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1.
Behafarid Ghalandari Atefeh Poursoleiman Mina Fekri Ali Komeili Adeleh Divsalar Mahboube Eslami Moghadam 《Journal of biomolecular structure & dynamics》2019,37(13):3422-3433
To perform biological evaluations of newly-designed Pt(II) and Pd(II) complexes, the present study was conducted with targeted protein human serum albumin (HSA) and HCT116 cell line as model of human colorectal carcinoma. The binding of Pt(II) and Pd(II) complexes to HSA was analyzed using fluorescence spectroscopy and molecular docking. The thermal stability and alterations in the secondary structure of HSA in the presence of Pt(II) and Pd(II) complexes were investigated using the thermal denaturation method and circular dichroism (CD) spectroscopy. The cytotoxicity of the Pt(II) and Pd(II) complexes was studied against the HCT116 cell line using MTT assay. The binding analysis revealed that the fluorescence findings were well in agreement with docking results such that there is only one binding site for each complex on HSA. Binding constants of 8.7?×?103 M?1, 2.65?×?103 M?1, 0.3?×?103 M?1, and 4.4?×?103 M?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 25?°C, respectively. Also, binding constants of 1.9?×?103 M?1, 15.17?×?103 M?1, 1.9?×?103 M?1, and 13.1?×?103 M?1 were determined for Pd(II) and Pt(II) complexes (I–IV) at temperature of 37?°C, respectively. The results of CD and thermal denaturation showed that the molecular structure of HSA affected by interaction with Pt(II) and Pd(II) complexes is stable. Cytotoxicity studies represented the growth suppression effect of the Pt(II) and Pd(II) complexes toward the human colorectal carcinoma cell line. Therefore, the results suggest that the new designed Pt(II) and Pd(II) complexes are well promising candidates for use in cancer treatment, particularly for human colorectal cancer.
Communicated by Ramaswamy H. Sarma 相似文献
2.
Katerina Duskova Sara Sierra María-José Fernández Lourdes Gude Antonio Lorente 《Bioorganic & medicinal chemistry》2012,20(24):7112-7118
A series of ethylenediamine platinum(II) complexes connected through semi-rigid chains of 1,2-bis(4-pyridyl)ethane to DNA intercalating subunits (naphthalene, anthracene or phenazine) has been synthesized, and their interactions with calf thymus (CT) DNA have been evaluated by viscometric titrations and equilibrium dialysis experiments. The parent ligands that contain anthracene or phenazine chromophores showed a monointercalative mode of DNA interaction (especially the anthracene derivative), with apparent association constants in the order of 104 M?1. The corresponding platinum(II) complexes bind CT DNA through bisintercalation, as established by the significant increase of DNA contour length inferred from viscosity measurements, and the association constants are in the order of 105 M?1. The naphthalene derivatives, however, exhibit a mixed mode of interaction, which suggests a partial contribution of both intercalation and groove binding for the ligand, and monointercalation in the case of the platinum(II) complex. Competition dialysis experiments carried out on the intercalative compounds have revealed a moderate selectivity towards GC DNA sequences for the derivatives containing the anthracene chromophore. 相似文献
3.
In acidic media, ibuprofen substantially enhanced the weak chemiluminescence (CL) produced by sodium sulfite and potassium permanganate. The increased signals were linearly correlated with ibuprofen concentrations ranging from 1.2 × 10‐3 to 4.8 μM, with a detection limit of 4.8 × 10‐4 μM. Two ultrafiltration (UF) membranes were used to construct a unit for trapping 0.15 and 0.75 μM human serum albumin (HSA) and coupled online with the CL system. At low HSA concentrations, the numbers of bound molecules per binding site were calculated to be 0.9 for Sudlow site I and 6.2 for Sudlow site II. The association constants on these binding sites were 5.9 × 105 and 3.4 × 104 M‐1, respectively. Our CL–UF protocol presents a rapid and sensitive method for studies on drug–protein interaction. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
4.
