Distinction of the two binding sites of serum transferrin by resonance Raman spectroscopy

Mecklenburg, Sandra L.; Mason, Anne B.; Woodworth, Robert C.; Donohoe, Robert J.

doi:10.1002/(sici)1520-6343(1997)3:6<435::aid-bspy2>3.0.co;2-#

Cited by 7 publications

(3 citation statements)

References 27 publications

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“…7a). The intensity pattern and frequencies of these bands correlate well with the ring deformation modes of a tyrosinate bound to Fe(III), as observed in other Fe(III)–tyrosinate proteins and related model complexes [23, 49, 61, 66–68]. Unfortunately, attempts to observe the Fe–O vibrations (500–600 cm −1 ) and the Fermi doublet (800–900 cm −1 ) characteristic of Fe(III)–phenolates were unsuccessful.…”

Section: Resultssupporting

confidence: 71%

Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions

et al. 2010

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The design, synthesis, and metal-binding properties of DF3, a new de novo designed di-iron protein model are described ("DF" represents due ferri, Italian for "two iron," "di-iron"). DF3 is the latest member of the DF family of synthetic proteins. They consist of helix-loop-helix hairpins, designed to dimerize and form an antiparallel four-helix bundle that encompasses a metal-binding site similar to those of non-heme carboxylate-bridged di-iron proteins. Unlike previous DF proteins, DF3 is highly soluble in water (up to 3 mM) and forms stable complexes with several metal ions (Zn, Co, and Mn), with the desired secondary structure and the expected stoichiometry of two ions per protein. UV-vis studies of Co(II) and Fe(III) complexes confirm a metal-binding environment similar to previous di-Co(II)-and di-Fe(III)-DF proteins, including the presence of a µ-oxo-di-Fe(III) unit. Interestingly, UV-vis, EPR, and resonance Raman studies © SBIC 2010 Correspondence to: Angela Lombardi. suggest the interaction of a tyro-sine adjacent to the di-Fe(III) center. The design of DF3 was aimed at increasing the accessibility of small molecules to the active site of the four-helix bundle. Indeed, binding of azide to the di-Fe(III) site demonstrates a more accessible metal site compared with previous DFs. In fact, fitting of the binding curve to the Hill equation allows us to quantify a 150% accessibility enhancement, with respect to DF2. All these results represent a significant step towards the development of a functional synthetic DF metalloprotein.

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Section: Resultssupporting

confidence: 71%

Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions

et al. 2010

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“…The majority of previous Raman spectroscopy studies of metal binding proteins, such as transferrin, have used resonance Raman to enhance the Raman signal arising from the presence of metal with wavenumbers below 900 cm −1 strongly influenced by the specific metal ligand. 11,12 More recently, iron saturation in transferrin 13 and differences between ferritin and magnetoferritin have been determined using the nonresonance Raman spectroscopy 14 and although both studies focused on Raman peaks arising from the presence of iron they also observed intense protein structure and polysaccharide associated peaks in the spectra.…”

Section: Introductionmentioning

confidence: 99%

Detection of glycosylation and iron-binding protein modifications using Raman spectroscopy

Ashton

Brewster

Correa

et al. 2017

Analyst

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In this study we demonstrate the use of Raman spectroscopy to determine protein modifications as a result of glycosylation and iron binding. Most proteins undergo some modifications after translation which can directly affect protein function. Identifying these modifications is particularly important in the production of biotherapeutic agents as they can affect stability, immunogenicity and pharmacokinetics. However, post-translational modifications can often be difficult to detect with regard to the subtle structural changes they induce in proteins. From their Raman spectra apo-and holo-forms of iron-binding proteins, transferrin and ferritin, could be readily distinguished and variations in spectral features as a result of structural changes could also be determined. In particular, differences in solvent exposure of aromatic amino acids residues could be identified between the open and closed forms of the iron-binding proteins. Protein modifications as a result of glycosylation can be even more difficult to identify. Through the application of the chemometric techniques of principal component analysis and partial least squares regression variations in Raman spectral features as a result of glycosylation induced structural modifications could be identified. These were then used to distinguish between glycosylated and non-glycosylated transferrin and to measure the relative concentrations of the glycoprotein within a mixture of the native non-glycosylated protein.

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“…While the free coordination sites in the apo-Tf skeleton are loaded with Fe 3+ -ions, additional vibration modes emerge in the Raman spectra of the protein. [25][26][27][28] The features from the spectra of Tf saturated with different Fe isotopes do not differ significantly which prevents the use of an isotope dilution approach. Therefore, the signal intensity ratio of the iron-related modes to the characteristic protein modes has now been evaluated for the first time in the quantitative analysis of the iron saturation in Tf.…”

Section: Introductionmentioning

confidence: 99%

Reference measurement procedures for the iron saturation in human transferrin based on IDMS and Raman scattering

et al. 2012

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Two reference measurement procedures are presented here that allow the determination of the iron saturation in human transferrin, based on different molecular properties. The results, directly derived from the number of ions bound to the protein molecule, are traceable to the SI. Up to now, the iron saturation has only been deduced indirectly from the amount-of-substance ratio of serum iron to transferrin in serum. Interlaboratory tests have shown the need for more accurate methods, as the results from many participant test samples for both parameters do not lie within the acceptable range of deviation given by relevant guidelines when different methods or kits are applied. Using isotope dilution, an HPLC ICP-MS procedure was developed in compliance with the requirements of a primary reference measurement procedure. In this manner, the iron saturation was measured with an associated relative expanded measurement uncertainty of 4%. Based on the results, a straightforward Raman procedure was evolved, which allows the determination of the iron saturation in transferrin with an associated relative expanded uncertainty of 7%.

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Distinction of the two binding sites of serum transferrin by resonance Raman spectroscopy

Cited by 7 publications

References 27 publications

Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions

Spectroscopic and metal-binding properties of DF3: an artificial protein able to accommodate different metal ions

Detection of glycosylation and iron-binding protein modifications using Raman spectroscopy

Reference measurement procedures for the iron saturation in human transferrin based on IDMS and Raman scattering

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