scholarly journals The binding of binuclear platinum(II)-terpyridine complexes to DNA

1987 ◽  
Vol 242 (1) ◽  
pp. 177-183 ◽  
Author(s):  
W D McFadyen ◽  
L P Wakelin ◽  
I A G Roos ◽  
B L Hillcoat
Keyword(s):  
Optical Absorption ◽  
Base Pair ◽  
Base Composition ◽  
Association Constant ◽  
Equilibrium Dialysis ◽  
Association Constants ◽  
Ligand Molecule ◽  
Aqueous Buffer ◽  
Scatchard Plots ◽  
Polyelectrolyte Theory

The binding of platinum (II)-terpyridine complexes to DNA was studied by using equilibrium dialysis. Optical absorption methods were used to measure the ability of the ligands to aggregate in aqueous buffer. Scatchard plots for the binding of the monomeric [Pt(terpy)SC4H9]+ cation to DNA at I0.01 are curvilinear, concave upwards, suggesting two modes of binding. The association constant decreases at higher ionic strengths, consistent with polyelectrolyte theory, and 1.1 cations are released per bound ligand molecule. The association constants of the binuclear ligands [Pt(terpy)S[CH2]4S(terpy)Pt]2+ and [Pt(terpy)S[CH2]6S(terpy)Pt]2+ are 8 and 23 times larger respectively than the affinity of the monomer. For the latter binuclear derivative the increase may be ascribed to bifunctional reaction. Differential dialysis experiments with DNAs of differing base composition show that [Pt(terpy)SC4H9]+ has a requirement for a single G X C base-pair at the highest-affinity site. However, in the binuclear ligands chromophore specificity is severely compromised. Similar experiments indicate that 9-aminoacridine and selected methylene-linked diacridines show no significant sequence selectivity.

scholarly journals Interaction of phenylthiolato-(2,2′,2′′-terpyridine)platinum(II) cation with DNA

1984 ◽  
Vol 222 (1) ◽  
pp. 203-215 ◽  
Author(s):  
L P G Wakelin ◽  
W D McFadyen ◽  
A Walpole ◽  
I A G Roos
Keyword(s):  
Base Pair ◽  
Equilibrium Dialysis ◽  
Platinum Complexes ◽  
Nucleotide Composition ◽  
Contour Length ◽  
Ligand Molecule ◽  
Experimental Conditions ◽  
Kinetic Measurements ◽  
Binding Isotherms ◽  
Scatchard Plots

The interaction between a novel aromatic thiolato derivative from the family of DNA-intercalating platinum complexes, phenylthiolato-(2,2′,2″-terpyridine)platinum(II)-[PhS(ter py)Pt+], and nucleic acids was studied by using viscosity, equilibrium-dialysis and kinetic measurements. Viscosity measurements with sonicated DNA provide direct evidence for intercalation, and show that at binding ratios below 0.2 molecules per base-pair PhS(terpy)Pt+ causes an increase in contour length of 0.2 nm per bound molecule. However, helix extension diminishes at greater extents of binding, indicating the existence of additional, non-intercalated, externally bound forms of the ligand. The ability of PhS(terpy)Pt+ to aggregate in neutral aqueous buffers at a range of ionic strengths and temperatures was assessed by using optical-absorption methods. Scatchard plots for binding to calf thymus DNA at ionic strength 0.01 (corrected for dimerization) are curvilinear, concave upward, providing further evidence for two modes of binding. The association constant decreases at higher ionic strengths, in accord with the expectations of polyelectrolyte theory, although the number of cations released per bound unipositive ligand molecule is substantially greater than 1. Stopped-flow kinetic measurements confirm the complexity of the binding reaction by revealing multiple bound forms of the ligand whose kinetic processes are both fast and closely coupled. Thermal denaturation of DNA radically alters the shapes of binding isotherms and either has little effect on, or enhances, the affinity of potential binding sites, depending on experimental conditions. Scatchard plots for binding to natural DNA species with differing nucleotide composition show that the ligand has a requirement for a single G X C base-pair at the highest-affinity intercalation sites.

