scholarly journals Ligand binding kinetics and dissociation of the human embryonic haemoglobins

1996 ◽  
Vol 315 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Oliver HOFMANN ◽  
Thomas BRITTAIN
Keyword(s):  
Ligand Binding ◽  
Flash Photolysis ◽  
Ph Dependence ◽  
Equilibrium Constants ◽  
General Scheme ◽  
Dissociation Rate ◽  
Oxygen Binding ◽  
Dissociation Kinetics ◽  
Oxygen Dissociation ◽  
Dissociation Rate Constants

The three human embryonic haemoglobins have been studied using a range of stopped-flow and flash photolysis experiments. The association and dissociation kinetics and equilibrium constants for the tetramer–dimer reactions of the deoxy and oxygenated forms have been investigated and found to be characterized by constants similar to those of the human adult protein. The rates of oxygen dissociation from the embryonic haemoglobins have been measured and appear to be responsible for the high oxygen-binding affinity associated with the embryonic proteins compared with the adult protein. The pH dependence of the oxygen dissociation rate constants also accounts for the rather unusual, previously described, Bohr effects characteristic of the embryonic haemoglobins. A general scheme has been developed coupling both the dimer–tetramer equilibria and ligand-binding steps observed following photolysis of the liganded forms of the human embryonic haemoglobins.

Temperature effects on xanthone–β-cyclodextrin binding dynamics

2011 ◽  
Vol 89 (3) ◽  
pp. 395-401 ◽  
Author(s):  
Tamara C. S. Pace ◽  
Cornelia Bohne
Keyword(s):  
Excited State ◽  
Complex Formation ◽  
Ground State ◽  
Rate Constants ◽  
Flash Photolysis ◽  
Equilibrium Constants ◽  
Dissociation Rate ◽  
Activation Entropy ◽  
Triplet Excited State ◽  
Dissociation Rate Constants

The complexation dynamics of the triplet excited state of xanthone with β-cyclodextrin were studied at various temperatures between 10 and 50 °C. Association and dissociation rate constants were determined using the laser flash photolysis quenching methodology with Cu2+ as a quencher. The rate constants for the association and dissociation of triplet xanthone with β-cyclodextrin increased with temperature, while the equilibrium constant for the triplet excited state remained relatively constant. Equilibrium constants for the ground-state complexation of xanthone with β-cyclodextrin were determined from fluorescence studies at various temperatures. The ground-state binding efficiency decreased with temperature and was markedly greater than that of the triplet excited state at all temperatures. The enthalpy and entropy for the β-cyclodextrin complex formation of the ground and triplet excited states fall on the enthalpy–entropy compensation relationship previously established for cyclodextrin complexes. The activation enthalpies for the association and dissociation rate constants for triplet xanthone are similar. The activation entropy is favorable for the association process, whereas a negative activation entropy was measured for the dissociation process, suggesting that solvation plays a key role in the complex formation between xanthone and β-cyclodextrin.

Oxygen affinity and amino acid sequence of myoglobins from endothermic and ectothermic fish

2001 ◽  
Vol 280 (4) ◽  
pp. R1123-R1133 ◽  
Author(s):  
David J. Marcinek ◽  
Joseph Bonaventura ◽  
Jonathan B. Wittenberg ◽  
Barbara A. Block
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rate Constants ◽  
Oxygen Affinity ◽  
Dissociation Rate ◽  
Low Oxygen ◽  
Dissociation Kinetics ◽  
Oxygen Dissociation ◽  
Dissociation Rate Constants

Myoglobin (Mb) buffers intracellular O2 and facilitates diffusion of O2 through the cell. These functions of Mb will be most effective when intracellular Po 2 is near the partial pressure of oxygen at which Mb is half saturated (P50) of the molecule. We test the hypothesis that Mb oxygen affinity has evolved such that it is conserved when adjusted for body temperature among closely related animals. We measure oxygen P50s tonometrically and oxygen dissociation rate constants with stopped flow and generate amino acid sequence from cDNA of Mbs from fish with different body temperatures. P50s for the endothermic bluefin tuna, skipjack tuna, and blue marlin at 20°C were 0.62 ± 0.02, 0.59 ± 0.01, 0.58 ± 0.04 mmHg, respectively, and were significantly lower than those for ectothermic bonito (1.03 ± 0.07 mmHg) and mackerel (1.39 ± 0.03 mmHg). Because the oxygen affinity of Mb decreases with increasing temperature, the above differences in oxygen affinity between endothermic and ectothermic fish are reduced when adjusted for the in vivo muscle temperature of the animal. Oxygen dissociation rate constants at 20°C for the endothermic species ranged from 34.1 to 49.3 s−1, whereas those for mackerel and bonito were 102 and 62 s−1, respectively. Correlated with the low oxygen affinity and fast dissociation kinetics of mackerel Mb is a substitution of alanine for proline that would likely result in a more flexible mackerel protein.

