Binding of the Volatile Anesthetic Chloroform to Albumin Demonstrated Using Tryptophan Fluorescence Quenching

Intra- and intermolecular beta-pleated sheet formation in glutamine-repeat inserted myoglobin as a model for polyglutamine diseases

Background : The site of action of general anesthesia remains controversial, but evidence in favor of specific protein target(s) is accumulating. Saturable binding of halothane to bovine serum albumin (BSA) has recently been reported using photoaffinity labeling and fluorine 19 nuclear magnetic resonance spectroscopy. We report a new approach to study anesthetic binding to soluble proteins, based on native tryptophan fluorescence. Methods : Thymol-free halothane and fatty acid-free BSA were equilibrated in gas-tight Hamilton syringes and dispensed into stoppered quartz cuvettes at predetermined dilutions. Steady-state fluorescence spectroscopy was used to study their interaction. Results : Halothane quenched the tryptophan fluorescence of BSA in a concentration-dependent, saturable manner with a dissociation constant = 1.8 ± 0.2 mM and a Hill number = 1.0 ± 0.1. The two optical isomers of halothane bound to BSA with equal affinity. The ability of halothane to quench BSA tryptophan fluorescence was markedly decreased at pH 3.0 (which causes full uncoiling of BSA), with loss of saturable binding. Diethyl ether displaced a portion of halothane from its binding sites. Circular dichroism spectroscopy revealed no significant effect of halothane or diethyl ether on the secondary structure of BSA. Conclusions : The results suggest that halothane binds in hydrophobic domains containing tryptophan in BSA. This approach may prove useful for studying the interaction of volatile anesthetics and proteins and has the advantage that the location of halothane in the protein is identified.

Binding of Halothane to Serum Albumin Demonstrated Using Tryptophan Fluorescence

The guanidinium chloride denaturation/renaturation of the holo- and apo-horseradish peroxidase isoenzyme c (HRP) has been studied by fluorescence and circular dichroism spectroscopies.



A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride (0.5 M). This intermediate has a secondary structure content like that of the native protein but a poorly defined tertiary structure. Renaturation of the apo-HRP is reversible and 100% activity could be obtained after addition of a twofold excess of free haem.



The denaturation of the holo-HRP is more complex and occurs in two distinct steps; unfolding of the protein backbone and loss of the haem. The denatured protein folds back to its native conformation but the incorporation of the haem occurs only after the secondary structure is formed.



Ca2+ appears to be important for the stability of the protein as the apo-HRP is more resistant to denaturation in the presence of Ca2+. The free-energy change during unfolding of the apo-HRP was determined in the absence and presence of Ca2+ and found to be 9.2 kJ/mol and 16.7 kJ/mol, respectively. The importance of Ca2+ to the protein stability was also supported by studies on the loss of the haem from the protoporphyrin-IX–apo-HRP complex.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1993.tb17654.x

A step towards understanding the folding mechanism of horseradish peroxidase. Tryptophan fluorescence and circular dichroism equilibrium studies.

https://www.ks.uiuc.edu/Publications/Papers/PDF/ERVI2002/ERVI2002.pdf

What causes hyperfluorescence: folding intermediates or conformationally flexible native states?

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.

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 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 less than 5.5 and pH greater than 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 less than 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 I325/I350 which diminishes at pH greater than 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 maintenance 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.

Fluorescence study of the conformational properties of myoglobin structure. 3. pH-dependent changes in porphyrin and tryptophan fluorescence of the complex of sperm whale apomyoglobin with protoporphyrin IX; the role of the porphyrin macrocycle and iron in formation of native myoglobin structure.

Myoglobin is known to be the reference protein that is why the knowledge of the conformational behavior of the myoglobin-like structure is of common importance for understanding the mechanism of protein functioning. In this work fluorescence was used to study the pH- and ligand-induced conformational changes in sperm whale myoglobin and related structures — apomyoglobin (apo-Mb) and the complex of apo-Mb with protoporphyrin IX (PPIX-apo-Mb) which is a iron-free analog of Mb. The structural changes at the N-terminal and adjacent protein region were followed by the fluorescence of two invariant tryptophans of the A-helix, Trp7(A5) and Trpl4(A12), while those in the “active site”, the heme crevice, were followed by emission of PPIX. As distinct from the non-fluorescing heme, PPIX has an extensive fluorescence in the visible spectral region.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1991.tb16007.x

Fluorescence study of the conformational properties of myoglobin structure. 2. pH- and ligand-induced conformational changes in ferric- and ferrousmyoglobins.

Conformational changes induced by ligands and pH in lupine ferrileghemoglobin selectively modified at Tyr-E16 by the imidazolide spin label has been studied by the method of electron spin resonance in the pH range 6–13. It is shown that in the alkaline pH region the bound spin label registers a local conformational transition which precedes the alkaline denaturation of the protein. In aquamet, cyanide and nicotinate complexes of ferrileghemoglobin this transition occurs with pK 10.5, in acetate and azide complexes with pK 11. In all these ligand derivatives the transition is induced by alteration in the ionization state of one group (ΔnH+∼ 1), most probably, the ɛ-amino group of Lys-GH3. The latter is linked with the Glu-A14 residue and this bond is essential for maintaining the native conformation of leghemoglobin. The ligand-induced conformational changes in the vicinity of the label are small and consist, most likely, in some alteration of the mutual arrangement of the AE and GH helical complexes. No correlation has been revealed between the spin state of the heme iron and the conformation of leghemoglobin in the region under study.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1985.tb08892.x

Elena M. Yumakova

Papers

Fluorescence study of the conformational properties of myoglobin structure. 1. pH-dependent changes of tryptophanyl fluorescence in intact and chemically modified sperm whale apomyoglobins.

Fluorescence study of the conformational properties of myoglobin structure. 3. pH-dependent changes in porphyrin and tryptophan fluorescence of the complex of sperm whale apomyoglobin with protoporphyrin IX; the role of the porphyrin macrocycle and iron in formation of native myoglobin structure.

Fluorescence study of the conformational properties of myoglobin structure. 2. pH- and ligand-induced conformational changes in ferric- and ferrousmyoglobins.

Spin-label study of conformational changes induced by pH and ligands in leghemoglobin from yellow lupine root nodules.