scholarly journals The binding of dihydronicotinamide–adenine dinucleotide and pyridine-3-aldehyde–adenine dinucleotide by yeast alcohol dehydrogenase

1970 ◽  
Vol 120 (4) ◽  
pp. 821-830 ◽  
Author(s):  
F. M. Dickinson
Keyword(s):  
Alcohol Dehydrogenase ◽  
Binding Sites ◽  
Ternary Complex ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Coenzyme Binding ◽  
Yeast Alcohol Dehydrogenase ◽  
Number Of Binding Sites ◽  
Established Fact

1. Yeast alcohol dehydrogenase has been found to react with NADH in the presence of acetamide to form a highly fluorescent ternary complex. Titration of the enzyme to form this complex has provided a method for the estimation of the number of binding sites on the enzyme. 2. The binding of NADH by the enzyme has been studied independently, with a modified form of equilibrium dialysis, by using gel filtration. 3. A third method, depending upon the formation of a ternary complex of enzyme, hydroxylamine and pyridine-3-aldehyde–adenine dinucleotide, has also been used to titrate the enzyme. 4. Values obtained with all three methods are substantially in agreement that only three coenzyme-binding sites are available. This is in contrast with the established fact that the enzyme is composed of four identical subunits.

scholarly journals The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

1969 ◽  
Vol 113 (1) ◽  
pp. 117-121 ◽  
Author(s):  
L. Stevens
Keyword(s):  
Ionic Strength ◽  
Ribosomal Rna ◽  
Ribonucleic Acid ◽  
Binding Sites ◽  
Bacillus Stearothermophilus ◽  
Gel Filtration ◽  
Ribosomal Ribonucleic Acid ◽  
Number Of Binding Sites ◽  
Filtration Technique

1. The total intracellular concentrations of Na+, K+, Mg2+, spermine, spermidine and RNA were measured in Bacillus stearothermophilus. 2. The binding of spermine to ribosomes and to ribosomal RNA from B. stearothermophilus was studied under various conditions by using a gel-filtration technique. 3. The affinity of spermine for ribosomes and for ribosomal RNA decreased with increasing ionic strength of the medium in which they were suspended. 4. The extent of spermine binding did not change appreciably in the temperature range 4–60°. 5. Optimum binding occurred at about pH7·0. 6. The number of binding sites for spermine on either ribosomes or ribosomal RNA was 0·10–0·13/RNA phosphate group. 7. A high proportion of the intracellular spermine is likely to be bound to the ribosomes in vivo; spermine competes with Mg2+ on equal terms for sites on the ribosomes.

The Protein Binding of Acetylsalicylic Acid and Salicylic Acid

1969 ◽  
Vol 15 (11) ◽  
pp. 1027-1038 ◽  
Author(s):  
M Amir Ali ◽  
Joseph I Routh
Keyword(s):  
Salicylic Acid ◽  
Human Serum Albumin ◽  
Acetylsalicylic Acid ◽  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Progressive Increase ◽  
Lower Percentage ◽  
Therapeutic Implications ◽  
Dialysis Cell

Abstract Quantitative experiments to study the binding of acetylsalicylic acid (ASA) and salicylic acid (SA) by human serum albumin (HSA) were carried out using 14C-labeled ASA or SA and gel filtration to separate the free from the bound forms. Two binding procedures were employed: the ASA or SA was incubated with the HSA, or the mixture was placed in an equilibrium dialysis cell. By withdrawing samples at intervals, the extent of binding or the attainment of equilibrium could be assessed. Evidence that filtration by the gel caused unbinding of the bound SA was obtained, with resultant lower percentage binding of SA by HSA than that obtained by equilibrium dialysis without gel filtration. In either case, binding equilibrium was reached in 4-8 hr. The binding of ASA by HSA was markedly different from that of SA. The experiments both with or without gel filtration demonstrated a progressive increase in binding of ASA in the 20- to 53-hr periods studied. In addition, ASA apparently displaces SA from its binding sites on albumin, an observation that may have therapeutic implications.

scholarly journals Oestrogen receptor of calf mammary gland. Purification by use of sodium bromide and heparin-sepharose

1979 ◽  
Vol 178 (3) ◽  
pp. 581-587 ◽  
Author(s):  
A Rotondi ◽  
F Auricchio
Keyword(s):  
Mammary Gland ◽  
Binding Sites ◽  
Oestrogen Receptor ◽  
Large Scale ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Receptor Aggregation ◽  
Low Salt ◽  
Number Of Binding Sites ◽  
Stokes Radius