scholarly journals The kinetics of antibody binding to membrane antigens in solution and at the cell surface

1980 ◽  
Vol 187 (1) ◽  
pp. 1-20 ◽  
Author(s):  
D W Mason ◽  
A F Williams
Keyword(s):  
Cell Surface ◽  
Rate Constants ◽  
Equilibrium Constants ◽  
Surface Antigens ◽  
Dissociation Rate ◽  
Simple Relationship ◽  
Dissociation Kinetics ◽  
Cell Surface Antigens ◽  
Dissociation Rate Constants ◽  
Kinetics Of

The reaction kinetics of 125I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab′)2 or Fab′ form and by using antigen in polymeric or monomeric forms. Association rate constants (k+1), dissociation rate constants (k-1) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-1 antigen were studied. The major findings were as follows. (i) With F(ab′)2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-1 for the univalent interaction, in which case all binding was bivalent. (ii) k+1 values were similar for F(ab′)2 or Fab′ antibody, and for the different antibodies were in the range 0.8 × 10(5)–1.1 × 10(6) M-1.s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently. However, the k-1 values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.

scholarly journals Mycobacterial and Human Ferrous Nitrobindins: Spectroscopic and Reactivity Properties

2021 ◽  
Vol 22 (4) ◽  
pp. 1674
Author(s):  
Giovanna De Simone ◽  
Alessandra di Masi ◽  
Alessandra Pesce ◽  
Martino Bolognesi ◽  
Chiara Ciaccio ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Ph Dependence ◽  
Dissociation Rate ◽  
Heme Pocket ◽  
Functional Relationships ◽  
Distal Side ◽  
Structural And Functional Properties ◽  
Marked Enhancement ◽  
Ph Range ◽  
Dissociation Rate Constants

Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H2O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural–functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

Hemoglobin in five genetically diverse Frankia strains

2002 ◽  
Vol 48 (12) ◽  
pp. 1048-1055 ◽  
Author(s):  
Jason Beckwith ◽  
John D Tjepkema ◽  
Robert E Cashon ◽  
Christa R Schwintzer ◽  
Louis S Tisa
Keyword(s):  
Hemoglobin Concentration ◽  
Dissociation Rate ◽  
Size Exclusion ◽  
Facilitated Diffusion ◽  
Truncated Hemoglobin ◽  
Oxygen Dissociation ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
N Treatment ◽  
Dissociation Rate Constants

Five strains of Frankia were selected to represent a wide range of genetic diversity and examined for presence of hemoglobin. All five strains produced hemoglobin when grown on media without (–N) or with (+N) combined nitrogen. This indicates that hemoglobin is common in Frankia and is not directly associated with nitrogen fixation. Frankia strain EAN1pec was examined in more detail. It showed greater hemoglobin concentration when grown at 2% O2 than at 20% O2 in the –N treatment but no effect of oxygen on hemoglobin concentration in the +N treatment. At both oxygen levels, it produced substantially more biomass in +N than in –N culture. It also produced significantly more biomass when the medium contained 0.2% CO2 than in the absence of CO2. The molecular mass of the hemo- globin as determined by size exclusion chromatography was 13.4 ± 0.2 kDa (mean ± SE, n = 3) and is consistent with that of a truncated hemoglobin. The hemoglobin had absorption spectra that were typical of a hemoglobin. The oxygen dissociation rate constants for the hemoglobin were 131.2 ± 5.8 s–1 for –N culture and 166 ± 8.2 s–1 for +N culture. These rapid rates are consistent with a function in facilitated diffusion of oxygen.Key words: Frankia, hemoglobin, truncated hemoglobin.

