Fluorescence study of the conformational properties of myoglobin structure. 1. pH-dependent changes of tryptophanyl fluorescence in intact and chemically modified sperm whale apomyoglobins.
The pH-dependent fluorescence of intact sperm whale apomyoglobin (apo-Mb) containing two tryptophans at positions 7 and 14, and of apo-Mb derivatives modified on Trp7 by 2-hydroxy-5-nitrobenzyl bromide (Koshland reagent) and o-nitrophenylsulphenyl chloride, has been studied. The fluorescence of apomyoglobins modified at His residues by iodoacetamide and bromoacetate, and at the N-terminal alpha-NH2 group by methylisothiocyanate, has also been investigated. The individual fluorescent properties of both tryptophans and their contributions to the total spectrum of apo-Mb have been resolved within the pH range 2-12.5. The quantum yield of the 'buried' Trp14 (lambda max at 326 nm) is shown to be twofold higher at pH greater than 8.5 than that of the 'exposed' Trp7 (lambda max at 333 nm). At pH 8.5-5.5 the fluorescence of Trp14 diminished approximately twofold due to quenching by the ionized His residue, most probably His119. The quenching is evidently dynamic because the fluorescence lifetime is shown to be linearly proportional to quantum yield in this pH range. The fluorescence of Trp7 practically does not change between pH 5.5 and 10.0 but increases 2.5-3-fold in the pH range 5.5-4.3 while the contribution of Trp14 remains constant. The conformational changes at the N-terminal and in the region adjacent to it, as well as in the whole apo-Mb molecule in acidic, alkaline and neutral pH ranges, are considered. A relationship is revealed between conformational states of the heme crevice and the N-terminal part of apo-Mb.
Postnikova
G B
GB
Laboratory of Biophysics of Redox Proteins, Institute of Biological Physics of the USSR Academy of Sciences, Moscow Region.
Komarov
Y E
YE
Yumakova
E M
EM
D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D009211:Myoglobin / Q000737:chemistry*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry*; D014907:Whales
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D014364:Tryptophan