scholarly journals Kinetic and spectroscopic studies of haemoglobin and myoglobin from Urechis caupo. Distal residue effects

1990 ◽  
Vol 269 (3) ◽  
pp. 739-747 ◽  
Author(s):  
T J DiFeo ◽  
A W Addison ◽  
J J Stephanos
Keyword(s):  
Bound States ◽  
Binding Constants ◽  
Thiol Group ◽  
Spectroscopic Studies ◽  
Human Haemoglobin ◽  
Equilibrium Binding ◽  
Urechis Caupo ◽  
The Fourier Transform ◽  
T State ◽  
Kinetics Of

Seven components of the tetrameric haemoglobin (Hbu) from Urechis caupo were separated by preparative isoelectric focusing and characterized by their absorption spectra and pI values. The helix content and Soret delta epsilon values are reported for several of the components. Temperature-jump O2-binding kinetics of the major components of Hbu show biphasic behaviour, with the majority species having kon = 1.57 x 10(9) mol-1.s-1 and koff = 3.32 x 10(4) s-1. The Fourier-transform i.r. spectrum of pooled Hbu(II)-CO displays a stretching frequency of 1942 cm-1. E.s.r. of Hbu(II)-NO demonstrates evidence of proximal strain similar to that encountered in T-state human haemoglobin. CO-driven reduction of U. caupo methaemoglobin, Hbu(III) and U. caupo metmyoglobin [Mbu(III)] shows much higher rates relative to haemoglobins and myoglobins known to possess a distal histidine residue. Nitrosyl auto-reduction kinetics of Hbu(III)-NO and Mbu(III)-NO are examined. The equilibrium binding constants of several ligands are reported for both Hbu and Mbu, and together with the above kinetic data suggest differences in haem pocket environments between Hbu and Mbu. Reaction of Hbu with 2-chloromercuri-4,6-dinitrophenol demonstrates the presence of one reactive thiol group per globin chain. lambda max. values and the respective molar absorption coefficients for selected ligand-bound states are reported for the major component of Hbu and for Mbu. The majority haem orientation in U. caupo haemoglobin is identical with that of human haemoglobin.

scholarly journals Cross-linking with O-raffinose lowers oxygen affinity and stabilizes haemoglobin in a non-cooperative T-state conformation

2004 ◽  
Vol 384 (2) ◽  
pp. 367-375 ◽  
Author(s):  
Yiping JIA ◽  
Somasundaram RAMASAMY ◽  
Francine WOOD ◽  
Abdu I. ALAYASH ◽  
Joseph M. RIFKIND
Keyword(s):  
Hyperfine Splitting ◽  
Oxygen Affinity ◽  
Thiol Group ◽  
Cross Linking ◽  
Native Protein ◽  
Epr Spectrum ◽  
Human Haemoglobin ◽  
Lower Oxygen ◽  
T State ◽  
Kinetics Of

O-R-polyHbA0 is an intra- and intermolecularly O-raffinose cross-linked derivative of deoxygenated human haemoglobin developed as an oxygen therapeutic. When compared with its native protein (HbA0), O-R-polyHbA0 was found to be locked in the T (tense) quaternary conformation with a lower oxygen affinity, a reduced Bohr effect (50% of HbA0) and no measurable cooperativity (h=1). The kinetics of oxygen and CO binding to the protein indicate lower ‘on’ rates and faster ‘off’ rates than HbA0 and the absence of effects of inositol hexaphosphate (IHP) on the kinetics. Other properties consistent with a T-like conformation are inaccessibility of the βCys-93 thiol group of O-R-polyHbA0, the hyperfine splitting from nitrogen in the EPR spectrum of the Fe(II)NO complex of O-R-polyHbA0 and decreased flexibility in the distal haem pocket, as indicated by low-spin bis-histidine complexes detected by EPR of oxidized chains. A comparison of the properties of O-R-polyHbA0 with those of HbA0 with and without IHP, as well as the reaction of nitrite with deoxygenated haemoglobin, provide additional insights into the variations in the conformation of T-state haemoglobin in solution (modifications of the T state produced by adding organic phosphates, like IHP and 2,3-diphosphoglycerate). Although the physiological ramifications of locking HbA0 in the T conformation with the O-raffinose are still unknown, valuable insights into haemoglobin function are provided by these studies of O-R-polyHbA0.

The conformational changes of haemoglobin on its binding to haptoglobin

1981 ◽  
Vol 46 (5) ◽  
pp. 1288-1295
Author(s):  
Barbora Dvořánková ◽  
Zdeněk Pavlíček
Keyword(s):  
Thin Layer ◽  
Conformational Changes ◽  
Peroxidase Activity ◽  
Quaternary Structure ◽  
Analogue Computer ◽  
Gel Chromatography ◽  
Human Haemoglobin ◽  
T State ◽  
Kinetics Of

The binding of human haemoglobin to human haptoglobin has been found to alter the conformation of haemoglobin. Spectrophotometric measurements, measuring of peroxidase activity, thin-layer gel chromatography and modelling on an analogue computer led to the conclusion that the binding of haemoglobin to haptoglobin was associated with a change in the quaternary structure of haemoglobin, with a transition from the R state to the T state. The kinetics of the conformational changes had an autocatalytic character.