M.I. Schimerlik D.A. Malencik S.R. Anderson Y. Shalitin 《Biochemical and biophysical research communications》1982,106(4):1331-1339
Anthroylcholine was utilized as an extrinsic fluorescent probe in rapid kinetic studies of calcium dissociation from calmodulin (koff = 10 S?1) and the calmodulin-troponin I complex (koff = 6 S?1). At concentrations lower than 70 μM, the mechanism of dye binding agreed with the simple kinetic scheme in which the dye binds exclusively to the respective calcium complexes of calmodulin and calmodulin-troponin I. The sensitivity of anthroylcholine also made possible the estimation of values for the association (1.0 ± 0.8) × 108 S?1) and dissociation rate constants (2 ± 170 S?1) for troponin I binding to the calcium4-calmodulin complex. 相似文献
5.
Miral V. Lunagariya Khyati P. Thakor Bharat H. Pursuwani 《Nucleosides, nucleotides & nucleic acids》2013,32(8):455-483
AbstractSquare planar mononuclear platinum(II) complexes having general formula [Pt(Ln)Cl2], (where, Ln?=?L1–4) were synthesized with neutral bidentate heterocyclic 1,3,5-trisubstituted bipyrazole based ligands. The synthesized compounds were characterized by physicochemical method such as TGA, molar conductance, micro-elemental analysis and magnetic moment, and spectroscopic method such as, FT-IR, UV–vis, 1H NMR, 13C NMR and mass spectrometry. Biological applications of the compounds were carried out using in vitro brine shrimp lethality bioassay, in vitro antimicrobial study against five different pathogens, and cellular level cytotoxicity against Schizosaccharomyces pombe (S. Pombe) cells. Pt(II) complexes were tested for DNA interaction activities using electronic absorption titration, viscosity measurements study, fluorescence quenching technique and molecular docking assay. Binding constants (Kb) of ligands and complexes were observed in the range of 0.23–1.07?×?105?M?1 and 0.51–3.13?×?105?M?1, respectively. Pt(II) complexes (I–IV) display an excellent binding tendency to biomolecule (DNA) and possess comparatively high binding constant (Kb) values than the ligands. The DNA binding study indicate partial intercalative mode of binding in complex-DNA. The gel electrophoresis activity was carried out to examine DNA nuclease property of pUC19 plasmid DNA. 相似文献
6.
—The major toxin of black widow spider venom, α-latrotoxin, can be iodinated with 125I with hardly any loss in biological activity. The radioactive toxin could bind specifically to a dog cerebral cortex synaptosomal membrane preparation but not to a dog liver plasma membrane preparation. The bound protein could be recovered from the neuronal membrane preparation in an unchanged form. Non-specific binding was only 6–10% of the total binding. The protein nature of the presumed receptor was indicated by the complete inhibition of the binding by either heating the membrane preparation at 70°C or treating the membrane with trypsin. Pre-incubation with 2%β-mercaptoethanol also completely inhibited the binding, while 70% inhibition was observed after pre-treatment with 10m M-EDTA or EGTA. From plots of the equilibrium binding data, it could be ascertained that the binding is non-cooperative, with an apparent equilibrium dissociation constant, K1, of 1.0 nM. Kinetic data gave an apparent association rate constant of 8.2 × 105 M?1 s?1. Dissociation followed a biphasic exponential with rate constants of 1.4 × 10?3 and 5.2 × 10?5s?1 corresponding to half-lives of 8.2 min and 3.7 h. Possible schemes for the binding interaction were proposed. Based on the present results and on previous results which indicated that α-latrotoxin causes the release of all neurotransmitters and a depletion of the synaptic vesicle population in vertebrate synapses, a hypothetical mechanism of the action for the toxin was proposed, involving the binding of the toxin to a membrane protein receptor which interacts with filamentous proteins linking the synaptic vesicles to the axolemma. 相似文献
7.
The binding of 45Ca2+ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg2+ (no ATP) binding was half-maximal at 1.4 · 10?6M Ca2+ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 106 M?1) and the Hill coefficient was 1.0. In the absence of Mg2+, binding was half-maximal at 3 · 10?7 M Ca2+ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 107 and 2 · 106 M?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg2+ binding to about half of the total Ca2+ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites. 相似文献
8.
