scholarly journals Evaluation of equilibrium constants by affinity chromatography

1974 ◽  
Vol 143 (2) ◽  
pp. 435-443 ◽  
Author(s):  
Lawrence W. Nichol ◽  
Alexander G. Ogston ◽  
Donald J. Winzor ◽  
William H. Sawyer
Keyword(s):  
Affinity Chromatography ◽  
Binding Site ◽  
Ricinus Communis ◽  
Equilibrium Constants ◽  
Quantitative Studies ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Glucose System ◽  
Matrix Bound ◽  
Sepharose 4B

Theoretical expressions are derived for affinity chromatography of systems comprising an acceptor A with one binding site for attachment to a functional group X on the column matrix and one site for interaction with a small ligand B that specifically affects its elution. From a general relationship covering all possible interactions between A, B and X simpler expressions are derived for affinity systems in which only two equilibria operate. Methods are suggested whereby these simpler systems may be characterized in terms of the two pertinent equilibrium constants and the concentration of matrix-bound constituent. The means by which the theory may be adapted to affinity chromatography of acceptors with multiple binding sites for ligand is also illustrated. Results of partition experiments on the Sephadex G-100–lysozyme–d-glucose system in acetate–chloride buffer (I=0.17m), pH5.4, are used to demonstrate the feasibility of evaluating quantitatively affinity-chromatography interactions. Values of 30m−1 and 1.2×106m−1 are obtained for the equilibrium constants for the reactions of lysozyme with glucose and Sephadex respectively, there being only an occasional binding site in the polysaccharide matrix (approximately 1 in 105 glucose residues). In a second experimental study the phytohaemagglutinin from Ricinus communis is subjected to frontal chromatography on Sepharose 4B in the presence of different concentrations of d-galactose, the results illustrating some of the difficulties and limitations that are likely to be encountered in quantitative studies of affinity-chromatographic systems.

Effects of Multiple Binding Sites on Studies of Hydrogen Bonding between Nitroxide Radicals and Solvent Molecules

1993 ◽  
Vol 58 (1) ◽  
pp. 47-52 ◽  
Author(s):  
Imad Al-Bala'a ◽  
Richard D. Bates
Keyword(s):  
Hydrogen Bonding ◽  
Magnetic Resonance ◽  
Binding Site ◽  
Binding Sites ◽  
Hyperfine Coupling Constant ◽  
Hydrogen Donor ◽  
Complex Formation Constants ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Solvent Molecules

The role of more than one binding site on a nitroxide free radical in magnetic resonance determinations of the properties of the complex formed with a hydrogen donor is examined. The expression that relates observed hyperfine couplings in EPR spectra to complex formation constants and concentrations of each species in solution becomes much more complex when multiple binding sites are present, but reduces to a simpler form when binding at the two sites occurs independently and the binding at the non-nitroxide site does not produce significant differences in the hyperfine coupling constant in the complexed radical. Effects on studies of hydrogen bonding between multiple binding site nitroxides and hydrogen donor solvent molecules by other magnetic resonance methods are potentially more extreme.

scholarly journals Binding of aldolase to actin-containing filaments. Evidence of interaction with the regulatory proteins of skeletal muscle

1980 ◽  
Vol 186 (1) ◽  
pp. 89-98 ◽  
Author(s):  
T P Walsh ◽  
D J Winzor ◽  
F M Clarke ◽  
C J Masters ◽  
D J Morton
Keyword(s):  
Skeletal Muscle ◽  
Binding Constant ◽  
Moving Boundary ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Affinity Constant ◽  
Quantitative Studies ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Troponin Complex

The interactions of aldolase with regulatory proteins of rabbit skeletal muscle were investigated by moving-boundary electrophoresis. A salt-dependent interaction of troponin, tropomyosin and the tropomyosin-troponin complex with aldolase was detected, the tropomyosin-troponin complex displaying a greater affinity for the enzyme than did either regulatory protein alone. The results indicate that aldolase possesses multiple binding sites (three or more) for these muscle proteins. Quantitative studies of the binding of aldolase to actin-containing filaments showed the interaction to be influenced markedly by the presence of these muscle regulatory proteins on the filaments. In imidazole/HCl buffer, I 0.088, pH 6.8, aldolase binds to F-actin with an affinity constant of 2 × 10(5) M-1 and a stoicheiometry of one tetrameric aldolase molecule per 14 monomeric actin units. Use of F-actin-tropomyosin as adsorbent results in a doubling of the stoicheiometry without significant change in the intrinsic association constant. With F-actin-tropomyosin-troponin a lower binding constant (6 × 10(4) M-1) but even greater stoicheiometry (4:14 actin units) are observed. The presence of Ca2+ (0.1 mM) decreases this stoicheiometry to 3:14 without affecting significantly the magnitude of the intrinsic binding constant.