1. Calf mammary-gland cytosol apparently has a single oestrogen receptor capable of auto- and/or hetero-association of varying complexity. Computation of the dissociation constant for oestradiol-17beta gives Kd = 0.5 nM. The number of binding sites is 40 fmol/mg of cytosol protein. The oestrogen receptor in the presence of NaBr, a chaotropic salt that inhibits the interaction of receptor with other cytosol components, sediments through sucrose density gradients as a single sharp peak at 4S, and it has a Stokes radius of 3.4 nm measured by gel filtration. 2. A large-scale purification procedure of the calf mammary-gland oestrogen receptor based on the inhibition of receptor aggregation by NaBr and interaction with heparin-Sepharose is reported. The receptor is purified more than 1500-fold over that in the 27,000g supernatant of the homogenate, with a 30% yield. In ‘low-salt’ buffer the purified receptor sediments through sucrose gradients at 4S and the Stokes radius, measured by gel filtration in the presence of heparin, is 3.4 nm. The mol.wt computed from these values is about 60,000, and the frictional ratio is 1.3.

scholarly journals Fluorescence-polarization studies on binding of 4-methylumbelliferyl β-d-galactopyranoside to Ricinus communis (castor-bean) agglutinin

1980 ◽  
Vol 191 (2) ◽  
pp. 395-400 ◽  
Author(s):  
M I Khan ◽  
M K Mathew ◽  
P Balaram ◽  
A Surolia
Keyword(s):  
Binding Sites ◽  
Fluorescence Polarization ◽  
Castor Bean ◽  
Ricinus Communis ◽  
Equilibrium Dialysis ◽  
Scatchard Analysis ◽  
Polarization Data ◽  
Appreciable Change ◽  
Number Of Binding Sites ◽  
Polarization Studies

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.

scholarly journals Comparison of the sedimentation and gel-filtration behaviour of human apotransferrin and its copper and iron complexes

1973 ◽  
Vol 133 (4) ◽  
pp. 749-754 ◽  
Author(s):  
Peter A. Charlwood
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Copper Binding ◽  
Metal Binding Sites ◽  
Stokes Radius ◽  
Effect Of Copper ◽  
Specific Metal

Equilibrium-dialysis experiments showed that Tris or citrate in the solution prevented copper from occupying completely the specific metal-binding sites on human transferrin. Differential measurements of sedimentation velocity under conditions where two atoms of copper per molecule of protein were bound showed an increase in s020,w, relative to that of the apoprotein, practically the same as that produced by two atoms of iron. Gel-filtration experiments made under the same conditions to investigate the effect of copper binding on the Stokes radius of the protein showed merely that it lost most of the metal as it passed down the column.

Equilibrium dialysis, ultrafiltration, and ultracentrifugation compared for determining the plasma-protein-binding characteristics of valproic acid.

1985 ◽  
Vol 31 (1) ◽  
pp. 60-64 ◽  
Author(s):  
J Barré ◽  
J M Chamouard ◽  
G Houin ◽  
J P Tillement
Keyword(s):  
Valproic Acid ◽  
Human Serum Albumin ◽  
Binding Sites ◽  
Plasma Proteins ◽  
Equilibrium Dialysis ◽  
Anticonvulsant Drug ◽  
Drug Binding ◽  
Binding Characteristics ◽  
Affinity Constants ◽  
Number Of Binding Sites

Abstract Equilibrium dialysis, ultrafiltration, and ultracentrifugation were compared to determine their reliability and applicability in the study of binding of an anticonvulsant drug, valproic acid, by plasma proteins. We studied drug binding with pooled serum and with solutions of human serum albumin at physiological concentrations. We compared binding characteristics such as number of binding sites, affinity constants, and percent of binding as measured by each method in the therapeutic range for valproic acid. Results by ultracentrifugation differed from those by equilibrium dialysis and ultrafiltration, which agreed reasonably well with each other.

