Scatchard Plot and Heterogeneity in Binding Affinity of Labeled and Unlabeled Ligand

1975 ◽  
Vol 21 (12) ◽  
pp. 1769-1773 ◽  
Author(s):  
H J G Hollemans ◽  
R M Bertina
Keyword(s):  
Binding Affinity ◽  
Binding Sites ◽  
Mathematical Expression ◽  
Molar Concentration ◽  
Scatchard Plot ◽  
Affinity Constant

Abstract In saturation analysis the Scatchard plot is a generally accepted method for calculation of the affinity constant, K, and the molar concentration, q, of the binder. However, in a system where the K's for the labeled and unlabeled ligand are unequal, a nonlinear plot can be obtained from which incorrect values for K and q may be calculated. This paper mathematically explains how the plot may deviate and under which conditions there will be a maximum in the curve. When the binding sites are homogeneous, the coordinates of this maximum can be used to calculate K and q. A general mathematical expression is derived on the basis of which a linear curve can be constructed for calculation of q and K, which is valid even when affinity for the labeled and unlabeled ligand is not identical.

MEASUREMENT OF TESTOSTERONE BINDING SITES

1970 ◽  
Vol 65 (1_Suppl) ◽  
pp. S225-S238 ◽  
Author(s):  
William H. Pearlman
Keyword(s):  
Serum Concentration ◽  
Binding Affinity ◽  
Adult Male ◽  
Binding Sites ◽  
Late Pregnancy ◽  
Molar Concentration ◽  
Total Testosterone ◽  
Model Calculations ◽  
Testosterone Concentration ◽  
Binding Component

ABSTRACT A Sephadex assay technique, in use in our laboratory for the study of steroid-protein interactions, allows expression of the molar concentration of a testosterone-binding component of human serum of high binding affinity in terms of the molar concentration of testosterone-binding sites: examples with model calculations are provided. The presence in the assay system of competing protein of low binding affinity (i. e. albumin) and of competing steroid (i. e. endogenous, radioinert testosterone) is taken into account when estimating the testosterone-binding component. With the aid of some approximations, the distribution of bound testosterone between the testosterone-binding component and albumin is readily calculated. From this estimate and the total testosterone concentration, the serum concentration of unbound testosterone (Su) is calculated. The concentration of the testosterone-binding component is thus estimated to be of the order of 8 × 10−7M and 1 × 10−7M in late pregnancy serum and adult male serum, respectively, assuming that there is only one binding site for testosterone in the protein molecule. The testosteronebinding component appears to play a role at least equal to that of albumin in binding endogenous testosterone in adult male serum; the testosterone-binding component is clearly dominant in this role in late pregnancy serum. The values for Su at 25° are calculated to be of the order of 3 × 10−10M and 1 × 10−11M in adult male serum and late pregnancy serum, respectively, corresponding to 1.7% and 0.27% of the total testosterone concentration, respectively. It is suggested that the physiologically effective level of circulating androgen may correlate better with the serum concentration of unbound testosterone (Su) than with that of total testosterone.

OESTROGEN RECEPTORS AND METABOLIC ACTIVITY IN THE GENITAL TRACT AFTER OVARIECTOMY OF EWES WITH PERMANENT INFERTILITY CAUSED BY EXPOSURE TO PHYTO-OESTROGENS

1981 ◽  
Vol 89 (3) ◽  
pp. 365-370 ◽  
Author(s):  
B. Y. TANG ◽  
N. R. ADAMS
Keyword(s):  
Binding Affinity ◽  
Metabolic Activity ◽  
Binding Sites ◽  
Genital Tract ◽  
Vaginal Epithelium ◽  
Affinity Constant ◽  
Oestrogen Receptors ◽  
Glycoprotein Synthesis

Characteristics of the uterus and cervix after ovariectomy of ewes with permanent phyto-oestrogenic infertility (PPI) were compared with controls. Ewes with PPI had more oestrogen-binding sites in the cervix, but not in the uterus. There was no difference between the two groups of ewes in the binding affinity constant of receptors from the uterus or cervix. There were more keratinized cells in the vaginal epithelium of ewes with PPI, and the rates of protein and glycoprotein synthesis in the uterus and cervix were higher in ewes with PPI. These results offer further evidence that PPI in adult ewes is similar to the 'persistent oestrus' syndrome in rodents oestrogenized neonatally.

scholarly journals Low-affinity transcription factor binding sites shape morphogen responses and enhancer evolution