scholarly journals Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

2009 ◽  
Vol 76 (3) ◽  
pp. 860-865 ◽  
Author(s):  
Mohammad Tofazzal Hossain Howlader ◽  
Yasuhiro Kagawa ◽  
Ai Miyakawa ◽  
Ayaka Yamamoto ◽  
Tetsuya Taniguchi ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Binding Site ◽  
Receptor Binding ◽  
Significant Loss ◽  
Target Tissue ◽  
Receptor Binding Site ◽  
Multiple Binding Sites ◽  
Mosquitocidal Activity ◽  
Multiple Binding ◽  
Loop 2

ABSTRACT Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.

A lactate dehydrogenase-immunoglobulin G1 complex, not blocked by anti-idiotype antibody, in a patient with IgG1-lambda type M-proteinemia.

1987 ◽  
Vol 33 (8) ◽  
pp. 1478-1483 ◽  
Author(s):  
K Fujita ◽  
I Sakurabayashi ◽  
M Kusanagi ◽  
T Kawai
Keyword(s):  
Lactate Dehydrogenase ◽  
Complex Formation ◽  
Affinity Chromatography ◽  
Binding Site ◽  
Binding Capacity ◽  
Isoenzyme Pattern ◽  
M Protein ◽  
Light Chains ◽  
Ldh Isoenzymes ◽  
Sepharose 4B

Abstract The serum of a patient with IgG1-lambda type M-proteinemia showed an abnormal isoenzyme pattern for lactate dehydrogenase (LDH, EC 1.1.1.27). By affinity chromatography, we showed that four isoenzymes (LDH2, LDH3, LDH4, and LDH5) were bound to the M-protein. This complex formation was not blocked by anti-idiotype antibody, even though the binding capacity of IgG was exclusively located in the Fab region of the molecule. Moreover, heavy and light chains of the patient's IgG, obtained by reduction, separately had affinities for each of the LDH isoenzymes. LDH-IgG complex was easily dissociated by affinity chromatography on 5'-AMP-Sepharose 4B or by added NADH. We propose the following hypothesis for the LDH-IgG complex formation: LDH can recognize the gamma-Fab region of IgG at the NAD+ binding site of the molecule, but the affinity of the LDH molecule for immunoglobulin is much weaker than that for NADH or 5'-AMP.

scholarly journals Hidden partners: Using cross-docking calculations to predict binding sites for proteins with multiple interactions

2018 ◽  
Author(s):  
Nathalie Lagarde ◽  
Alessandra Carbone ◽  
Sophie Sacquin-Mora
Keyword(s):  
Nucleic Acid ◽  
Binding Site ◽  
Protein Interactions ◽  
Binding Sites ◽  
Protein Protein Interactions ◽  
Protein Binding Sites ◽  
Cross Docking ◽  
Multiple Binding Sites ◽  
Multiple Binding ◽  
Docking Calculations

