scholarly journals The binding of cupric ions to 1-carboxymethylhistidine-119-ribonuclease

1968 ◽  
Vol 108 (4) ◽  
pp. 583-586 ◽  
Author(s):  
R. H. Saundry ◽  
W D Stein
Keyword(s):  
Binding Site ◽  
Binding Constant ◽  
Metal Ion ◽  
Binding Constants ◽  
Enzymic Activity

Binding of Cu2+ by 1-carboxymethylhistidine-119-ribonuclease was investigated by using diligand metal ion buffers. A single Cu2+-binding site was found over the Cu2+ concentration range studied. The binding constants for this site were 8·33×105 (±2%)m−1 and 1·57×104 (±6%)m−1 at pH7·0 and 6·1 respectively. An estimate of the pH-independent Cu2+-binding constant suggests that the most avid Cu2+-binding site has disappeared after carboxymethylation. This is consistent with an earlier report that binding of Cu2+ at the most avid site is associated with the loss of enzymic activity.

Binding Properties of 2-Acetylnaphthalene with Hydroxypropyl Cyclodextrins from Fluorescence Quenching Experiments

2005 ◽  
Vol 59 (5) ◽  
pp. 691-695 ◽  
Author(s):  
Maximilian Seel ◽  
T. C. Werner
Keyword(s):  
Complex Formation ◽  
Fluorescence Quenching ◽  
Binding Site ◽  
Thermodynamic Parameters ◽  
Binding Constant ◽  
Binding Constants ◽  
Dispersion Forces ◽  
Binding Properties ◽  
Hydroxypropyl Cyclodextrins

The quenching of 2-acetylnaphthalene (2-AN) fluorescence by hydroxypropyl cyclodextrins (HP-CD) has been analyzed using modified Stern–Volmer plots to obtain binding constants as a function of temperature for 2-AN:HP-CD complexes. The HP-CDs were commercially available and contained 4–7 HP groups per CD molecule for α-CD, β-CD, and γ-CD. HP substitution causes a 12 to over 40% increase in binding constant ( Kave) for 2-AN compared to that for unsubstituted CDs, although the Kave value is not strongly dependent on the extent of HP substitution for β-CD. No evidence of formation of a 2:2 complex, such as that observed with 2-AN and γ-CD, is observed with 2-AN and HP-γ-CD. Thermodynamic parameters (ΔH°o and ΔS°o) suggest that the increase in Kave with HP substitution is due to an enlarged binding site for the HP-CDs that allows greater motional freedom for 2-AN. Comparison is made to the binding of 2-methylnaphthoate (2-MN) to CDs and HP-CDs, and the larger Kave values for 2-MN over 2-AN are attributed to greater dispersion forces for 2-MN complex formation.

scholarly journals Revisiting the Rate-Limiting Step of the ANS–Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 420
Author(s):  
Chikashi Ota ◽  
Shun-ichi Tanaka ◽  
Kazufumi Takano
Keyword(s):  
Protein Binding ◽  
Binding Site ◽  
Binding Constant ◽  
Fluorescence Probe ◽  
Binding Constants ◽  
Intensity Change ◽  
Binding Process ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS–protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS–protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective.

scholarly journals Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

2006 ◽  
Vol 400 (3) ◽  
pp. 385-392 ◽  
Author(s):  
Erdeni Bai ◽  
Federico I. Rosell ◽  
Bao Lige ◽  
Marcia R. Mauk ◽  
Barbara Lelj-Garolla ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Metal Ions ◽  
Binding Site ◽  
Metal Ion ◽  
Association Constants ◽  
Ion Binding ◽  
Ferric Uptake Regulator ◽  
Metal Ion Binding ◽  
Dimerization Domain ◽  
High Affinity

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.

A conserved scaffold with heterogeneous metal ion binding site: the multifaceted example of HD-GYP proteins

2022 ◽  
Vol 450 ◽  
pp. 214228
Author(s):  
Francesca Cutruzzolà ◽  
Alessandro Paiardini ◽  
Chiara Scribani Rossi ◽  
Sharon Spizzichino ◽  
Alessio Paone ◽  
...  
Keyword(s):  
Binding Site ◽  
Metal Ion ◽  
Ion Binding ◽  
Metal Ion Binding

scholarly journals Interaction of 2′R-ochratoxin A with Serum Albumins: Binding Site, Effects of Site Markers, Thermodynamics, Species Differences of Albumin-binding, and Influence of Albumin on Its Toxicity in MDCK Cells

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 353 ◽  
Author(s):  
Zelma Faisal ◽  
Diána Derdák ◽  
Beáta Lemli ◽  
Sándor Kunsági-Máté ◽  
Mónika Bálint ◽  
...  
Keyword(s):  
Binding Site ◽  
Ochratoxin A ◽  
Mdck Cells ◽  
Binding Constants ◽  
Albumin Binding ◽  
Serum Albumins ◽  
Site Markers ◽  
Fetal Bovine ◽  
Toxic Concentrations ◽  
Insight Into

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. Roasting of OTA-contaminated coffee results in the formation of 2′R-ochratoxin A (2′R-OTA), which appears in the blood of coffee drinkers. Human serum albumin (HSA) binds 2′R-OTA (and OTA) with high affinity; therefore, albumin may influence the tissue uptake and elimination of ochratoxins. We aimed to investigate the binding site of 2′R-OTA (verses OTA) in HSA and the displacing effects of site markers to explore which molecules can interfere with its albumin-binding. Affinity of 2′R-OTA toward albumins from various species (human, bovine, porcine and rat) was tested to evaluate the interspecies differences regarding 2′R-OTA-albumin interaction. Thermodynamic studies were performed to give a deeper insight into the molecular background of the complex formation. Besides fluorescence spectroscopic and modeling studies, effects of HSA, and fetal bovine serum on the cytotoxicity of 2′R-OTA and OTA were tested in MDCK kidney cell line in order to demonstrate the influence of albumin-binding on the cellular uptake of ochratoxins. Site markers displaced more effectively 2′R-OTA than OTA from HSA. Fluorescence and binding constants of 2′R-OTA-albumin and OTA-albumin complexes showed different tendencies. Albumin significantly decreased the cytotoxicity of ochratoxins. 2′R-OTA, even at sub-toxic concentrations, increased the toxic action of OTA.

scholarly journals Single Molecule Investigation of Ag+ Interactions with Single Cytosine-, Methylcytosine- and Hydroxymethylcytosine-Cytosine Mismatches in a Nanopore

2014 ◽  
Vol 4 (1) ◽  
Author(s):  
Yong Wang ◽  
Bin-Quan Luan ◽  
Zhiyu Yang ◽  
Xinyue Zhang ◽  
Brandon Ritzo ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Binding Site ◽  
Single Molecule ◽  
Metal Ion ◽  
Detection Method ◽  
Direct Detection ◽  
Alpha Hemolysin ◽  
Duplex Stability ◽  
Dynamics Simulations ◽  
Direct Detection Method

Abstract Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications.

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