Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo)resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins

Kocyła, Anna; Pomorski, Adam; Krężel, Artur

doi:10.1016/j.jinorgbio.2015.08.024

Cited by 77 publications

(99 citation statements)

References 79 publications

(119 reference statements)

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“…For this we took advantage of the ability of the compound 4-(2-pyridylazo)-resorcinol (PAR) to form a colored 2:1 complex with zinc with high affinity (effective dissociation constant of the Zn(PAR) 2 complex, at pH 7.0, 2.1 x 10 −12 M 2 [17]), which presents an absorption maximum at 492 nm and can be measured spectrophotometrically. In turn, EDTA forms a 1:1 complex with zinc of extremely high affinity: 6 x 10 −14 M at pH 7.0 [18].…”

Section: Resultsmentioning

confidence: 99%

Drawbacks of Dialysis Procedures for Removal of EDTA

et al. 2017

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Ethylenediaminetetraacetic acid (EDTA) is a chelating agent commonly used in protein purification, both to eliminate contaminating divalent cations and to inhibit protease activity. For a number of subsequent applications EDTA needs to be exhaustively removed. Most purification methods rely in extensive dialysis and/or gel filtration in order to exchange or remove protein buffer components, including metal chelators. We report here that dialysis protocols, even as extensive as those typically employed for protein refolding, may not effectively remove EDTA, which is reduced only by approximately two-fold and it also persists after spin-column gel filtration, as determined by NMR and by colorimetric methods. Remarkably, the most efficient removal was achieved by ultrafiltration, after which EDTA became virtually undetectable. These results highlight a potentially widespread source of experimental variability affecting free divalent cation concentrations in protein applications.

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Section: Resultsmentioning

confidence: 99%

Drawbacks of Dialysis Procedures for Removal of EDTA

et al. 2017

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“…At each condition, concentrations of the ZnH x (PAR) 2 complex and total protein were calculated from absorbance values at 492 and 280 nm, respectively, and K d values were derived from fits as described elsewhere. 48 …”

Section: Methodsmentioning

confidence: 99%

Clinical Variants of New Delhi Metallo-β-Lactamase Are Evolving To Overcome Zinc Scarcity

Stewart¹,

Bethel

VanPelt

et al. 2017

ACS Infect. Dis.

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Use and misuse of antibiotics has driven the evolution of serine β-lactamases to better recognize new generations of β-lactam drugs, but the selective pressures driving evolution of metallo-β-lactamases are less clear. Here, we present evidence that New Delhi Metallo-β-lactamase (NDM) is evolving to overcome the selective pressure of zinc(II) scarcity. Studies of NDM-1, NDM-4 (M154L), and NDM-12 (M154L, G222D) demonstrate that the point mutant M154L, contained in 50% of clinical NDM variants, selectively enhances resistance to the penam ampicillin at low zinc(II) concentrations relevant to infection sites. Each of the clinical variants is shown to be progressively more thermostable and to bind zinc(II) more tightly than NDM-1, but a selective enhancement of penam turnover at low zinc(II) concentrations indicates that most of the improvement derives from catalysis rather than stability. X-ray crystallography of NDM-4 and NDM-12, as well as bioinorganic spectroscopy of dizinc(II), zinc(II)/cobalt(II), and dicobalt (II) metalloforms probe the mechanism of enhanced resistance and reveal perturbations of the dinuclear metal cluster that underlie improved catalysis. These studies support the proposal that zinc(II) scarcity, rather than changes in antibiotic structure, is driving the evolution of new NDM variants in clinical settings.

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“…The concentration of the purified protein was obtained spectrophotometrically, via DTNB and PAR assays regarding thiol and Zn(II) concentration, respectively. 35,36 Additionally, samples were analysed by ICP (ICP-AES iCAP 7400, Thermo Scientific) to confirm the spectrophotometric results.…”

Section: Reconstitution Of Mt2 and The B-domain With Zn(ii)mentioning

confidence: 99%

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

et al. 2018

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a Metallothioneins (MTs), small cysteine-rich proteins, present in four major isoforms, are key proteins involved in zinc and copper homeostasis in mammals. To date, only one X-ray crystal structure of a MT has been solved. It demonstrates seven bivalent metal ions bound in two structurally independent domains with M 4 S 11 (a) and M 3 S 9 (b) clusters. Recent discoveries indicate that Zn(II) ions are bound with MT2 with the range from nano-to picomolar affinity, which determines its cellular zinc buffering properties that are demonstrated by the presence of partially Zn(II)-depleted MT2 species. These forms serve as Zn(II) donors or acceptors and are formed under varying cellular free Zn(II) concentrations. Due to the lack of appropriate methods, knowledge regarding the structure of partially-depleted metallothionein is lacking. Here, we describe the Zn(II) binding mechanism in human MT2 with high resolution with respect to particular Zn(II) binding sites, and provide structural insights into Zn(II)-depleted MT species. The results were obtained by the labelling of metal-free cysteine residues with iodoacetamide and subsequent top-down electrospray ionization analysis, MALDI MS, bottom-up nanoLC-MALDI-MS/MS approaches and molecular dynamics (MD) simulations. The results show that the a-domain is formed sequentially in the first stages, followed by the formation of the b-domain, although both processes overlap, which is in contrast to the widely investigated cadmium MT. Independent ZnS 4 cores are characteristic for early stages of domain formation and are clustered in later stages. However, Zn-S network rearrangement in the b-domain upon applying the seventh Zn(II) ion explains its lower affinity. Detailed analysis showed that the weakest Zn(II) ion associates with the b-domain by coordination to Cys21, which was also found to dissociate first in the presence of the apo-form of sorbitol dehydrogenase. We found that Zn(II) binding to the isolated b-domain differs significantly from the whole protein, which explains its previously observed different Zn(II)-binding properties. MD results obtained for Zn(II) binding to the whole protein and isolated b-domain are highly convergent with mass spectrometry data. This study provides a comprehensive overview of the crosstalk of structural and zinc buffering related-to-thermodynamics properties of partially metal-saturated mammalian MT2 and sheds more light on other MT proteins and zinc homeostasis. Significance to metallomicsThe growing interest in metallomics within metal-dependent biological systems provides an opportunity to explore the structural and buffering properties of metallothioneins (MTs). The wide relevance of MTs is obvious due to their multiple biological functions such as participation in zinc and copper homeostasis. However, the MT metalloproteome is still not fully explored and is rather complicated in vivo or in vitro because of its highly dynamic structure, which lacks secondary structural elements and metamorphic behaviour. We have undertaken compreh...

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Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo)resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins

Cited by 77 publications

References 79 publications

Drawbacks of Dialysis Procedures for Removal of EDTA

Drawbacks of Dialysis Procedures for Removal of EDTA

Clinical Variants of New Delhi Metallo-β-Lactamase Are Evolving To Overcome Zinc Scarcity

Crosstalk of the structural and zinc buffering properties of mammalian metallothionein-2

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