Michael W. Piepkorn David Lagunoff Gottfried Schmer 《Biochemical and biophysical research communications》1978,85(2):851-856
Heparin fractions of different molecular weights and anticoagulant activities were prepared by chromatography on protamine-Sepharose, and the association constants and stoichiometry for binding to antithrombin III were determined by measurement of enhancement of tryptophan fluorescence. A 7,900 molecular weight heparin preparation bound to antithrombin III with a stoichiometry of close to 2:1, whereas 14,300 and 21,600 molecular weight fractions bound at approximately 1:1 with the protein. Apparent association constants were 0.66 × 106 M?1 for the low molecular weight preparation and 2.89 × 106 M?1 for the high molecular weight material. Maximal fluorescence enhancement was greater with the higher molecular weight heparin. These results suggest a model of heparin-antithrombin III binding in which two sites on antithrombin III can accommodate one large heparin molecule with high affinity or two smaller molecules with low affinity. 相似文献
9.
A procedure for the isolation of highly purified sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been described using sucrose gradient centrifugation in zonal rotors. The yield of our purest fraction was 300 mg of sarcoplasmic reticulum protein using 1 kg muscle. The sarcoplasmic reticulum vesicles were relatively simple in composition. The Ca2+-pump protein accounted for most (approx. two-thirds) of the sarcoplasmic reticulum protein. Two other protein components, a Ca2+-binding protein and a M55 protein (approx. 55 000 daltons) each accounted for about 5–10% of the protein. Enrichment in the level of phosphoenzyme by the Ca2+-pump protein was regarded as an important index of the purification of sarcoplasmic reticulum vesicles. The sarcoplasmic reticulum vesicles were capable of forming 6.4 nmoles of 32P-labelled phosphoenzyme per mg protein and had a high capacity of energized Ca2+ uptake. The Ca2+-dependent formation of phosphoenzyme has been used to estimate the sarcoplasmic reticulum protein content in rabbit skeletal muscle and found to be about 2.5% of the total muscle protein.The Ca2+-pump and Ca2+-binding proteins were isolated with a purity of 90% or more by treating the purified sarcoplasmic reticulum vesicles with bile acids in the presence of salt. The solubilized Ca2+-pump protein reaggregated during dialysis together with phospholipid to form membranous vesicles which were capable of forming approx. 9 nmoles 32P-labelled phosphoenzyme per mg protein. The Ca2+-binding protein was water soluble and contained a high percentage of acidic amino acids (35% of total residues).Ca2+ binding by sarcoplasmic reticulum vesicles and by the Ca2+-pump and Ca2+-binding proteins was studied by equilibrium dialysis. Sarcoplasmic reticulum vesicles and Ca2+-pump protein contained nonspecific high-affinity Ca2+ binding sites with a capacity of 90–100 and 55–70 nmoles Ca2+ per mg protein, respectively. Both of them specifically bound 10–15 nmoles Ca2+ per mg protein. The binding constants for nonspecific and specific Ca2+ binding by both preparations were approx. 1 μM?1. The Ca2+-binding protein nonspecifically bound 900–1000 nmoles Ca2+ per mg protein with a binding constant of about 0.25 μM?1. 相似文献
10.
Ion binding constants for phosphatidylserine membranes have been derived from the variation of the surface potential of phosphatidylserine monolayers with divalent cation concentrations in the presence of various monovalent salts in the aqueous subphase. The observed surface potential data for the monolayers, analyzed by use of the Gouy-Chapman diffuse potential theory, together with a simple binding reaction formula, yield, for Ca2+, Mg2+, Na+ and (Me)4N+ binding constant values of 30 M?1, 10 M?1, 0.6 M?1 and 0.05 M?1, respectively. The effect of pH on surface potential of phosphatidylserine monolayers was found to be dependent upon ionic species other than H+ in the subphase solution. The distinction between apparent and intrinsic dissociation constants of H+ for biomolecules was made in terms of ion binding due to other ions at the same site as for H+ in biomolecules. 相似文献
11.