Binding of Ca++ to Human Prothrombin

1975 ◽  
Author(s):  
R. Benarous ◽  
J. Elion
Keyword(s):  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Specific Property ◽  
Binding Properties ◽  
Binding Ability ◽  
Ph Variation ◽  
Maximum Value ◽  
Number Of Binding Sites ◽  
Scatchard Plots

The Ca++ binding properties of human prothrombin were studied by equilibrium dialysis using 45 calcium chloride at +4° C with prothrombin concentration of about 1 mg/ml equilibrated in 0.025 M Tris HCl, 0.12 M NaCl buffer pH 7.4. Scatchard plots obtained were similar to those described by Steenflo (1973) for bovine prothrombin, suggesting a positive cooperativity in the binding of Ca++ with a maximum ratio of bound Ca++/free Ca++ of 3 moles of Ca++ bound per mole of protein.The total number of binding sites was found to be at about 7, less than 10 to 12 found for bovine prothrombin. Ca++ binding was dependent on pH variation of the buffer with a maximum value for pH 8.5. Chemical modifications of carboxyl groups of prothrombin according to Hoare and Koshland (1967) abolished the Ca++ binding ability of the molecule confirming the essential role of these residues in this specific property of prothrombin.

Spectrometric, Thermodynamic, pH Metric and Viscometric Studies on the Binding of TEALS as Surfactant with Albumin as Biopolymer

2020 ◽  
Vol 10 (1) ◽  
pp. 47-64
Author(s):  
Shveta Acharya ◽  
Arun Kumar Sharma
Keyword(s):  
Binding Sites ◽  
Equilibrium Dialysis ◽  
Protein Molecule ◽  
Structure Characterization ◽  
Binding Interaction ◽  
Practical Applications ◽  
Egg Protein ◽  
Binding Data ◽  
Number Of Binding Sites ◽  
Lower Temperature

Background:: Since the interactions of small anions with protein are very important in their transportation and distribution processes in biological systems, it is helpful to study these interactions to understand the nature of the transportation and distribution processes. Therefore, it is aimed to study the interaction of albumin with surfactant molecule by different physical methods. Objective:: Present work attempts to work on assessing the structure, characterization of the surfactants as TEALS (tri-ethanalamine lauryl sulphate) binding sites, with albumin involved in various process of living being are discussed. Method:: The binding of surfactant TEALS to egg protein has been studied at different pH values and temperatures by spectrophotometric and equilibrium dialysis methods. The binding data were found to be pH and temperature dependent. The binding data studied by the absorbance method, were found approximately identical with those obtained from the equilibrium dialysis method. Results:: The association constants and the number of binding sites were calculated from Scatchard plots and found to be at maximum at lower pH and at lower temperature. The free energy of the combining sites was lowest at higher pH and highest at low pH. Therefore, a lower temperature and a lower pH offered more sites in the protein molecule for interaction with surfactant. The ΔG (free energies of aggregation) associated with the binding interaction of the surfactants and protein were calculated. The negative values of the ΔG confirm the feasibility of interaction between the surfactant and protein. All the observations recorded in this paper indicate that the TEALS has a good affinity of binding with egg protein and the number of binding sites is dependent on various physical and chemical factors. Conclusion:: On the basis of the results of the experiments which were conducted to examine the interaction between anionic surfactant and protein by measuring the various parameters of the solutions, it is concluded that the interaction of surfactant and protein gives an idea of fundamental understanding of the structure of surfactant-protein complex and their practical applications in every field.

COMPETITIVE BINDING OF FREE AND CONJUGATED OESTROGENS TO PLASMA PROTEINS

1969 ◽  
Vol 44 (2) ◽  
pp. 219-230 ◽  
Author(s):  
ELEONORA P. GIORGI ◽  
P. G. CROSIGNANI
Keyword(s):  
Human Plasma ◽  
Binding Sites ◽  
Plasma Proteins ◽  
Equilibrium Dialysis ◽  
Competitive Binding ◽  
Physiological Significance ◽  
Ovarian Vein ◽  
Oestrone Sulphate ◽  
Graafian Follicle ◽  
Number Of Binding Sites

SUMMARY Human plasma was dialysed against increasing concentrations of [4-14C]oestradiol-17β, [4-14C]oestrone, Na[6,7-3H]oestradiol-17β-glucuronide and Na[6,7-3H]oestrone sulphate. When the oestrogens were dialysed separately, binding to a protein with a limited number of binding sites (probably a globulin) could be shown. At comparable concentrations oestrone sulphate was bound to a greater extent than the free compounds and the glucuronide. When free and conjugated oestrogens were dialysed together a displacement of the conjugates from globulin to albumin was demonstrated. This observation was confirmed by the results of the equilibrium dialysis of plasma of ovarian vein blood obtained from a mare and a donkey after injection of [4-14C]oestradiol-17β and Na[6,7-3H]oestradiol-177β-glucuronide into a Graafian follicle of the homolateral ovary. The possible physiological significance of this competition between free and conjugated oestrogens is discussed.

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