Quantum yield of near-infrared bioluminescence with firefly luciferin analog: AkaLumine

Abstract

We investigated bioluminescence with d-AkaLumine, which is a firefly luciferin analog producing 675-nm light through chemical reaction with O2, ATP, and Mg2+ in native firefly luciferase, and is useful for in vivo imaging. Using a total-photon flux spectrometer system calibrated for 450–950 nm, we evaluated the bioluminescence quantum yield for d-AkaLumine, characterizing the concentration of d-AkaLumine solutions using qNMR spectroscopy and HPLC measurements. We found that the bioluminescence quantum yield for d-AkaLumine with Photinus pyralis is insensitive to pH and that its value is 4.0 ± 0.5 %. The bioluminescence spectrum for d-AkaLumine is well fit by two simple Gaussian components. The decay rate for d-AkaLumine is very sensitive to the pH value, and it increases to 7.0×10−2sec−1 at pH 8.0. For stable absorption spectra for 4–20-μM AkaLumine aqueous solutions, we determined the molar extinction coefficient at 490 nm to be 3.33×104 L/mol cm.