AFFINITY AND SPECIFICITY OF ANTISERA AGAINST HUMAN FSH AND TSH OBTAINED BY IMMUNIZING RABBITS WITH HIGHLY PURIFIED HORMONE PREPARATIONS

1975 ◽  
Vol 78 (1) ◽  
pp. 22-31 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand
Keyword(s):  
Pregnant Women ◽  
Association Constant ◽  
Association Constants ◽  
The Other ◽  
Specific Antiserum ◽  
Α Subunit ◽  
Specific Antisera ◽  
Scatchard Plots

ABSTRACT Two groups of 4 rabbits were immunized according to the method described by Vaitukaitis et al. (1971). One group was given 55 μg (900 IU) FSH/animal and the other 100 μg (0.5 IU) TSH/animal. Two highly specific antisera against FSH and one specific antiserum against TSH were obtained. The FSH antisera showed affinities towards LH and TSH of 0.13 and 0.25% respectively as compared to that of FSH. The TSH antisera showed affinities towards LH and FSH of 2.2 and 1.7 % respectively as compared to that of TSH. The specific antisera showed no or minor affinities towards the LH-α subunit and they were sufficient specific to be used in the radioimmunoassay of serum from pregnant women without pre-absorption with HCG. Scatchard plots showed the association constants of the FSH antisera to be 1.3 × 1011 and 1.2 × 1011 1/mol respectively, and the association constant of the TSH antiserum to be 6 × 1010 l/mol. All 3 antisera showed high titers.

Binding of zinc to bovine and human milk proteins

1989 ◽  
Vol 56 (2) ◽  
pp. 235-248 ◽  
Author(s):  
Harjinder Singh ◽  
Albert Flynn ◽  
Patrick F. Fox
Keyword(s):  
Ionic Strength ◽  
Binding Sites ◽  
Binding Capacity ◽  
Equilibrium Dialysis ◽  
Whey Proteins ◽  
Milk Proteins ◽  
Association Constants ◽  
Zn Concentration ◽  
Β Lactoglobulin ◽  
Scatchard Plots

SummaryZn binding by whole bovine and human casein and by purified bovine caseins and whey proteins was investigated by equilibrium dialysis. Bovine αs1 casein had the greatest Zn-binding capacity (˜ 11 atoms Zn/mol). Protein aggregation was observed as Zn concentration was increased and- the protein precipitated at a free Zn concentration of 1·7 mM. Zn binding increased with increasing pH in the range 5·4–7·0 and decreased with increasing ionic strength. Competition between Zn and Ca was observed for binding to αs1-casein indicating common binding sites for these two metals. Bovine β-casein bound up to 8 atoms Zn/ mol and precipitated at a free Zn concentration of ˜ 2·5 mM, while K-casein bound 1–2 atoms Zn/mol. Whole bovine and human casein bound 5–8 atoms Zn/mol and precipitated at a free Zn concentration of ˜ 2·0 mM. Scatchard plots for Zn binding to caseins showed upward convexity, possibly due to Zn-induced association of caseins. Apparent average association constants (K¯app) for all caseins were similar (log K¯app 3·0–3·2). Enzymic dephosphorylation of αs1- or whole bovine casein markedly reduced, but did not eliminate, Zn binding. Thus, phosphoserine residues appeared to be the primary Zn-binding sites in caseins. With the exception of bovine serum albumin. which bound over 8 atoms Zn/mol, the bovine whey proteins, β-lactoglobulin, α-lactalbumin and lactotransferrin, had little capacity for Zn binding.

scholarly journals Interaction of rice (Oryza sativa) lectin with N-acetylglucosaminides. Fluorescence studies

1985 ◽  
Vol 229 (3) ◽  
pp. 687-692 ◽  
Author(s):  
F Tabary ◽  
J P Frénoy
Keyword(s):  
Oryza Sativa ◽  
Binding Sites ◽  
Wheat Germ ◽  
Equilibrium Dialysis ◽  
Blue Shift ◽  
Association Constants ◽  
Fluorescence Studies ◽  
Saccharide Binding ◽  
Binding Enthalpy

The interaction of lectin isolated from rice (Oryza sativa) embryos with N-acetylglucosaminides was studied by equilibrium dialysis and fluorescence. Equilibrium dialysis with 4-methylumbelliferyl-(GlcNac)2 showed that rice lectin (Mr 38000) contains four equivalent saccharide-binding sites. Addition of the N-acetylglucosaminides GlcNac, (GlcNac)2 and (GlcNac)3 enhanced the intrinsic fluorescence of rice lectin and this was accompanied by a 10nm blue-shift of its maximum fluorescence with (GlcNac)2 and (GlcNac)3. These changes in intensity allowed determination of the association constants, which increased with the number of saccharide units: at 20 degrees C, Ka = (1.3 +/- 0.1) X 10(3), (5.1 +/- 0.4) X 10(4) and (2.6 +/- 0.1) X 10(5) M−1 for GlcNac, (GlcNac)2 and (GlcNac)3 respectively. The binding enthalpy, delta H0, for the three glucosaminides were very low and ranged from −12.1 to −20.6 kJ X mol-1. The results are compared with those obtained with wheat-germ agglutinin, another GlcNac-specific gramineaous lectin.