AbstractProtein-protein interactions control a large range of biological processes and their identification is essential to understand the underlying biological mechanisms. To complement experimental approaches, in silico methods are available to investigate protein-protein interactions. Cross-docking methods, in particular, can be used to predict protein binding sites. However, proteins can interact with numerous partners and can present multiple binding sites on their surface, which may alter the binding site prediction quality. We evaluate the binding site predictions obtained using complete cross-docking simulations of 358 proteins with two different scoring schemes accounting for multiple binding sites. Despite overall good binding site prediction performances, 68 cases were still associated with very low prediction quality, presenting individual area under the specificity-sensitivity ROC curve (AUC) values below the random AUC threshold of 0.5, since cross-docking calculations can lead to the identification of alternate protein binding sites (that are different from the reference experimental sites). For the large majority of these proteins, we show that the predicted alternate binding sites correspond to interaction sites with hidden partners, i.e. partners not included in the original cross-docking dataset. Among those new partners, we find proteins, but also nucleic acid molecules. Finally, for proteins with multiple binding sites on their surface, we investigated the structural determinants associated with the binding sites the most targeted by the docking partners.AbbreviationsANOVA: ANalysis Of Variance; AUC: Area Under the Curve; Best Interface: BI; CAPRI: Critical Assessment of Prediction of Interactions; CC-D: Complete Cross-Docking; DNA: DesoxyriboNucleic Acid; FDR: False Discovery Rate; FRIres(type): Fraction of each Residue type in the Interface; FP: False Positives; GI: Global Interface; HCMD: Help Cure Muscular Dystrophy; JET: Joint Evolutionary Tree; MAXDo: Molecular Association via Cross Docking; NAI: Nucleic Acid Interface; NPV: Negative Predicted Value; PDB: Protein Data Bank; PIP: Protein Interface Propensity; PiQSi: Protein Quaternary Structure investigation; PPIs: Protein-Protein Interactions; PPV: Positive Predicted Value; Prec.: Precision; PrimI: Primary Interface; RNA: RiboNucleic Acid; ROC: Receiver Operating Characteristic; SecI: Secondary Interface; Sen.: Sensitivity; Spe.: Specificity; TN: True Negatives; TP: True Positives; WCG: World Community Grid.

scholarly journals Possible multiple binding sites for o-aminophenol on uridine diphosphate glucuronyltransferase

1977 ◽  
Vol 163 (1) ◽  
pp. 125-131 ◽  
Author(s):  
R D Howland ◽  
L D Bohm
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Fold Increase ◽  
Prior Treatment ◽  
Uridine Diphosphate ◽  
Adult Rats ◽  
Ph Optimum ◽  
Acceptor Substrate ◽  
Multiple Binding Sites ◽  
Multiple Binding

1. Hepatic microsomal UDP-glucuronyltransferase (EC 2.4.1.17) derived from either weanling or adult rats exhibits three pH optima, at pH 5.4, 7.2 and 9.2, when o-aminophenol is the acceptor substrate, whereas p-nitrophenol is the acceptor substrate only on pH optimum is observed, at pH 5.4.2. Prior treatment of rats of either age with 3-methylcholanthrene results in a 2-3-fold increase in o-aminophenol conjugation at pH 5.4 and a 6-9-fold increase at pH 9.2. At pH 7.2, the induced enzyme is 2 to 3 times more active towards o-aminophenol than the control enzyme, but no pH optimum is demonstrable. 3. o-Aminophenol conjugation at pH 5.4 and 9.2 is inhibited competitively by both p-nitrophenol and p-nitrophenyl glucuronide, suggesting that the two phenolic aglycones share the same binding site. At pH 7.2, however, p-nitrophenyl glucuronide does not inhibit o-aminophenol conjugation, suggesting that the binding site at this pH is not shared by the two phenols. These data are consistent with the existence of more than one binding site for o-aminophenol on UDP-glucuronyltransferase.

scholarly journals Ethylene Binding Site Affinity in Ripening Apples

1993 ◽  
Vol 118 (5) ◽  
pp. 609-612 ◽  
Author(s):  
Sylvia M. Blankenship ◽  
Edward C. Sisler
Keyword(s):  
Binding Site ◽  
Malus Domestica ◽  
Binding Sites ◽  
Multiple Binding Sites ◽  
Ethylene Binding ◽  
Multiple Binding ◽  
Apple Tissue ◽  
Scatchard Plots

Scatchard plots for ethylene binding in apples (Malus domestica Borkh.), which were harvested weekly for 5 weeks to include the ethylene climacteric rise, showed C50 values (concentration of ethylene needed to occupy 50% of the ethylene binding sites) of 0.10, 0.11, 0.34, 0.40, and 0.57 μl ethylene/liter-1, respectively, for each of the 5 weeks. Higher ethylene concentrations were required to saturate the binding sites during the climacteric rise than at other times. Diffusion of 14C-ethylene from the binding sites was curvilinear and did not show any indication of multiple binding sites. Ethylene was not metabolized by apple tissue.

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