Development of a heme sensor using fluorescently labeled heme oxygenase-1

Koga, Shinya; Yoshihara, Shun; Bando, Hiroki; Yamasaki, Kazuki; Higashimoto, Yuichiro; Noguchi, Masato; Sueda, Shinji; Komatsu, Hideyuki; Sakamoto, Hiroshi

doi:10.1016/j.ab.2012.10.002

Cited by 17 publications

(27 citation statements)

References 36 publications

(42 reference statements)

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“…The F 0 eF value was plotted against the hemin concentration, and the K d values were then estimated by nonlinear curve fitting as described previously (Fig. 2A, inset) [2]. This analysis indicated that K18C/D140HeA555 had 1:1 binding stoichiometry with heme.…”

Section: Contents Lists Available At Sciencedirectmentioning

confidence: 95%

“…The Cys residue of K18C was used for modification with thiol-reactive fluorescent dyes [2]. The K18C was further mutated at Asp140 by His or Ala; these double mutants are referred to as K18C/D140H and K18C/D140A, respectively.…”

Section: Contents Lists Available At Sciencedirectmentioning

confidence: 99%

“…The K18C was further mutated at Asp140 by His or Ala; these double mutants are referred to as K18C/D140H and K18C/D140A, respectively. The desired proteins were purified from Escherichia coli cells transfected with the respective expression plasmids according to a procedure reported previously [2]. Detailed information on construction of the expression plasmids and purification of proteins is described in the online supplementary material.…”

Section: Contents Lists Available At Sciencedirectmentioning

confidence: 99%

“…To overcome the problem, Alexa Fluor 555 (A555; Invitrogen Life Technologies, Carlsbad, CA, USA), which has longer wavelengths of excitation and emission at 545 and 565 nm, respectively, than those of AGEs, was employed and then incorporated into K18C/D140H to yield K18C/D140HeA555. The procedures for fluorescent dye labeling were described in our previous study [2]. The fluorescence quenching of K18C/ D140HeA555 depending on the hemin concentration was measured to confirm whether K18C/D140HeA555 functioned as a heme sensor.…”

Section: Contents Lists Available At Sciencedirectmentioning

confidence: 99%

“…Sassa mentioned that the concentration of free heme in cells should not be greater than 10 À9 M [1]. We recently developed a novel heme sensor based on rat heme oxygenase-1 (HO-1) [2] that allowed for detection of heme at a concentration level lower than 10 nM in a microplate-based assay. Thus, the sensor could become a powerful diagnostic tool in diseases such as colon cancer and Alzheimer's disease where free heme needs to be determined [3e5].…”

Section: Introductionmentioning

confidence: 99%

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Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

Taira

Nakashima

Yoshihara

et al. 2015

Analytical Biochemistry

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Section: Contents Lists Available At Sciencedirectmentioning

confidence: 95%

Section: Contents Lists Available At Sciencedirectmentioning

confidence: 99%

Section: Contents Lists Available At Sciencedirectmentioning

confidence: 99%

Section: Contents Lists Available At Sciencedirectmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

Taira

Nakashima

Yoshihara

et al. 2015

Analytical Biochemistry

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Heme Binding Induces Dimerization and Nitration of Truncated β‐Amyloid Peptide Aβ16 Under Oxidative Stress

et al. 2013

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Several observations suggest a link between (ferric) heme and Alzheimers disease (AD), for example, an increase in heme oxygenase 1, [1,2] a loss of complex IV, [3][4][5][6] and abnormal iron metabolism. These observations suggest that heme metabolism is altered in age-related disorders. As a possible explanation, hemin has been proposed to interact with b-amyloid peptides (Ab), [7] and it is now well established that Ab peptides bind ferric heme. [8] The adducts of hemin-Ab peptides also exhibit increased peroxidase activity with respect to free hemin, [9] but these complexes have not been characterized. Herein we provide thermodynamic and spectroscopic data showing that hemin b can coordinate two Ab16 molecules to form the low-spin, six-coordinated complex [hemin(Ab16) 2 ], and this is in equilibrium with the high-spin [hemin(Ab16)] species, the relative amounts of which strongly depend on peptide concentration and temperature. In the presence of H 2 O 2 , hemin-Ab16 complexes produce dimerization of the peptide through dityrosine cross-linking, and in addition the peptide undergoes endogenous nitration at Tyr10 when nitrite is also present in solution. This result is important because both cross-linking and nitration at Tyr10 critically enhance Ab aggregation and plaque formation, [10] thus showing that the binding of heme to Ab can indeed be a factor contributing to neuroinflammation and Ab aggregation. The endogenous peroxidative (H 2 O 2 ) and nitrative (H 2 O 2 /NO 2 À ) activities exhibited by the hemin-Ab16 peptide add to, and are likely more deleterious than, the oxidation [9] and nitration [11] of external substrates which have been previously reported.Upon adding Ab16 to a 50 mm aqueous phosphate buffer solution of hemin b at pH 7.4 and 300 K, the broad Soret band of the complex near l = 385 nm progressively changes to give a sharp, red-shifted band at l = 414 nm, with additional visible bands at l = 535 and 566 nm (Figure 1 C, and see the Supporting Information). The conversion from the high-spin to the low-spin hemin species is complete after addition of 20 equivalents of Ab16, and spectrophotometric data analysis (see the Supporting Information) confirms a 2:1 Ab16/hemin stoichiometry, thus indicating the formation of the [hemin-(Ab16) 2 ] complex. The Mçssbauer and 1 H NMR spectra are consistent with a low-spin (S = 1/2) iron(III) center coordinated by two axial imidazole ligands. However, below saturating amounts of Ab16, the Mçssbauer spectrum clearly indicates the presence of a minor high-spin species (Figure 2). Figure 1. UV/vis spectra recorded in 50 mm phosphate buffer solution at pH 7.4. A) Hemin b (1.2 10 À5 m). B) Hemin b in the presence of an excess (20 equiv) of Ab16 peptide at 319 K. C) The same as (B) but at 300 K, the spectral changes are fully reversible. The D symbol indicates an increase in temperature.Figure 2. Mçssbauer spectrum of hemin b (2 mm), in the presence of 6 equivalents of the Ab16 peptide, recorded at 78 K. Quadrupole doublet simulation reveals the presence of a major...

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Heme Binding Induces Dimerization and Nitration of Truncated β‐Amyloid Peptide Aβ16 Under Oxidative Stress

Thiabaud¹,

Pizzocaro²,

García-Serres³

et al. 2013

Angew Chem Int Ed

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Development of a heme sensor using fluorescently labeled heme oxygenase-1

Cited by 17 publications

References 36 publications

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

Heme Binding Induces Dimerization and Nitration of Truncated β‐Amyloid Peptide Aβ16 Under Oxidative Stress

Heme Binding Induces Dimerization and Nitration of Truncated β‐Amyloid Peptide Aβ16 Under Oxidative Stress

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