Random Chemical Modification of Hemoglobin to Identify Chloride Binding Sites in the Central Dyad Axis: Their Role in Control of Oxygen Affinity

1994 ◽  
Vol 22 (2) ◽  
pp. 199-205 ◽  
Author(s):  
James M. Manning
Keyword(s):  
Chemical Modification ◽  
Binding Sites ◽  
Oxygen Affinity ◽  
Chloride Binding

Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function

1992 ◽  
Vol 72 (4) ◽  
pp. 1611-1615 ◽  
Author(s):  
R. E. Weber
Keyword(s):  
Binding Sites ◽  
Ionic Composition ◽  
Oxygen Affinity ◽  
Chloride Concentration ◽  
Anion Binding ◽  
Chloride Binding ◽  
Ph Values ◽  
Two Temperatures ◽  
Ethanesulfonic Acid ◽  
Chloride Concentrations

The functional characteristics of hemoglobin (Hb) depend on oxygenation-linked proton and anion binding and thus on solvent buffer groups and ionic composition. This study compares the oxygenation properties of human Hb in ionic [tris(hydroxymethyl)aminomethane (Tris) and BisTris] buffers with those in zwitterionic N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer under strictly controlled chloride concentrations at different pH values, two temperatures, and in the absence and presence of the erythrocytic cofactor, 2,3-diphosphoglycerate (DPG). In contrast to earlier studies (carried out at the same or different chloride concentrations) it shows only small buffer effects that are manifested at low chloride concentration and high pH. These observations suggest chloride binding to the Tris buffers, which reduces the interaction with specific chloride binding sites in the Hb. The findings indicate that HEPES allows for more accurate assessment of Hb-oxygen affinity and its anion and temperature sensitivities than ionic buffers and advocates standard use of HEPES in studies on Hb function. Precise oxygen affinities of Hb dissolved in both buffers are defined under standard conditions.

scholarly journals The Regulation of Haemocyanin Oxygen Affinity During Emersion of the Crayfish Austropotamobius Pallipes: III. The Dependence of Ca2+-Haemocyanin Binding on the Concentration Of L-Lactate

1987 ◽  
Vol 133 (1) ◽  
pp. 339-352
Author(s):  
S. MORRIS ◽  
C.R. BRIDGES ◽  
M. K. GRIESHABER
Keyword(s):  
Biological Sciences ◽  
Binding Sites ◽  
Lactate Concentration ◽  
Oxygen Affinity ◽  
Calcium Lactate ◽  
Worst Case ◽  
Austropotamobius Pallipes ◽  
University Of Calgary ◽  
Lactate Formation ◽  
The Relationship

The binding of Ca2+ to the haemocyanin of the crayfish Austropotamobius pallipes was investigated. The amount of bound Ca2+ was determined using an ultrafiltration technique to produce haemocyanin-free solutions, the Ca2+ concentration of which could then be compared with that of the original, unfiltered solution. Any difference between the two values would indicate the amount of calcium bound by haemocyanin. The effect of L-lactate on Ca2+ binding was investigated by determining the amount of bound ion at different concentrations of L-lactate. In addition, oxygen equilibrium curves were constructed for some of the solutions to verify that the haemocyanin oxygen affinity remained sensitive to L-lactate and to determine whether the haemocyanin was functionally similar to that used in previous investigations. With 17 mmol 1−1 total Ca2+ and approximately 1 mmol 1−1 L-lactate the number of Ca2+ binding sites was estimated to be between eight and nine per haemocyanin molecule. Without taking into account the formation of calcium lactate, the observed dependency of Ca2+-haemocyanin binding on L-lactate concentration could best be described by the equation: Ca2+/Hc = 8.64-0.32[lactate−]. A ‘worst case’ estimate for maximum calcium lactate formation, assuming Ca2+ to be the only counterion available to lactate, altered the relationship slightly to: Ca2+/Hc = 8.65-0.35[lactate−]. Note: Present address: Department of Biological Sciences, University of Calgary, 2500 University Drive N/V, Calgary, Alberta, Canada T2N 1N4.

N-Ethylmaleimide-modified actin filaments do not bundle in the presence of α-actinin

1995 ◽  
Vol 73 (1-2) ◽  
pp. 116-122 ◽  
Author(s):  
Aldo Milzani ◽  
Isabella DalleDonne ◽  
Roberto Colombo
Keyword(s):  
Polyethylene Glycol ◽  
Chemical Modification ◽  
Molecular Interactions ◽  
Binding Sites ◽  
Actin Filaments ◽  
Actin Assembly ◽  
Chemically Modified ◽  
C Terminus ◽  
Spatial Reorganization ◽  
Polyethylene Glycol 6000

We show that the modification of actin subdomain 1 by N-ethylmaleimide (NEM), which binds Cys-374 close to the C-terminus of the molecule, inhibits the α-actinin-induced bundling of actin filaments. This effect is not merely related to the block of Cys-374, since N-(1-pyrenyl)iodoacetamide (pyrene-IA) is unable to prevent bundling. Considering that NEM (but not pyrene-IA) influences actin assembly, we suggest that the inhibition of the actin – α-actinin interaction is due to the chemical modification of actin Cys-374 which, by inducing a marked spatial reorganization of actin monomers, is able to modify both the intra- and inter-molecular interactions of this protein. Finally, NEM-modified actin filaments form bundles in the presence of polyethylene glycol 6000 since, in this case, the side by side association of actin filaments does not depend on the accessibility of binding sites nor on the formation of chemical bonds.Key words: chemically modified actin, N-ethylmaleimide, pyrene-IA, Cys-374, actin bundles, α-actinin.

Chemical modification of the dihydropyridines binding sites by lysine reagent, pyridoxal 5′-phosphate

1998 ◽  
Vol 32 (4) ◽  
pp. 361-364 ◽  
Author(s):  
Barbara Costa ◽  
Laura Giusti ◽  
Claudia Martini ◽  
Antonio Lucacchini
Keyword(s):  
Chemical Modification ◽  
Binding Sites
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