scholarly journals Transient kinetic studies of Neurospora crassa cytochrome c oxidase

1983 ◽  
Vol 215 (2) ◽  
pp. 425-427 ◽  
Author(s):  
M Brunori ◽  
M C Silvestrini ◽  
M T Wilson ◽  
H Weiss
Keyword(s):  
Cytochrome C ◽  
Neurospora Crassa ◽  
Cytochrome C Oxidase ◽  
Flash Photolysis ◽  
Kinetic Studies ◽  
Dissociation Rate ◽  
Steady State Kinetic ◽  
Kinetic Investigations ◽  
Dissociation Rate Constants

The reaction of Neurospora crassa cytochrome c oxidase with CO was studied by flash-photolysis and rapid-mixing experiments, leading to the determination of the association and dissociation rate constants (7 X 10(4) M-1 X s-1 and 0.02s-1 respectively). Pre-steady-state kinetic investigations of the catalytic properties of the enzyme showed that under proper conditions Neurospora cytochrome c oxidase can be ‘pulsed’, i.e. activated, like the mammalian enzyme. The ‘pulsed’ species is spectroscopically different from the ‘resting’ one, and the decay into the ‘resting’ state is fast (t1/2 approx. 3 min).

scholarly journals Hemoglobin in Frankia, a Nitrogen-Fixing Actinomycete

2002 ◽  
Vol 68 (5) ◽  
pp. 2629-2631 ◽  
Author(s):  
John D. Tjepkema ◽  
Robert E. Cashon ◽  
Jason Beckwith ◽  
Christa R. Schwintzer
Keyword(s):  
Optical Absorption ◽  
Rate Constant ◽  
Dissociation Rate ◽  
Oxygen Binding ◽  
Association Rate Constant ◽  
Association Rate ◽  
Absorption Bands ◽  
Oxygen Dissociation ◽  
Equilibrium Oxygen ◽  
Strain Cci3

ABSTRACT Frankia strain CcI3 grown in culture produced a hemoglobin which had optical absorption bands typical of a hemoglobin and a molecular mass of 14.1 kDa. Its equilibrium oxygen binding constant was 274 nM, the oxygen dissociation rate constant was 56 s−1, and the oxygen association rate constant was 206 μM−1 s−1.

scholarly journals Evidence for a kinetic heterogeneity in ligand binding to R-state haemoglobin Kempsey [Asp-G1(99) β→Asn]

1986 ◽  
Vol 238 (2) ◽  
pp. 353-357
Author(s):  
M Coletta ◽  
T Brittain ◽  
M Brunori
Keyword(s):  
Ligand Binding ◽  
Time Course ◽  
Flash Photolysis ◽  
Activation Parameters ◽  
Binding Kinetics ◽  
Slow Phase ◽  
Kinetic Properties ◽  
Dissociation Kinetics ◽  
Binding Isotherms ◽  
O2 Binding

Thermodynamic and kinetic properties of O2 and CO binding to haemoglobin (Hb) Kempsey [Asp-G1(99) beta----Asn] were investigated and the activation parameters for the two ligands were determined. At every temperature the O2-binding isotherms display a weak co-operativity, n ranging between 1.1 and 1.2, and dissociation kinetics show a single-exponential behaviour. O2-binding kinetics were studied at 25 degrees C by temperature jump and are characterized at each saturation (from Y = 0.31 to Y = 1.0) by two processes, a fast bimolecular one and a slow monomolecular one (tau -1 = 20 s-1), which contributes to approx. 30% of the whole relaxation amplitude at every Y. CO-binding kinetics to Hb Kempsey were followed at several temperatures by flash photolysis and stopped flow. The process is biphasic, as reported elsewhere [Bunn, Wohl, Bradley, Cooley & Gibson (1974) J. Biol. Chem. 249, 7402-7409], and the relative contributions of the two bimolecular rates to the whole process are only slightly affected by temperature. On taking account for the fraction of dimers at every protein concentration, the slow phase corresponds to approx. 50% of the ligand binding to tetramers. Correlation of these results with previous spectroscopic data leads to the hypothesis that the biphasic time course of CO binding may be attributed to alpha/beta heterogeneity of the R-state of tetrameric Hb Kempsey.

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