Dietmar Pörschke 《Biophysical chemistry》1976,4(4):383-394
The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 107 s?1, whereas the dissociation rate is 1.0 × 106 s?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg++ and Ca++ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 104 M?1 at medium ionic strengths up to 2.7 × 107 M?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg++ binding are characterised over a broad range of Mg++ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg++ and Ca++ association to the polymer is close to the limit of diffusion control (kR = 1 × 1010 to 2 × 1010 M?1 s?1). This high rate demonstrates that Mg++ and Ca++ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg++ or Ca++, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg++ and Ca++ surround the polynucleotides in the form of a mobile ion cloud without site binding. 相似文献
12.
Tetrahydropalmatine (THP) is one of the active alkaloid ingredients of Rhizoma Corydalis. THP has a chiral center, and the stereoselective pharmacokinetics and tissue distribution have been reported. The aim of the present article is to study the stereoselective protein binding of THP using equilibrium dialysis followed by HPLC‐UV analysis. The results showed that THP stereoselectively binds to human serum albumin (HSA), α1‐acid glycoprotein (AGP), and proteins in human plasma. The fraction binding of (+)‐THP was significantly higher than that of (?)‐THP, whereas such stereoselectivity was not found in rat plasma. The affinity of HSA and AGP to (+)‐THP, expressed as nKA, were 9.0 × 103 M?1 and 2.34 × 105 M?1, respectively, which were notablely higher than to (?)‐THP, with the nKA of 3.4 × 103 M?1 and 1.44 × 105 M?1, respectively. The binding site of HSA for (?)‐THP was Site I, whereas for (+)‐THP was both Site I and Site II. The F1/S variants of AGP were proved to be the key variants (?)‐ and (+)‐THP binding to both. Finally, the AGP binding drugs, such as mifepristone, were demonstrated to reduce the fraction binding of (?)‐ and (+)‐THP with pure AGP (1 mg/ml) but did not affect the fraction binding of both (?)‐ and (+)‐THP with proteins in human plasma. It can be concluded that protein binding of THP is species dependent and stereoselective, both HSA and AGP contribute to the stereoselective binding to THP enatiomers, and AGP binding drugs may not cause the drug–drug interaction on THP in healthy human plasma. Chirality, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
13.
The kinetics of uptake and retention of β-ecdysone by imaginal discs from late third instar larvae of Drosophila melanogaster correspond well with those of the first synthetic response of discs to hormone, an increase in RNA synthesis.Competition studies indicate the presence of two types of hormone binding sites, specific and non-specific. The specific sites are saturated at hormone concentrations which fully induce morphogenesis. Results are consistent with the hypothesis that analogs which induce morphogenesis at differing concentrations bind to the same sites. Experiments with the inhibitors N-ethylmaleimide, actinomycin d, and cycloheximide suggest that the binding sites are pre-existing in the cell and require functional sulfhydryl groups for binding.Specific binding, binding that is competed by excess unlabeled β-ecdysone, is saturable (70–80 nM). Kinetic rate constants for this specific binding were estimated to be ka = 1.5 × 105M?1 min?1, kd = 3 × 10?2 min?1. The equilibrium dissociation constant calculated from the kinetic rate constants was Keq = 2 × 10?7M compared to 1.7 × 10?7M β-ecdysone required to induce morphogenesis in vitro and 2.5 × 10?7M determined to be the in vivo concentration at the time of induction of morphogenesis. 相似文献
14.
Lymphocyte plasma membranes bind 45Ca2+ with three affinity sites: KAl = 4.0 . 106 M?1, KA2 = 8.5 . 104 M?1 and KA3 = 4.2 . 102 M?1, and Ca2+ binding capacities are 0.10, 1.2 and 85 nmoles Ca2+/mg protein. In the presence of 15 μg/ml ConA the Ca2+ binding constants were KA1 = 4.6 . 106 M?1, KA2 = 4.4 . 104 M?1 and KA3 = 4.2 . 102 M?1. The Ca2+ binding capacity was increased by ConA, to 0.13, 2.4 and 91 nmoles/mg protein. The Ca2+ ATPase activity of lymphocyte membranes was increased by ConA from 1 to 2 μmol P/protein × h. The 45Ca2+ uptake was stimulated by ConA and PHA to about 16 %. 相似文献
15.