Water Proton Relaxation Rate Enhancements and Association Constants for Mn(II) to Serum Proteins Determined by NMR T1 Measurements

2005 ◽  
Vol 60 (9-10) ◽  
pp. 807-812 ◽  
Author(s):  
Hatice Budak
Keyword(s):  
Serum Albumin ◽  
Human Serum ◽  
Relaxation Rate ◽  
Association Constant ◽  
Serum Proteins ◽  
Association Constants ◽  
Water Proton ◽  
Proton Relaxation ◽  
Rate Enhancement ◽  
Human Serum Proteins

Abstract The water proton relaxation rate enhancement of Mn(II) bound to bovine serum albumin (BSA) and the association constant for manganese to BSA have already been determined, but such determinations have not been done for human serum albumin (HSA) and other human serum proteins and also for human serum. In this work, NMR T1 values in aqueous solutions of serum proteins and serum were measured versus increasing concentration of Mn(II). Proton relaxation rate enhancements (ε*) caused by different manganese concentrations were determined for each solution and 1/ε* was fitted against concentrations of Mn(II). Proton relaxation rate enhancements (εb) of Mn(II) bound to albumin, γ-globulin, (α+β)- globulins and serum were found to be 13.69, 3.09, 8.62, and 10.87, respectively. Free and bound manganese fractions, resulted from each addition of Mn(II) to the sample, were determined by using corresponding (ε*) and the εb values. Association constants for Mn(II) to HSA and γ-globulin were calculated as 1.84 x 104 ᴍ-1 and 2.35 x 104 ᴍ-1, respectively. Present data suggest that the proton relaxation rate enhancement of Mn(II) in serum is caused by Mn(II) bound to various serum constituents. Data also suggest that association constants for Mn(II) to γ-globulin are nearly the same as that to HSA.

Effect of anti-thyroxin autoantibodies on radioimmunoassay of free thyroxin in serum.

1982 ◽  
Vol 28 (6) ◽  
pp. 1389-1391 ◽  
Author(s):  
J Konishi ◽  
Y Iida ◽  
T Kousaka ◽  
K Ikekubo ◽  
T Nakagawa ◽  
...  
Keyword(s):  
Elderly Patient ◽  
Association Constant ◽  
Binding Capacity ◽  
Equilibrium Dialysis ◽  
Graves Disease ◽  
The Third ◽  
Commercial Kits

Abstract Serum thyroxin was nearly or completely undetectable by radioimmunoassay in an elderly patient with Graves' disease being treated with methimazole. Abnormal binding of thyroxin to antibodies of the IgG variety was shown, the association constant of the complex being 8.0 x 10(8) L/mol and the binding capacity 6.3 nmol/g of IgG. The effect of the antibody on results of radioimmunoassay of free thyroxin was studied with three commercial kits, two of which (Clinical Assays and Damon Diagnostics) gave essentially the same values as did equilibrium dialysis. The third (Amersham International) gave falsely high results because the 125I-labeled thyroxin derivative used in the kit was bound by the autoantibody.

Hydrolysis of aryl hydrogen maleate esters mediated by cyclodextrins — Effect on the intramolecular catalysis

2005 ◽  
Vol 83 (9) ◽  
pp. 1281-1286 ◽  
Author(s):  
Gabriel O Andrés ◽  
O Fernando Silva ◽  
Rita H de Rossi
Keyword(s):  
Maleic Anhydride ◽  
Transition State ◽  
Rate Constant ◽  
Association Constant ◽  
Kinetic Studies ◽  
Association Constants ◽  
Bulk Solution ◽  
Intramolecular Catalysis ◽  
Hydrolysis Of ◽  
Nonlinear Fashion