Kinetics of the reaction OH + NOCl as a possible source of ClOH for spectroscopic studies

1981 ◽  
Vol 17 (1) ◽  
pp. 142-143
Author(s):  
G. Poulet ◽  
J.L. Jourdain ◽  
G. Laverdet ◽  
G. Le Bras
Keyword(s):  
Spectroscopic Studies ◽  
Kinetics Of

A Fluorescence Quenching Analysis of the Binding of Fluoroquinolones to Humic Acid

2017 ◽  
Vol 71 (11) ◽  
pp. 2512-2518 ◽  
Author(s):  
Ryan P. Ferrie ◽  
Gregory E. Hewitt ◽  
Bruce D. Anderson
Keyword(s):  
Humic Acid ◽  
Fluorescence Quenching ◽  
Water Solubility ◽  
Binding Constants ◽  
High Water ◽  
Static Quenching ◽  
Temperature Range ◽  
Equilibrium Binding ◽  
Quenching Analysis ◽  
High Water Solubility

Fluorescence quenching was used to investigate the interaction of six fluoroquinolones with humic acid. Static quenching was observed for the binding of ciprofloxacin, enoxacin, fleroxacin, levofloxacin, norfloxacin, and ofloxacin to humic acid. The equilibrium binding constants were found from Stern–Volmer plots of the data. The quenching experiments were repeated over a temperature range of 25–45 ℃ and van’t Hoff plots were generated. From these linear plots, thermodynamic values were calculated for Δ H, Δ G, and Δ S for each of the fluoroquinolones. The equilibrium binding constants were found to be <1 for all the antibiotics studied. The calculated ΔH values were all negative and ranged from −9.5 to −27.6 kJ/mol. The high water solubility of the antibiotics and low ΔH of binding suggests that the antibiotics will be transported easily through the environment. Finally, whether the fluoroquinolones are in a protonated, deprotonated, or partially protonated state is found to correlate to the strength of binding to humic acid.

scholarly journals The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

1993 ◽  
Vol 290 (1) ◽  
pp. 15-19 ◽  
Author(s):  
A Lewendon ◽  
W V Shaw
Keyword(s):  
Chemical Modification ◽  
Ph Dependence ◽  
Thiol Group ◽  
Chloramphenicol Acetyltransferase ◽  
Histidine Residue ◽  
Pka Values ◽  
Primary Hydroxy Group ◽  
Pka Value ◽  
Catalytic Role ◽  
Kinetics Of

A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.

Bonding of molecular oxygen to T state human haemoglobin

Nature ◽  
1984 ◽  
Vol 307 (5946) ◽  
pp. 74-76 ◽  
Author(s):  
Andrzej Brzozowski ◽  
Zygmunt Derewenda ◽  
Eleanor Dodson ◽  
Guy Dodson ◽  
Mieczyslaw Grabowski ◽  
...  
Keyword(s):  
Molecular Oxygen ◽  
Human Haemoglobin ◽  
T State

Binding Sites, Singly Bound States, and Conformation Coupling Shape GABA-Evoked Currents

2003 ◽  
Vol 89 (2) ◽  
pp. 871-883 ◽  
Author(s):  
Jerzy W. Mozrzymas ◽  
Andrea Barberis ◽  
Katarzyna Mercik ◽  
Ewa D. Z˙arnowska
Keyword(s):  
Binding Sites ◽  
Bound States ◽  
Time Course ◽  
Functional Coupling ◽  
Binding Properties ◽  
The Impact ◽  
Kinetics Of ◽  
Source Of Information ◽  
Respective Rate ◽  
Gating Scheme

The time course of GABA-evoked currents is the main source of information on the GABAAreceptor gating. Since the kinetics of these currents depends on the transitions between several receptor conformations, it is a major challenge to define the relations between current kinetics and the respective rate constants of the microscopic gating scheme. The aim of this study was to further explore the impact of different GABAA receptor conformations on the kinetics of currents elicited by ultra-fast GABA applications. We show that the rising phase and amplitude of GABA-evoked currents depend on desensitization and singly bound states. The occupancy of bound receptors depends not only on binding properties but also on opening/closing and desensitization. The impact of such functional coupling between channel states is critical in conditions of high non-equilibrium typical for synaptic transmission. The concentration dependence of the rising phase of the GABA-elicited current indicates positive cooperativity between agonist binding sites. We provide evidence that preequilibration at low GABA concentrations reduce GABA-evoked currents due to receptor trapping in a singly bound desensitized state.

