Tryptophanyl fluorescence heterogeneity of apomyoglobins. Correlation with the presence of two distinct structural domains

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Abstract
The individual fluorescence of the 2 tryptophan residues (Trp-7 and Trp-14) of mammalian apomyoglobins was resolved by comparing the fluorescence properties of these proteins to those of bluefin tuna apomyoglobin, which contains only Trp-14. The 2 trypotphan residues had different emission maxima, i.e., 321 for Trp-14 and 333 for Trp-7. The fluorescence of Trp-14 depends on the protonation of a sterically related histidyl residue in the pH range between 8.3 and 5.6, where no conformational change was detected. This residue was identified as His-119. At pH 8.3 the quantum yield of Trp-7 is lower than that of Trp-14. An increase of the fluorescence intensity of Trp-7 occurs when the heme binding site of apomyoglobin is destroyed by acid or a low concentration of guanidine hydrochloride. An independent unfolding of the N-terminal district of the apomyoglobin molecule occurs on increasing the guanidine concentration. The 2 distinct structural transitions were discussed in terms of 2 domains of tertiary structure.