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Leontieva O. V., Vlasova T. N., Ugarova N. N.
Interaction of the Fire-Fly Luceferase with Dimethyloxyluciferin
Abstract
Analysis of dymethyloxyluciferin (DMOL) absorption and fluorescence
spectra in pH range 3.9 - 12.0 showed that at pH 12.0 lmax of absorption
shifted from 485 nm (pH 9.6) to 356 nm. At pH 3.9 – 8.0 fluorescence lmax for DMOL is 639 nm. At higher pH the
additional fluorescence peak with l max = 537 nm (lex = 383 nm) was
observed. Its intensity grew in time.
It was shown that this peak corresponded to the product of DMOL chemical
transformation at pH > 9. This
transformation can be described by the first order reaction (k = 0.02 min-1).
Firefly luciferase catalytic and fluorescent properties were examined at the
presence of DMOL (20 – 140 mM) at pH 7.8. From
the data on the quenching of protein own fluorescence by DMOL the binding
constant Ks =45.7 mM was determined.
It was shown that DMOL is strong luciferase inhibitor (Ki = 10 mM ).
Copyright (C) Chemistry Dept., Moscow State University, 2002
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