scholarly journals Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue β-93

1977 ◽  
Vol 165 (1) ◽  
pp. 141-148 ◽  
Author(s):  
C C Winterbourn ◽  
R W Carrell
Keyword(s):  
Electron Transfer ◽  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Thiol Group ◽  
Thiol Groups ◽  
Human Haemoglobin ◽  
High Affinity ◽  
Rate Determining Step ◽  
Haemoglobin Molecule

Addition of Cu(II) ions to human oxyhaemoglobin caused the rapid oxidation of the haem groups of the beta-chain. Oxidation required binding of Cu(II) to sites involving the thiol group of beta-93 residues and was prevented when these groups were blocked with iodoacetamide or N-ethylmaleimide. Equilibrium-dialysis studies showed three pairs of binding sites, two pairs with high affinity for Cu(II) and one pair with lower affinity. It was the second pair of high-affinity sites that were blocked with iodoacetamide and were involved in haem oxidation. Cu(II) oxidized deoxyhaemoglobin at least ten times as fast as oxyhaemoglobin, and analysis of rates suggested that binding rather than electron transfer was the rate-determining step. No thiol-group oxidation to disulphides occurred during the period of haem oxidation, although it did occur subsequently in the presence of oxygen, or when Cu(II) was added to methaemoglobin. It is proposed that thiol oxidation did not occur because there exists a pathway of electron transfer between the haem group and copper bound to the beta-93 thiol groups. The route for this electron transfer is discussed, as well as the implications as to the function of the beta-93 cysteine in the haemoglobin molecule.

Kinetics and Mechanism of Cationic Micelle/Flexible Nanoparticle Catalysis: A Review

2018 ◽  
Vol 43 (1) ◽  
pp. 1-20 ◽  
Author(s):  
Mohammad Niyaz Khan ◽  
Ibrahim Isah Fagge
Keyword(s):  
Rate Increase ◽  
Molecular Level ◽  
Binding Constants ◽  
Exchange Constant ◽  
Interior Region ◽  
Bimolecular Reactions ◽  
Exterior Region ◽  
Different Shapes ◽  
Molecular Origin ◽  
Kinetics Of

The aqueous surfactant (Surf) solution at [Surf] > cmc (critical micelle concentration) contains flexible micelles/nanoparticles. These particles form a pseudophase of different shapes and sizes where the medium polarity decreases as the distance increases from the exterior region of the interface of the Surf/H2O particle towards its furthest interior region. Flexible nanoparticles (FNs) catalyse a variety of chemical and biochemical reactions. FN catalysis involves both positive catalysis ( i.e. rate increase) and negative catalysis ( i.e. rate decrease). This article describes the mechanistic details of these catalyses at the molecular level, which reveals the molecular origin of these catalyses. Effects of inert counterionic salts (MX) on the rates of bimolecular reactions (with one of the reactants as reactive counterion) in the presence of ionic FNs/micelles may result in either positive or negative catalysis. The kinetics of cationic FN (Surf/MX/H2O)-catalysed bimolecular reactions (with nonionic and anionic reactants) provide kinetic parameters which can be used to determine an ion exchange constant or the ratio of the binding constants of counterions.

Kinetic and spectrophotometric studies of binding of iron(III) by glutathione

1976 ◽  
Vol 54 (20) ◽  
pp. 3192-3199 ◽  
Author(s):  
Tahir R. Khan ◽  
Cooper H. Langford
Keyword(s):  
Complex Formation ◽  
Kinetic Method ◽  
Binding Constants ◽  
Chelating Agent ◽  
Formation Reaction ◽  
Spectrophotometric Data ◽  
Natural Water Chemistry ◽  
Kinetics Of ◽  
Sulfur Containing

In this report, determination of unbound aquo iron species is accomplished by a kinetic method involving reaction with sulfosalicylic acid (SSA) on a time scale which is very short with respect to reaction of SSA with the glutathione complexes of iron. The data are used to calculate conditional binding constants for Fe(III) to glutathione. Binding constants in 0.1 M ionic strength media were obtained between pH 1 and 2.4 by the kinetic method, and near pH = 3 by spectrophotometry and by examination of the ratio of rate of complex formation and dissociation. The conditional binding 'constant' between pH 1 and 3 is represented as pK = −1.96 – 0.50pH. This is consistent with the importance of reactions involving only very limited proton release. Spectrophotometric data show that the —OH group on Fe(OH)2+ is lost on glutathione complexing. Kinetics of the complex formation reaction between aquo iron(III) species and glutathione are slower than rates of reaction of iron(III) with simple ligands.The glutathione system is regarded as a model system important to natural water chemistry because it is a widely distributed biological sulfur-containing chelating agent.

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