Fluorescence study of the conformational properties of myoglobin structure

Abstract

The porphyrin and tryptophan fluorescence of sperm whale apomyoglobin complexed with protoporphyrin IX has been studied in the pH range 2-13. It has been shown that the fluorescence and absorption spectra of protoporphyrin incorporated into the heme crevice remain constant in the pH range 5.5 -10.8 but change significantly at pH < 5.5 and pH > 10.8, due to the acid and alkaline denaturation, respectively, of the complex accompanied by dissociation of protoporphyrin IX. At the same pH ranges, the quantum yield of tryptophanyl fluorescence increases sharply as a result of removal of protoporphyrin, acting as a quencher, from the complex. Other parameters of tryptophanyl fluorescence (maximum position, halfwidth and spectrum shape) change in the alkaline region as well. In the acidic pH range, these parameters change only at pH < 4.3, indicating that the Trp surroundings are more stable to denaturation than the heme crevice region. Between pH 5.5 and 10.9, where the complex of apomyoglobin with protoporphyrin IX is in its native state, the main parameters of tryptophan fluorescence remain unchanged except for the ratio 1325/1350 which diminishes at pH > 9.5. Its alteration precedes the alkaline denaturation of the complex and can be explained by a local conformational change induced by the break of the 'salt bridges' essential for the maintaince of the native Mb structure in the N-terminal region. The fluorescence data obtained for apomyoglobin, myoglobin and the complex between protoporphyrin IX and apomyoglobin enable one to compare their structures and to evaluate the role of the porphyrin macrocycle and the iron atom in the formation of the native myoglobin structure and its functioning.In the previous parts of this work (see two preceding papers), we studied conformational changes in apomyoglobin and ferrous-and ferricmyoglobins at the N-terminal and in the region adjacent to it, by means of tryptophanyl fluorescence. In this paper, using the same technique, we have investigated pH-induced conformational changes both at the periphery of the myoglobin structure where both tryptophan residues, Trp7 (A5) and Trpl4 (A12), are localized, and at the 'active site' of myoglobin in the heme crevice. Since the ironporphyrin complex (heme) does not fluorescence, a metal-free analog of myoglobin, the complex of the apoprotein with protoporphyrin IX (PPIX-apo-Mb) was used which shows a strong emission in the visible spectral region. As has been shown earlier, PPIX readily enters the heme cavity of different heme proteins like myoglobin [l -31, hemoglobin [4-71, horseradish peroxidase [8] and cytochrome c [9].Correspondence to G. B. Postnikova, Institute of Biological Physics of the USSR Academy of Sciences, 142292, Pushchino, Moscow Region, USSR Ahhreviations. PPIX, protoporphyrin IX; PPIX-apo-Mb, complex of PPIX with apomyoglobin; MITC-apo-Mb, apomyoglobin modified by methyisothiocyanate (MITC); PPIX-apo-HRP, complex of PPIX with horseradish peroxidase; PPIX-apo-Hb, complex of PPIX with hemoglobin; apo-Mb, myoglobin con...

Interactions of Two Amphiphilic Penicillins with Myoglobin in Aqueous Buffered Solutions: A Thermodynamic and Spectroscopy Study

“…This is in agreement with the fact that at the isoelectric point the electrostatic interactions are small and, therefore, the free energy of unfolding is due to the solvophobic effect during the denaturation process . Similar results were obtained in the denaturation studies of different proteins, such as bovine catalase, myoglobin, and HSA . It has been also seen that dicloxacillin has a larger denaturant effectivity in agreement with light scattering data …”

A Dye-Decolorizing Peroxidase from Vibrio cholerae

“…Furthermore, the peaks of the Soret and Q bands of Vc DyP as purified were almost identical to those of Vc DyP reconstituted with 1 equiv of PPIX (405, 506, 544, 577, and 630 nm). The fluorescence spectrum of Vc DyP as purified with excitation at 407 nm exhibited an intense band at 624 nm (Figure B), similar to that of the PPIX complexed with apomyoglobin . These results indicate that some percentage of Vc DyP was purified as a complex with PPIX, but not with heme.…”

“…The absorption spectrum shows the Soret band at 406 nm and Q-bands at 510, 548, 569, and 623 nm. This absorption spectrum is not typical of those of heme-containing proteins, but rather similar to those of apomyoglobin-containing protoporphyrin IX (PPIX) with bands at 409, 508, 543, 574, and 626 nm, or IsdC containing PPIX with bands at 406, 509, 546, 570, and 625 nm . Furthermore, the peaks of the Soret and Q bands of Vc DyP as purified were almost identical to those of Vc DyP reconstituted with 1 equiv of PPIX (405, 506, 544, 577, and 630 nm).…”

“…The fluorescence spectrum of VcDyP as purified with excitation at 407 nm exhibited an intense band at 624 nm (Figure 1B), similar to that of the PPIX complexed with apomyoglobin. 28 These results indicate that some percentage of VcDyP was purified as a complex with PPIX, but not with heme. The ratio of absorbance at 406 nm to that at 280 nm suggested that the PPIX-bound protein was estimated to be at most 10% of the total protein when the protein was expressed in LB medium, but it was reduced to ∼5% when M9 minimal medium was used.…”

Red GFP and endogenous porphyrins

“…On the other hand, this analysis demonstrated a fragmentation pattern very similar to that of uroporphyrin III (our unpublished data). The fluorescence lifetime of the red chromophore, measured by phase and modulation fluorometry [7], is 15.5 ± 0.1 ns, which is considerably longer than that of conventional fluorescent chromophores, but is remarkably close to that of porphyrins, for example, protoporphyrin IX has a fluorescence lifetime of 13.7 ± 0.3 ns (see also [8]).…”