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.

scholarly journals The effect of quaternary structure on the kinetics of conformational changes and nanosecond geminate rebinding of carbon monoxide to hemoglobin.

1988 ◽  
Vol 85 (7) ◽  
pp. 2151-2155 ◽  
Author(s):  
L. P. Murray ◽  
J. Hofrichter ◽  
E. R. Henry ◽  
M. Ikeda-Saito ◽  
K. Kitagishi ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Kinetics Of

scholarly journals Effect of pH on the Hydrolytic Kinetics of Gamma-Glutamyl Transferase fromBacillus subtilis

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Sharath Balakrishna ◽  
Asmita Prabhune
Keyword(s):  
Conformational Changes ◽  
Quaternary Structure ◽  
Gamma Glutamyl Transferase ◽  
Effect Of Ph ◽  
Gamma Glutamyl ◽  
Steady State Kinetics ◽  
Kinetic Transformation ◽  
Ph Range ◽  
Glutamyl Transferase ◽  
Kinetics Of

The effect of pH on the steady state kinetics of gamma-glutamyl transferase (GGT) fromBacillus subtiliswas examined using glutamyl-(3-carboxyl)-4-nitroanilide as the chromogenic reporter substrate. The enzyme was active in the pH range 7.0–11.0 with the optimum activity at pH 11.0. We noticed a pH dependent transformation in the nature of substrate consumption kinetics. The substrate saturation curves were hyperbolic in the pH range 7.0–9.0 but changed into sigmoid form at pH 10.0 and 11.0. Hill’s coefficients were >1. We also analysed the effect of pH on the structure of the enzyme. The circular dichroism spectra of the enzyme sample at pH 9.0 and 11.0 were coincidental in both far and near UV regions indicating conservation of the secondary and tertiary structures, respectively. The molecular weight of the enzyme sample was the same in both pH 7.0 and 11.0 indicating conservation of the quaternary structure. These results show that the kinetic transformation does not involve significant conformational changes. Cooperative binding of multiple substrate molecules may not be the basis for the sigmoid kinetics as only one substrate binding site has been noticed in the reported crystal structures ofB. subtilisGGT.

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.

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.

Electron Microscopy and image reconstruction reveal the structural basis for spectrin's elastic properties

1992 ◽  
Vol 50 (1) ◽  
pp. 510-511
Author(s):  
Amy M. McGough ◽  
Robert Josephs
Keyword(s):  
Electron Microscopy ◽  
Elastic Properties ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Projection Algorithm ◽  
Structural Basis ◽  
Iterative Approximation ◽  
Membrane Skeletons

The remarkable deformability of the erythrocyte derives in large part from the elastic properties of spectrin, the major component of the membrane skeleton. It is generally accepted that spectrin's elasticity arises from marked conformational changes which include variations in its overall length (1). In this work the structure of spectrin in partially expanded membrane skeletons was studied by electron microscopy to determine the molecular basis for spectrin's elastic properties. Spectrin molecules were analysed with respect to three features: length, conformation, and quaternary structure. The results of these studies lead to a model of how spectrin mediates the elastic deformation of the erythrocyte.Membrane skeletons were isolated from erythrocyte membrane ghosts, negatively stained, and examined by transmission electron microscopy (2). Particle lengths and end-to-end distances were measured from enlarged prints using the computer program MACMEASURE. Spectrin conformation (straightness) was assessed by calculating the particles’ correlation length by iterative approximation (3). Digitised spectrin images were correlation averaged or Fourier filtered to improve their signal-to-noise ratios. Three-dimensional reconstructions were performed using a suite of programs which were based on the filtered back-projection algorithm and executed on a cluster of Microvax 3200 workstations (4).

Kinetics of temperature-programmed reactivation of a hydrodesulphurization catalyst

1983 ◽  
Vol 48 (12) ◽  
pp. 3340-3355 ◽  
Author(s):  
Pavel Fott ◽  
Pavel Šebesta
Keyword(s):  
Carbon Dioxide ◽  
Thermal Analysis ◽  
Differential Thermal Analysis ◽  
Thin Layer ◽  
Kinetic Parameters ◽  
Diffusion Region ◽  
Reaction Heat ◽  
Gaseous Products ◽  
Temperature Programmed ◽  
Kinetics Of

The kinetic parameters of reactivation of a carbonized hydrodesulphurization (HDS) catalyst by air were evaluated from combined thermogravimetric (TG) and differential thermal analysis (DTA) data. In addition, the gaseous products leaving a temperature-programmed reactor with a thin layer of catalyst were analyzed chromatographically. Two exothermic processes were found to take part in the reactivation, and their kinetics were described by 1st order equations. In the first process (180-400 °C), sulphur in Co and Mo sulphides is oxidized to sulphur dioxide; in the second process (300-540 °C), in which the essential portion of heat is produced, the deposited carbon is oxidized to give predominantly carbon dioxide. If the reaction heat is not removed efficiently enough, ignition of the catalyst takes place, which is associated with a transition to the diffusion region. The application of the obtained kinetic parameters to modelling a temperature-programmed reactivation is illustrated on the case of a single particle.

Thin-layer gel chromatography of dyed proteins

1975 ◽  
Vol 105 (2) ◽  
pp. 317-321 ◽  
Author(s):  
J.N. Miller ◽  
O. Erinle ◽  
Janet M. Roberts ◽  
Christine Thirkettle
Keyword(s):  
Thin Layer ◽  
Gel Chromatography

scholarly journals Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 475-488 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Katja Kohler ◽  
Andrea Bacconi ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Reaction Rates ◽  
Transport Pathway ◽  
Extracellular Medium ◽  
Voltage Range ◽  
Substituted Cysteine Accessibility ◽  
Voltage Dependent ◽  
Opposite Behavior ◽  
Rate Limiting ◽  
Kinetics Of

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

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