The kinetics of t-[3H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acidA (GABAA) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABAA antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [3H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation kapp=koff±kon [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B1a (AVM B1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC50, 7.8 nM) followed by displacement at high concentrations (IC50 6.3 μM) of AVM B1a. AVM B1a resulted in similar biphasic effects on t- [35S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [3H]TBOB binding (IC50 125 μM DIDS) leaving the enhancement by AVM B1a unaffected. 相似文献
16.
《Animal reproduction science》1998,51(3):249-257
Receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) have been identified in porcine, rabbit, rat, and human myometrium. To determine the estrous cycle and pregnancy related changes in the receptor capacity and affinity, radioreceptor assays were performed with membrane homogenates of porcine uterine tissues. Cycling gilts were divided into four experimental groups: I (n=6), day 1–2; II (n=5), day 6–7; III (n=5), day 11–12; and IV (n=6), day 18–20 of the estrous cycle. Pregnant pigs were divided into three experimental groups: I (n=5), day 35–40; II (n=5), day 65–70; and III (n=4), day 95–105 of pregnancy. The concentrations [femtomoles/mg protein (fmol/mg protein)] and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. Receptor concentrations were highest (P<0.01) in groups II and III (19.3±2.5 and 35.8±2.1 fmol/mg protein, respectively), and was lowest in groups I and IV (5.3±1.4 and 7.5±0.7 fmol/mg protein, respectively). Receptor affinity constants (Ka) were consistent (P>0.05) throughout the estrous cycle [I, (5.1±1.5)×109; II, (3.0±0.8)×109; III, (3.2±0.9)×109; IV, 5.5±0.7×109 lm−1]. Plasma hormone concentrations of progesterone, estrogen and LH were typical of values noted at these times. During pregnancy, receptor concentrations were greatest (P<0.05) in group II (85.4±18.5 fmol/mg protein). In groups I and III receptor numbers were 10.8±2.3 and 26.7±6.6 fmol/mg protein, respectively. The Ka in group I was 10 times greater (P<0.05) than Ka in groups II and III, (I, 3.1±0.9×1010 lm−1; II, 3.4±0.3×109 lm−1; III, 3.3±1.1×109 lm−1). Plasma hormone concentrations typically found during pregnancy were noted. The function of these LH/hCG binding sites remains unknown; however, changes in receptor capacity during the estrous cycle and pregnancy support a role for modulation of the receptor by hormonal factors. 相似文献
17.
18.
Pratyusa Das Sunil Kumar Chaudhari Asmita Das Somashree Kundu 《Journal of biomolecular structure & dynamics》2019,37(6):1414-1426
Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure–activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 107 M?1 and 4.2 × 106 M?1, respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA–quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA–quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds. 相似文献
19.
The effect of magnesium ions on the parameters of the DNA helix-coil transition has been studied for the concentration range 10?6–10?1M at the ionic strengths of 10?3M Na+. Special attention has been given to the region of low ion concentrations and to the effect of polyvalent metallic impurities present in DNA. It has been shown that binding with Mg++ increases the DNA stability, the effect being observed mainly in the concentration range 10?6–10?4M. At[Mg++]>10?2M the thermal stability of DNA starts to decrease. The melting range extends to concentrations ~10?5M and then decreases to 7–8°C at the ion content of 10?3M. Asymmetry of the melting curves is observed at low ionic strengths ([Na+] = 10?3M) and [Mg++] ? 10?5M. The results, analyzed in terms of the statistical thermodynamic theory of double-stranded homopolymers melting in the presence of ligands, suggest that the effects observed might be due to the ion redistribution from denatured to native DNA. An experimental DNA–Mg++ phase diagram has been obtained which is in good agreement with the theory. It has been shown that thermal denaturation of the system may be an efficient method for determining the ion-binding constants for both native and denatured DNA. 相似文献
20.
Two simple colorimetric receptors PS and PP (thiophene and pyridine appended derivative) were prepared and their cation sensing properties were investigated. Receptors PS and PP displayed a selective colorimetric change (from colorless to orange) upon binding to Fe3+ in MeOH solution. The association constants for receptors PS–F e3+ and PP–F e3+ in MeOH were determined to be 1.15 × 106 and 4.31 × 106 M?1, respectively, using Hill plots. The detection limits of PS and PP were 490.7 ppm and 393.7 ppm, respectively. 相似文献