1.Applications of luciferin biosynthesis: Bioluminescence assays for l-cysteine and luciferase.
Niwa K1, Nakajima Y, Ohmiya Y. Anal Biochem. 2010 Jan 15;396(2):316-8. doi: 10.1016/j.ab.2009.09.014. Epub 2009 Sep 11.
Based on the biosynthetic pathway of firefly bioluminescence substrate D-luciferin, the concentration of L-cysteine can be quantified using a simple protocol and a conventional luminescence detector. The lower limit of quantification (signal/noise ratio [S/N]=10) was 0.26 microM. Using our method, the total amount of free/reduced and disulfide/oxidized L-cysteine could be measured successfully in human serum. In addition, biosynthetic precursors such as 2-cyano-6-hydroxybenzothiazole and L-luciferin could replace d-luciferin in the cell-based luciferase assay. Our results suggest that the bioluminescence reaction associated with the biosynthesis of bioluminescence substrates can provide a fast and cost-effective assay method.
2.Enhancement of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hydrazides by 6-hydroxybenzothiazoles.
Thorpe GH, Kricka LJ, Gillespie E, Moseley S, Amess R, Baggett N, Whitehead TP. Anal Biochem. 1985 Feb 15;145(1):96-100.
6-Hydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, and 2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid (dehydroluciferin) dramatically enhance light emission from the horseradish peroxidase conjugate catalyzed oxidation of luminol, isoluminol, N-(6-aminobutyl)-N-ethyl isoluminol, and 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide by either peroxide or perborate. Light emission is enhanced by up to 1000-fold, which is an improvement over the enhancement previously observed using firefly luciferin (4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid). Enhancement is influenced by enhancer concentration and pH. Spectral scans of light emitted in enhanced and unenhanced reactions are similar, suggesting that aminophthalate products, and not the enhancers, are the emitters.
3.Oxyluciferin, a luminescence product of firefly luciferase, is enzymatically regenerated into luciferin.
Gomi K1, Kajiyama N. J Biol Chem. 2001 Sep 28;276(39):36508-13. Epub 2001 Jul 16.
The activity regenerating luciferin from the luminescent product oxyluciferin was found in the protein fraction of a lantern extract from Photinus pyralis. The protein, luciferin-regenerating enzyme (LRE), was purified to homogeneity by ammonium sulfate precipitation followed by successive column chromatography on Ultrogel AcA34, S-Sepharose FF, Q-Sepharose FF, TSKgel super Q 5pw and TSKgel G3000 SW(XL). This enzyme was a single polypeptide with a molecular mass of 38 kDa. LRE converted oxyluciferin to 2-cyano-6-hydroxybenzothiazole and thioglycolic acid. In the presence of d-cysteine, 2-cyano-6-hydroxybenzothiazole was turned over into luciferin. The same activities were detected in the extracts from two Japanese fireflies, Luciola cruciata and Luciola lateralis. We have cloned a cDNA encoding LRE from poly(A)+ RNA of the lantern of P. pyralis using reverse transcription-polymerase chain reaction, 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE.