An experiment was proposed and accomplished that was based on the hypothesis of the dissociation of the luciferase-luciferin complex in photoexcitation. A pump-probe experiment was performed with the use of picosecond laser pulses and was based on the effect of quenching of enzyme tryptophan fluorescence caused by luciferin binding. A photoinduced increase of the tryptophan fluorescence intensity was detected. Experimental results were interpreted on the basis of the assumptions on photoinduced dissociation of the luciferin-luciferase complex and Forster energy transfer from tryptophan to luciferin. Under the assumption on the photoinduced dissociation and stationary quenching of tryptophan fluorescence the rate of propagation of the conformational changes in the protein caused by the complex dissociation was estimated to be >20 m/s.
An experiment providing evidence in support of the hypothesis that the bioluminescent reaction is dissociative in character was proposed and carried out. The processes occurring in the native bioluminescent system were modelled by the luciferase — luciferin complex. The experiment was performed in accordance with the pump-and-probe scheme involving probing by picosecond laser pulses and was based on the steady-state quenching of the intrinsic tryptophan fluorescence of luciferase when the latter is bound in a complex with luciferin. An increase in the intensity of the tryptophan fluorescence on photoexcitation of luciferin was regarded as confirmation of the hypothesis of the dissociation of the complex and it made it possible to estimate the rate of propagation of the corresponding conformational changes through the protein molecule.
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