Apohorseradish Peroxidase Unfolding and Refolding: Intrinsic Tryptophan Fluorescence Studies

Lasagna, Mauricio; Gratton, Enrico; Jameson, David M.; Brunet, Juan E.

doi:10.1016/s0006-3495(99)77211-5

Cited by 32 publications

(14 citation statements)

References 42 publications

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“…Two-Photon UV Fluorescence Excitation of Amino Acids The output of the intra-cavity doubled optical parametric oscillator around 590 nm is used to excite three different amino acids, tryptophan, tyrosine, and glycine. All of them possess auto-fluorescence in the UV regime (Lasagna et al, 1999;Weber and Teale, 1957). The benzene ring structure within tryptophan and tyrosine (Davies, 1979;Lodish et al, 2000) is believed to be the origin of UV fluorescence (Rehms and Callis, 1993;Toshifumi and Ohshima, 2001).…”

Section: Harmonic Generation Microscopy Ofmentioning

confidence: 99%

The use of optical parametric oscillator for harmonic generation and two‐photon UV fluorescence microscopy

Kao

2004

Microscopy Res & Technique

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Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1,050-1,300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation. In addition, through intra-cavity doubling wavelengths from 525-650 nm are made available for effective two-photon (2-p) excitation with the equivalent photon energy in the UVB range (290-320 nm) and beyond. This new capacity allows UV (auto-) fluorescence excitation and imaging, for example, from some amino acids, such as tyrosine, tryptophan, and glycine.

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Section: Harmonic Generation Microscopy Ofmentioning

confidence: 99%

The use of optical parametric oscillator for harmonic generation and two‐photon UV fluorescence microscopy

Kao

2004

Microscopy Res & Technique

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“…As a consequence the observed emission spectra of individual proteins (and of their corresponding conformational states) do not even vary with respect to l max but also can show different spectral shapes and widths (7,8). However, upon unfolding transitions generally distinct shifts of the emission spectra are reported (see for example (4,(9)(10)(11)(12)(13)(14)). To derive thermodynamic parameter from spectroscopic data it is crucial whether the signal changes during the unfolding process are proportional to the molar fractions of the folded and unfolded protein.…”

Section: Introductionmentioning

confidence: 97%

How Aggregation and Conformational Scrambling of Unfolded States Govern Fluorescence Emission Spectra

Duy

Fitter

2006

Biophysical Journal

147

103

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In a case study on five homologous alpha-amylases we analyzed the properties of unfolded states as obtained from treatments with GndHCl and with elevated temperatures. In particular the wavelength of the tryptophan fluorescence emission peak (lambda(max)) is a valuable parameter to characterize properties of the unfolded state. In all cases with a typical red shift of the emission spectrum occurring during structural unfolding we observed a larger magnitude of this shift for GndHCl-induced unfolding as compared to thermal unfolding. Although a quantitative relation between aggregation and reduction of the unfolding induced red shifts cannot be given, our data indicate that protein aggregation contributes significantly to smaller magnitudes of red shifts as observed during thermal unfolding. In addition, other properties of the unfolded states, most probable structural compactness or simply differences in the conformational scrambling, also affect the magnitude of red shifts. For the irreversible unfolding alpha-amylases studied here, transition temperatures and magnitudes of red shifts are strongly depending on heating rates. Lower protein concentrations and smaller heating rates lead to larger red shifts upon thermal unfolding, indicating that under these conditions the protein aggregation is less pronounced.

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“…Hemin is a complex structure obtained by oxidation of the ferrous ion near the porphyrin ring of heme group to a ferric ion, along with its interaction with chloride; it has been widely used as an iron source in HRP refolding. HRP refolding yield can be increased by addition of hemin to the refolding buffer at the beginning or at the end of the refolding reaction since it also binds to apo‐HRP .…”

Section: Discussionmentioning

confidence: 99%

Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids

Bae

Eom

Lan

et al. 2016

Biotechnology Journal

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The effects of various refolding additives, including metal cofactors, organic co-solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well-known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion-containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca(2+) and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3-fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea-containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor-containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein.

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Apohorseradish Peroxidase Unfolding and Refolding: Intrinsic Tryptophan Fluorescence Studies

Cited by 32 publications

References 42 publications

The use of optical parametric oscillator for harmonic generation and two‐photon UV fluorescence microscopy

The use of optical parametric oscillator for harmonic generation and two‐photon UV fluorescence microscopy

How Aggregation and Conformational Scrambling of Unfolded States Govern Fluorescence Emission Spectra

Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids

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