* To whom correspondence should be addressed.
Received September 10, 2004; Revision received October 12, 2004
The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambdaem = 639 nm. At higher pH values an additional emission maximum appears at lambdaem = 500 nm (wavelength of excitation maximum lambdaex = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambdaabs = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambdaabs = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambdaabs = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambdaex = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.
KEY WORDS: bioluminescence, fluorescence, firefly luciferase, luciferin, oxyluciferin, monomethyloxyluciferin, dimethyloxyluciferin