Abstract
Acid-induced unfolding of horse apomyoglobin is studied with fluorescence of tryptophan residues and extrinsic probes 8-anilino-1-naphthalenesulfonate (ANS) and pyrene. Two-dimensional statistical correlation coefficient analysis of tryptophan, ANS and pyrene fluorescence reveals that apomyoglobin unfolds in two steps via a folding intermediate. Both tryptophan and ANS fluorescence show microenvironment-sensitive emission maximum and fluorescence intensity. Hetero-spectral 2D correlation analysis reveals that the “red” tryptophan fluorescence correlates with the “red” ANS fluorescence, and the “blue” correlates with the “blue” in the acid-induced unfolding process. Correlation curves are established between the hydrophobicity of the probe-binding site(s) as disclosed by ANS and pyrene fluorescence and the folding extent of apomyoglobin as indicated by tryptophan fluorescence. Two anion-induced refolding intermediates of apomyoglobin: Cl −-induced A-1, and trichloroacetate (TCA)-induced I-2 do not follow the correlation trajectory in the acid-induced unfolding. This suggests that the two anion-induced intermediates do not belong to the low-salt acid-unfolding pathway of apomyoglobin.
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