The color variations of light emitted by some natural and mutant luciferases are normally attributed to collective factors referred to as microenvironment effects; however, the exact nature of these interactions between the emitting molecule (oxyluciferin) and the active site remains elusive. Although model studies of noncomplexed oxyluciferin and its variants have greatly advanced the understanding of its photochemistry, extrapolation of the conclusions to the real system requires assumptions about the polarity and proticity of the active site. To decipher the intricate excited-state dynamics, global and target analysis is performed here for the first time on the steady-state and time-resolved spectra of firefly oxyluciferin complexed with luciferase from the Japanese firefly (Luciola cruciata). The experimental steady-state and time-resolved luminescence spectra of the oxyluciferin/luciferase complex in solution are compared with the broadband time-resolved firefly bioluminescence recorded in vivo. The results demonstrate that de-excitation of the luminophore results in a complex cascade of photoinduced proton transfer processes and can be interpreted by the pH dependence of the emitted light. It is confirmed that proton transfer is the central event in the spectrochemistry of this system for which any assignment of the pH-dependent emission to a single chemical species would be an oversimplification.
A spectrofluorimeter system has been developed which uses an on-line general purpose digital computer to both collect and correct fluorescence and luminescence spectra; no servosystems or complex analog circuitry are employed. Spectra are represented by a series of up to 500 individual data points, each of which is, in turn, the average of 16 samplings of the signal photomultiplier. The signal produced by a reference photomultiplier is also collected and used to make corrections appropriate to the type of spectra being collected. Wavelength drive is accomplished with a reversible stepping motor driven by commands from the computer; thus the wavelength position can be automatically and precisely controlled by the computer without recourse to cumbersome mechanical components. Corrections to collected spectra are made in real time on the basis of correction factors derived from fluorescence standards or a standard lamp. The complete system can collect a full spectrum (500 points) in 8 sec, average the results of any number of automatically repeated scans, and furnish the operator with both corrected and uncorrected spectra on either a wavelength or a wavenumber scale. In addition, the data can be retained on punched paper tape or presented graphically in a format acceptable for publication. Details of the system are presented with examples of its performance.
Extracts prepared from Gonyaulax cells harvested late in the 12-hour dark period of an artificial 24-hour day contain as much as four times more luciferin activity than similar extracts prepared during the light period. If cultures are not treated with heat or light to reduce the flashing associated with handling, the time of maximum activity is obscured.
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