reaction. 

To measure the luciferin precursors, 0.1 ml of 0.4 M decylamine hydrochloride (made in 75% methanol) is added to the above mixture before the addition of FeSCAp This mixture is left at room temperature for 20-30 min, then 15 p,l of 50mM FeSCA is added to trigger luminescence. 

Properties of luciferin precursors. About one dozen of the luciferin precursors of M. citricolor isolated by HPLC had a strong tendency of isomerization, as mentioned above. Their molecular weights could not be established by mass spectrometry, which is probably due to isomerization, although they appear to be in a range of 300-600. The precursors showed an absorption peak at about 369 nm in methanol and aqueous acetonitrile (Fig. 9.13). According to an NMR study, all precursors probably contain the following common partial structure (personal communication from Dr. H. Nakamura, 1998). 

Treatment of the precursors with decylamine resulted in a high level of chemiluminescence activity. Taking the activity obtained with decylamine as 100%, the activities obtained with other amines were methylamine, 5% hexylamine, 23% octylamine, 39% and dode-cylamine, 55%. For comparison, the Panellus precursors, PS-A and PS-B, are best activated with methylamine, and a synthetic model compound, K-l, is best activated with hexylamine. 

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