2013 ◽  
Vol 368 (1632) ◽  
pp. 20130018 ◽  
Author(s):  
Andrea I. Ramos ◽  
Scott Barolo
Keyword(s):  
Transcription Factor ◽  
Binding Affinity ◽  
Binding Sites ◽  
Target Genes ◽  
Gene Activation ◽  
Transcriptional Response ◽  
Morphogen Gradients ◽  
Weak Binding ◽  
Initial Hypothesis

In the era of functional genomics, the role of transcription factor (TF)–DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. Here, we investigate the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients . We noted that enhancers of several Hh-responsive Drosophila genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to our initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of dpp , wingless and stripe , by transplanting optimal sites from the patched gene, did not result in ectopic responses to Hh signalling. Instead, we found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, we observed patterning defects in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. We propose that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes.

Interaction of epidermal growth factor with receptors on human and porcine thyroid membranes

1984 ◽  
Vol 102 (1) ◽  
pp. 57-61 ◽  
Author(s):  
H. Humphries ◽  
S. MacNeil ◽  
D. S. Munro ◽  
S. Tomlinson
Keyword(s):  
Enzyme Activity ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Sites ◽  
Affinity Constant ◽  
Maximal Inhibition ◽  
High Affinity ◽  
Epidermal Growth ◽  
And Function ◽  
Bovine Tsh

ABSTRACT Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1·4 × 109 l/mol); the density of binding sites was low compared with the TSH receptor. At 37 °C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0·5 nmol/l) using 0·5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity. J. Endocr. (1984) 102, 57–61

scholarly journals Surface Epitope Coverage Affects Binding Characteristics of Bisphenol-A Functionalized Nanoparticles in a Competitive Inhibition Assay

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Yang Lu ◽  
Joshua Richard Peterson ◽  
Erwann Luais ◽  
John Justin Gooding ◽  
Nanju Alice Lee
Keyword(s):  
Bisphenol A ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Limit Of Detection ◽  
Molar Concentration ◽  
Inhibition Assay ◽  
Binding Characteristics ◽  
Sensor Performance ◽  
Reliable Detection ◽  
Minimum Number

The biomolecule interface is a key element in immunosensor fabrication, which can greatly influence the sensor performance. This paper explores the effects of surface epitope coverage of small molecule functionalized nanoparticle on the apparent affinity (avidity) of antibody in a competitive inhibition assay using bisphenol-A (BPA) as a model target. An unconventional two-antibody competitive inhibition ELISA (ci-ELISA) using thiolated BPA modified gold nanoparticles (cysBPAv-AuNP) as a competing reagent was devised for this study. It was shown that the antibody complexation with cysBPAv-AuNPs required a minimum number of surface epitopes on the nanoparticle to form a sufficiently strong interaction and reliable detection. The binding of cysBPAv-AuNP to anti-BPA antibodies, for limited antibody binding sites, was enhanced by a greater number of epitope-modified nanoparticles (cysBPAv-AuNP) as well as with higher epitope coverage. Increasing the molar concentration of epitope present in an assay enhanced the binding between anti-BPA antibodies and cysBPAv-AuNP. This implies that, to increase the limit of detection of a competitive inhibition assay, a reduced molar concentration of epitope should be applied. This could be achieved by either lowering the epitope coverage on each cysBPAv-AuNP or the assay molar concentration of cysBPAv-AuNP or both of these factors.

Supramolecular self-assemblies of inverted cucurbit[7]uril with biogenic amines

2019 ◽  
Vol 43 (1) ◽  
pp. 407-412
Author(s):  
Pei-Hui Shan ◽  
Zhi-Rui Zhang ◽  
Dong Bai ◽  
Bing Bian ◽  
Zhu Tao ◽  
...  
Keyword(s):  
Biogenic Amines ◽  
Binding Affinity ◽  
Biogenic Amine ◽  
Binding Sites ◽  
Experimental Results ◽  
Binding Interactions ◽  
Strong Binding

The binding interactions between six biogenic amine guests and the iQ[7] host were investigated. The experimental results have revealed that iQ[7] shows strong binding affinity towards five of the studied biogenic amines, but not histamine, and that the binding sites are different depending on the structure of the biogenic amine.

