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.

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

2005 ◽  
Vol 385 (3) ◽  
pp. 839-839
Author(s):  
Y. JIA ◽  
S. RAMASAMY ◽  
F. WOOD ◽  
A. I. ALAYASH ◽  
J. M. RIFKIND
Keyword(s):  
Oxygen Affinity ◽  
Cross Linking ◽  
T State

scholarly journals Rat haemoglobin heterogeneity. Two structurally distinct α chains and functional behaviour of selected components

1975 ◽  
Vol 149 (1) ◽  
pp. 245-258 ◽  
Author(s):  
L M Garrick ◽  
V S Sharma ◽  
M J McDonald ◽  
H M Ranney
Keyword(s):  
Rattus Norvegicus ◽  
Deae Cellulose ◽  
Oxygen Affinity ◽  
Amino Acid Sequences ◽  
Cellulose Acetate Electrophoresis ◽  
Human Haemoglobin ◽  
Globin Chains ◽  
Lower Oxygen ◽  
Alkaline Bohr Effect ◽  
Starch Gels

Six haemoglobins were separated analytically from haemolysates of adult Wistar rats (Rattus norvegicus) by cellulose acetate electrophoresis and preparatively by DEAE-cellulose chromatography. The globin chains were separated from unfractionated haemolysates by CM-cellulose chromatography by using a non-linear formic acid-pyridine gradient followed by CM-cellulose chromatography in 8M-urea by using a gradient of increasing Na+ concentration in phosphate buffer, pH 6.7. Two α chains and three non-α chains were identified. Chains isolated from purified haemoglobins were correlated with chains isolated from unfractionated haemolysates by electrophoresis on urea-starch gels to make presumptive assignments of the subunit composition of the six haemoglobin tetramers. Partial amino acid sequences were determined for the major and minor α chains. The oxygen equilibria of two of the major haemoglobin components and of the unfractionated haemolysate were examined at pH 7.5 and 8.0. The two purified haemoglobins exhibited similar oxygen affinities; the haemolysate, however, had a lower oxygen affinity than either of the two purified haemoglobins. Both the haemolysate and the two haemoglobins showed an alkaline Bohr effect larger than that of human haemoglobin A.

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.

Polarographic studies on renaturation of urea-denatured human serum albumin

1989 ◽  
Vol 54 (2) ◽  
pp. 536-543 ◽  
Author(s):  
Josef Chmelík ◽  
Pavel Anzenbacher ◽  
Vítěz Kalous
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Irreversible Process ◽  
State Model ◽  
Native Protein ◽  
Main Components ◽  
Two State Model ◽  
Kinetics Of

The renaturation of the two main components of human serum albumin, i.e. of mercaptalbumin and nonmercaptalbumin, was studied polarographically. It has been demonstrated that renaturation of both proteins after 1-min denaturation in 8M urea is reversible. By contrast, renaturation after 200 min denaturation in 8M urea is an irreversible process; the characteristics of renatured mercaptalbumin differ more from the properties of the native protein than the characteristics of nonmercaptalbumin. The studies of the kinetics of renaturation of both proteins have shown that the renaturation can be represented by a two-state model. This means that the existence of stable intermediary products during the renaturation process was not determined polarographically.

scholarly journals Kinetics of Cross-Linking of Peptidoglycan in Bacillus megaterium

1974 ◽  
Vol 249 (8) ◽  
pp. 2478-2482
Author(s):  
William D. Fordham ◽  
Charles Gilvarg
Keyword(s):  
Bacillus Megaterium ◽  
Cross Linking ◽  
Kinetics Of

scholarly journals Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A0 fully stripped of any anions

1999 ◽  
Vol 55 (11) ◽  
pp. 1914-1916 ◽  
Author(s):  
F. A. V. Seixas ◽  
W. F. de Azevedo ◽  
M. F. Colombo
Keyword(s):  
Diffraction Data ◽  
Oxygen Affinity ◽  
X Ray Diffraction ◽  
Structural Explanation ◽  
Human Haemoglobin ◽  
X Ray ◽  
High Resolution Structure ◽  
Allosteric Properties

In this work, initial crystallographic studies of human haemoglobin (Hb) crystallized in isoionic and oxygen-free PEG solution are presented. Under these conditions, functional measurements of the O2-linked binding of water molecules and release of protons have evidenced that Hb assumes an unforeseen new allosteric conformation. The determination of the high-resolution structure of the crystal of human deoxy-Hb fully stripped of anions may provide a structural explanation for the role of anions in the allosteric properties of Hb and, particularly, for the influence of chloride on the Bohr effect, the mechanism by which Hb oxygen affinity is regulated by pH. X-ray diffraction data were collected to 1.87 Å resolution using a synchrotron-radiation source. Crystals belong to the space group P21212 and preliminary analysis revealed the presence of one tetramer in the asymmetric unit. The structure is currently being refined using maximum-likelihood protocols.

scholarly journals Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors.

1980 ◽  
Vol 85 (2) ◽  
pp. 414-428 ◽  
Author(s):  
S D MacLean-Fletcher ◽  
T D Pollard
Keyword(s):  
Crude Extract ◽  
Cross Linking ◽  
Cross Link ◽  
Gelation Process ◽  
Optimum Ph ◽  
Kinetics Of ◽  
Low Shear

We have studied the kinetics of the gelation process that occurs upon warming cold extracts of Acanthamoeba using a low-shear falling ball assay. We find that the reaction has at least two steps, requires 0.5 mM ATP and 1.5 mM MgCl2, and is inhibited by micromolar Ca++. The optimum pH is 7.0 and temperature, 25 degrees-30 degrees C. The rate of the reaction is increased by cold preincubation with both MgCl2 and ATP. Nonhydrolyzable analogues of ATP will not substitute for ATP either in this "potentiation reaction" or in the gelation process. Either of two purified or any one of four partially purified Acanthamoeba proteins will cross-link purified actin to form a gel, but none can account for the dependence of the reaction in the crude extract on Mg-ATP or its regulation by Ca++. This suggests that the extract contains, in addition to actin-cross-linking proteins, factors dependent on Mg-ATP and Ca++ that regulate the gelation process.

scholarly journals Single-sided NMR for the measurement of the degree of cross-linking and curing

2018 ◽  
Vol 7 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Norbert Halmen ◽  
Christoph Kugler ◽  
Eduard Kraus ◽  
Benjamin Baudrit ◽  
Thomas Hochrein ◽  
...  
Keyword(s):  
Relaxation Times ◽  
Curing Kinetics ◽  
Cross Linking ◽  
Scanning Calorimetry ◽  
First Results ◽  
Non Destructive ◽  
Kinetics Of ◽  
Good Agreement

Abstract. The degree of cross-linking and curing is one of the most important values concerning the quality of cross-linked polyethylene (PE-X) and the functionality of adhesives and resin-based components. Up to now, the measurement of this property has mostly been time-consuming and usually destructive. Within the shown work the feasibility of single-sided nuclear magnetic resonance (NMR) for the non-destructive determination of the degree of cross-linking and curing as process monitoring was investigated. First results indicate the possibility of distinguishing between PE-X samples with different degrees of cross-linking. The homogeneity of the samples and the curing kinetics of adhesives can also be monitored. The measurements show good agreement with reference tests (wet chemical analysis, differential scanning calorimetry, dielectric analysis). Furthermore, the influence of sample temperature on the characteristic relaxation times can be observed.

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