Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state
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Abstract
The conformational transitions starting with the native protein, passing the molten
globule state and finally approaching the unfolded state of proteins was investigated
for bovine carbonic anhydrase B (BCAB) and human alpha-lactalbumin (alpha-HLA) by
means of fluorescence decay time measurements of the dye 8-anilinonaphthalene-1-sulphonic
acid (8-ANS). Stepwise denaturation was realized by using the denaturant guanidinium
chloride (GdmCl). It was shown that 8-ANS bound with protein yields a double-exponential
fluorescence decay, where both decay times considerably exceed the decay time of free
8-ANS in water. This finding reflects the hydrophobic environment of the dye molecules
attached to the proteins. The fluorescence lifetime of the short-time component is
affected by protein association and can be effectively quenched by acrylamide, indicating
that 8-ANS molecules preferentially bind at the protein surface. The fluorescence
lifetime of the long-time component is independent of the protein and acrylamide concentration
and may be related to protein-embedded dye molecules. Changes of the long lifetime
component upon GdmCl-induced denaturation and unfolding of BCAB and alpha-HLA correlate
well with overall changes of the protein conformation. The transition from native
protein to the molten globule state is accompanied by an increase of the number of
protein-embedded 8-ANS molecules, while the number of dye molecules located at the
protein surface decreases. For the transition from the molten globule to the unfolded
state was the opposite behaviour observed.