Platelet 3H-imipramine binding in major depressive episodes

1991 ◽  
Vol 6 (2) ◽  
pp. 73-78
Author(s):  
G Faludi ◽  
K Tekes
Keyword(s):  
Clinical Practice ◽  
Binding Sites ◽  
Biological Marker ◽  
Affinity Constant ◽  
Major Depressive ◽  
State Dependent ◽  
Imipramine Binding ◽  
Major Depressive Episodes ◽  
Depressive Episodes ◽  
Age And Sex

SummaryDensity (Bmax) and affinity constant (Kd) values of tritiated imipramine binding sites were determined on platelets from patients suffering from major depression and from healthy, age-and sex-matched controls. Significantly lower Bmax values were found in the depressed patients, while the Kd values did not differ from those of controls. The results suggest that, in accordance with data from the literature, decrease in 3H-imipramine binding sites may be used as a state-dependent biological marker of depression in clinical practice.

Enhanced testosterone secretion in adult rams after establishment of a high-frequency, low-amplitude pattern of LH pulses in the nonbreeding season occurs without changes in the number or binding affinity of testicular LH receptors

1990 ◽  
Vol 122 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Lee M. Sanford ◽  
Susan J. Baker
Keyword(s):  
Binding Affinity ◽  
Binding Sites ◽  
High Frequency ◽  
Leydig Cells ◽  
Jugular Vein ◽  
Pulse Amplitude ◽  
Nonbreeding Season ◽  
Testosterone Secretion ◽  
Low Amplitude ◽  
Amplitude Pattern

Abstract When the LH signal in the ram is changed from one of large and infrequent pulses to one of small and frequent pulses, the testes quickly become more responsive to LH and testosterone secretion is elevated, perhaps because the number and (or) binding affinity of testicular LH receptors have increased. An experiment was undertaken in the nonbreeding season (July) with 10 adult Dorset × Leicester × Suffolk rams that were about 3.5 years of age and 69 ± 2 kg in body weight. Rams were given injections into the jugular vein of either 5 μg NIH-LH-S24 (in 1 ml saline) or vehicle every 80 min for 6 days. LH treament produced a series of LH pulses that occurred three times more frequently and were 70% less in amplitude than pulses in the control rams, without causing mean LH concentration to increase. Endogenously produced LH pulses were not evident in the treated rams after LH injection began. The modified LH-pulse pattern elevated mean testosterone concentration by 150% (assessed on days 2 and 5), and caused the cumulative testosterone response to LH pulses, estimated by multiplying testosterone-pulse amplitude by frequency per 6 h, to increase progressively by 180% (days −2 through 5). Enhanced testicular steroidogenic activity, presumably due to greater enzymatic activity and cholesterol availability within Leydig cells, was not associated with increases in either the concentration or affinity of LH-binding sites in the testis (assessed on days 3 and 6).

TRIIODOTHYRONINE BINDING TO LYMPHOCYTES FROM EUTHYROID SUBJECTS AND A PATIENT WITH PERIPHERAL RESISTANCE TO THYROID HORMONE

1976 ◽  
Vol 83 (1) ◽  
pp. 64-70 ◽  
Author(s):  
Kristian Liewendahl
Keyword(s):  
Thyroid Hormone ◽  
Binding Sites ◽  
Binding Capacity ◽  
Human Lymphocytes ◽  
Peripheral Resistance ◽  
Subsequent Treatment ◽  
Affinity Constant ◽  
Prolonged Incubation ◽  
Resistance To Thyroid Hormone ◽  
The Mean

ABSTRACT Triiodothyronine (T3) binding to Ficoll-Isopaque purified human lymphocytes was studied. During incubation of lymphocytes with [125I]T3 in a calcium-free medium at 37°C, maximal uptake of T3 in nuclei occurred after 2 h and declined after prolonged incubation. Incubation of lymphocytes with T3 concentrations ranging from 1 × 10−11 to 1 × 10−9 mol/l and subsequent treatment with Triton X-100 to strip off [125I]T3 bound with low affinity was used for the estimation of affinity and capacity of nuclear T3 binding sites. The mean equilibrium affinity constant (Ka) estimated with the Scatchard method in 11 euthyroid healthy subjects was 4.5 × 109 1/mol, and the mean maximal binding capacity 25 × 10−15 mol/100 μg DNA. In a female patient with peripheral resistance to thyroid hormone action, the estimated Ka was 3.5 × 109 1/mol and the number of T3 binding sites 37 × 10−15 mol/100 μg DNA. Although not statistically different from the mean value in euthyroid subjects, this Ka value was outside the range of control values observed and was considered presumptive evidence that the nuclear T3 receptors in this patient have abnormally low affinity for its ligand. The nuclear T3 binding capacity in this patient was significantly increased.

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