Fluorescence Studies with Tryptophyl Peptides<sup>*</sup>

Edelhoch, Harold; Brand, Ludwig; Wilchek, M.

doi:10.1021/bi00854a024

Cited by 139 publications

(60 citation statements)

References 29 publications

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“…The protonated form of histidine (Shinitzky & Goldman, 1967;Loewenthal et al, 1991) and unionized carboxylic acid groups (White, 1959;Cowgill, 1963) have been demonstrated to be efficient quenchers of tryptophan fluorescence. Both protonated and deprotonated amino groups (White, 1959;Cowgill, 1963;Edelhoch et al, 1967;Shinitzky & Goldman, 1967) have the ability to quench tryptophan fluorescence, the distance of the amine group from the indole in the excited state dictating which form is the more efficient quencher (Edelhoch et al, 1967). In the case of peptide H1, His-33 was shown to be partially responsible for this quenching because replacement of His-33 with an alanine residue (in peptide Hla) reduced quenching from 30% to 15% in the random coil conformation and from 63 to 45% in the presence of SDS, which seems to induce a-helix in this system.…”

Section: Discussionmentioning

confidence: 99%

Effect of pH and denaturants on the folding and stability of murine interleukin‐6

et al. 1993

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The conformation and stability of a recombinant mouse interleukin-6 (mlL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near-and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of a-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide HI alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6. Keywords: circular dichroism; denaturation; fluorescence studies; interleukin-6; protein structure Interleukin-6 is a multifunctional cytokine that acts on a wide variety of tissues to elicit a broad spectrum of biological functions including terminal differentiation of B cells, growth and differentiation of T cells, and regulation of the acute-phase responses. IL-6 is also capable of inducing the differentiation of hematopoietic progenitor cells and megakaryocytes (for reviews see Kishimoto [1989], Van Snick [19901, and Gordon& Hoffman [1992]). Abbreviutions: IL-6, interleukin-6; m-, murine; h-, human; GdnHC1, guanidine hydrochloride; SDS, sodium dodecyl sulfate; MES, 2-(N-morpho1ino)ethane sulfonic acid; S, weight average sedimentation coefficient; [ B ] M~~, mean residue ellipticity; HEPES, N-2-hydroxyethylpiperazine-N1-2-ethane sulfonic acid; Tris, Tris(hydroxymethy1)aminomethane; CAPS, 3-(cyclohexylamino)-I-propane sulfonic acid; TFE, trifluoroethanol; GM-CSF, granulocyte-macrophage colony-stimulating factor.

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Section: Discussionmentioning

confidence: 99%

Effect of pH and denaturants on the folding and stability of murine interleukin‐6

et al. 1993

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“…Of two 'salt bridges', GluA2 -LysEF2 and GluA4-LysH10, which attach the Nterminal of the A helix to the E F fragment and to the H helix, the first occurs in all animal myoglobins and the second, which is probably of particular importance, is invariant for all the globins from various species [14]. The location of TrpA5 between LeuNA2 and LysEF2 seems to be preserved in apo-Mb since the emission of this Trp is quenched twofold compared to that of TrpA12, which could be due to the E-NH, group of the lysine [15].…”

Section: Comparison Of the Structures Of Apomyoglobin Upo-mb Complexmentioning

confidence: 96%

Fluorescence study of the conformational properties of myoglobin structure

Postnikova

Yumakova

1991

European Journal of Biochemistry

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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...

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“…Edelhoch et al (83) have examined the quenching of tryptophan fluorescence by ionized tyrosine in water at 25°C for the series of compounds Trp-(GlykTyr, where n = 0-4. The maximum degree of quenching falls from 85 % for Trp-Tyr to 50 % for Trp-(GlYk Tyr.…”

Section: Intramolecular Transfer In Oligopeptides Containing Tryptophmentioning

confidence: 99%

The Luminescence of the Aromatic Amino Acids

Weinryb

Steiner

1971

Excited States of Proteins and Nucleic Acids

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Fluorescence Studies with Tryptophyl Peptides^*

Cited by 139 publications

References 29 publications

Effect of pH and denaturants on the folding and stability of murine interleukin‐6

Effect of pH and denaturants on the folding and stability of murine interleukin‐6

Fluorescence study of the conformational properties of myoglobin structure

The Luminescence of the Aromatic Amino Acids

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Fluorescence Studies with Tryptophyl Peptides*

Cited by 139 publications

References 29 publications

Effect of pH and denaturants on the folding and stability of murine interleukin‐6

Effect of pH and denaturants on the folding and stability of murine interleukin‐6

Fluorescence study of the conformational properties of myoglobin structure

The Luminescence of the Aromatic Amino Acids

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Fluorescence Studies with Tryptophyl Peptides^*