Kinetic studies of the hydrolysis of Z-aryl hydrogen maleates (Z = H, p-CH3, m-CH3, p-Cl, m-Cl) were carried out in the presence and absence of hydroxypropyl-β-cyclodextrin (HPCD) at variable pH from 1.00 to 3.00. The reaction involves the formation of maleic anhydride as an intermediate and the rate of its formation is strongly dependent on the pH. This is because the neighboring carboxylate group is a better catalyst than the carboxylic group. The rate constant for the formation of maleic anhydride decreases as the HPCD concentration increases in a nonlinear fashion. The results were interpreted in terms of the formation of a 1:1 inclusion complex of the esters with HPCD. The neutral (HA) and anionic (A) species of the substrate have different association constants (K[Formula: see text] and K[Formula: see text]). In all cases studied, K[Formula: see text] is higher than K[Formula: see text] for the same substrate. This difference is responsible for a decrease in the amount of the anionic substrate (reactive species) in the presence of HPCD, which results in a diminution of the observed rate constant. Besides, the rate constant for the reaction of the complexed substrate is smaller than that in the bulk solution indicating that the transition state of the cyclodextrin mediated reaction is less stabilized than the anionic substrate. The values of ΔΔG‡ are almost independent of the substituent on the aryl ring and range within 0.48 and 1.05 kcal mol–1 (1 cal = 4.184 J). There is no correlation between KTS and the association constant of the substrate indicating that the factors stabilizing the transition state are different from those that stabilize the substrate. Key words: cyclodextrins, intramolecular catalysis, hydrolysis, inhibition.

scholarly journals The interactions of haem with ligandin and aminoazo-dye-binding protein A.

1976 ◽  
Vol 157 (2) ◽  
pp. 461-467 ◽  
Author(s):  
E Tipping ◽  
B Ketterer ◽  
L Christodoulides ◽  
G Enderby
Keyword(s):  
Association Constant ◽  
Protein Complexes ◽  
Binding Protein ◽  
Protein A ◽  
Association Constants ◽  
Dye Binding ◽  
Cotton Effects ◽  
Haem Protein ◽  
Haem Iron ◽  
Soret Region

1. The interactions of ferriprotoporphyrin IX with ligandin and aminoazo-dye-binding protein A result in absorption spectra in the Soret region characteristic of the ligand in its monomeric state. 2. Both proteins are able to bind ferrous as well as ferric haem. 3. Ferriprotoporphyrin IX is bound at a single site on both proteins. At pH7.0, I 0.16M, difference-spectrophotometric measurements gave association constants of 10(7) and 4 × 10(6) LITRE/MOL FOR LIGANDIN AND PROTEin A respectively. Under the same conditions fluorescence-quenching experiments gave an association constant of 2 × 10(7) litre/mol for ligandin. 4. Bilirubin, bromosulphophthalein and oesterone sulphate each compete with haem for binding by the two proteins. 5. Ferriprotoporphyrin IX bound to both ligandin and protein A is able to form co-ordination complexes with CN-, but not, to any measurable extent, with either N3- or F-. From these results it is suggested that binding by the two proteins may not involve the haem iron atom. 6. Both haem-protein complexes give rise to measurable extrinsic Cotton effects in the Soret region. 7. The formation and properties of the ligandin- and protein A-haem complexes are compared with those of haem-albumin, haemoglobin, myoglobin and other haemoproteins.

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.

Identification and glucan-binding properties of a new carbohydrate-binding module family

2001 ◽  
Vol 361 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Alisdair B. BORASTON ◽  
Mazyar GHAFFARI ◽  
R. Antony J. WARREN ◽  
Douglas G. KILBURN
Keyword(s):  
Association Constant ◽  
Association Constants ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Bacillus Sp ◽  
Binding Properties ◽  
Alkalophilic Bacillus ◽  
Binding Subsites

The C-terminal 191-residue module of Cel5A from the alkalophilic Bacillus sp. 1139 comprises a carbohydrate-binding module (CBM) belonging to a previously unidentified family that we have classified as CBM family 28. This example, called CBM28, bound specifically to cello-oligosaccharides and mixed β-(1,3)(1,4)-glucans (barley β-glucan) with association constants of approximately (1–4)×104 M−1. Its binding to barley β-glucan was remarkably insensitive to pH between 7.0 and 10.9, in keeping with its alkalophilic source. CBM28 bound to cellulose having a significant non-crystalline content with an association constant similar to that for its binding to soluble glucans. CBM17 (CBM family 17) and CBM28 modules naturally occur as tandems. The CBM17/CBM28 tandem from Cel5A bound with apparent co-operativity to barley β-glucan. The association of CBM28 with cello-oligosaccharides was driven enthalpically and marked by the different thermodynamic contribution of three putative binding subsites that accommodate a cellohexaose molecule.

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