共查询到20条相似文献,搜索用时 31 毫秒
1.
The behaviour of human serum albumin in the presence of three chemically distinct ligands: oxyphenylbutazone, dansylsarcosine and hemin, has been compared in buffer and in reverse micelles of isooctane, water, and either sodium bis(2-ethylhexyl)sulfosuccinate or hexadecyl trimethylammonium bromide, systems selected to mimic the membrane-water interface. Upon micellar incorporation, the dansylsarcosine-albumin complex dissociated, as evidenced by fluorescence emission spectroscopy (red shift from 485 nm to 570 nm) and by fluorescence polarization measurements. In contrast, the hemin-albumin complex remained stable in reverse micelles, as judged from the Soret absorption band at 408 nm and the molar absorption coefficient of 8.4 x 10(4) M-1 cm-1. The oxyphenylbutazone to albumin binding curves reveal that while the association constant remained unchanged (Ka approximately 1.0 x 10(5) M-1), only a fraction of the albumin molecules present reacted with the ligand. The results were unaffected by the nature and the concentration of the surfactant. These findings can be interpreted in the light of conformational changes induced in human serum albumin by the large micellar inner surface area. The blue shift of the fluorescence emission maximum from 344 nm in buffer to 327 nm in sodium bis(2-ethylhexyl)sulfosuccinate micelles and the lesser reactivity/accessibility of the fluorophore to oxidation by N-bromosuccinimide, indicate perturbations of the sole tryptophan-214 microenvironment. However, the distance between the indole residue and tyrosine-411 does not seem substantially modified by the 15% decrease affecting the alpha helices of the albumin molecule. It is proposed that the results reported herein reflect the interactions of albumin with a membrane-like interface which generates two protein subpopulations differing in their membrane-surface and ligand affinities. Overall and local conformational changes, originating from this surface-induced effect, may thus constitute a ligand-release facilitating mechanism acting at cellular membrane levels. 相似文献
2.
J Oravcová V Mlynárik S Bystricky L Soltés P Szalay L Bohácik T Trnovec 《Chirality》1991,3(5):412-417
The interaction of pirprofen enantiomers with human serum albumin (HSA) was investigated by means of high-performance liquid chromatography (HPLC), circular dichroism (CD), and 1H NMR spectroscopy. HPLC experiments indicated that both pirprofen enantiomers were bound to one class of high-affinity binding sites (n(+) = 1.91 +/- 0.13, K(+) = (4.09 +/- 0.64) x 10(5) M-1, n(-) = 2.07 +/- 0.13, K(-) = (6.56 +/- 1.35) x 10(5) M-1) together with nonspecific binding (n'K'(+) = (1.51 +/- 0.21) x 10(4) M-1, n'K'(-) = (0.88 +/- 0.13) x 10(-4) M-1). Slight stereoselectivity in specific binding was demonstrated by the difference in product n(+)K(+) = (0.77 +/- 0.08) x 10(6) M-1 vs. n(-)K(-) = (1.30 +/- 0.21) x 10(6) M-1, i.e., the ratio n(-)K(-)/n(+)K(+) = 1.7. CD measurements showed changes in the binding sites located on the aromatic amino acid side chains (a small positive band at 315 nm and a pronounced negative extrinsic Cotton effect in the region 250-280 nm). The protein remains, however, in its predominantly alpha-helical conformation. The 1H NMR difference spectra confirmed that both pirprofen enantiomers interacted with HSA specifically, most probably with site II on the albumin molecule. 相似文献
3.
From fluorescence measurements we could analyse the binding of cyclic adenosine 3',5'-monophosphate receptor protein (CRP) from Escherichia coli to its specific site on a 301 base-pair long DNA fragment containing the control region of the lactose operon. At physiological ionic strength selection of the specific site is strictly dependent on the allosteric effector cAMP, and binding of the cAMP . CRP complex to its specific site is favoured over the non-specific binding by 5 kcal/mol with Kass (specific) = 10(8) M-1 at 37 degrees C. 相似文献
4.
Characteristics of the binding of 3-carbethoxipsoralen (3CPS) to human serum albumin (HSA) and serum proteins have been studied. An electrophoretic study showed that the predominant binding protein fraction was albumin, with small binding to globulins. Binding to HSA, studied by equilibrium dialysis, is 75% and characterized by a small saturable number of binding sites (N = 0.27) with a moderate affinity constant (K = 8 X 10(4) M-1). Free fatty acids were shown to decrease 3CPS binding to HSA by a non competitive process. 相似文献
5.
The binding of rhein with human serum albumin (HSA) has been studied in detail by spectroscopic method including circular dichroism (CD), Fourier transformation infrared spectra (FT-IR), fluorescence spectra. The binding parameters for the reaction have been calculated according to Scatchard equation at different temperatures. The plots indicated that the binding of HSA to rhein at 303, 310 and 318 K is characterized by one binding site with the affinity constant K at (4.93+/-0.16)x10(5), (4.02+/-0.16)x10(5) and (2.69+/-0.16)x10(5) M-1, respectively. The secondary structure compositions of free HSA and its rhein complexes were estimated by the FT-IR spectra. FT-IR and curve-fitted results of amide I band are in good agreement with the analyses of CD spectra. Molecular Modeling method was used to calculate the interaction modes between the drug and HSA. 相似文献
6.
6-mercaptopurine (MP) conjugates of bovine gamma globulin (BGG) and human serum albumin (HSA) with increasing numbers of MP-groups per protein molecule were prepared and some physicochemical and antigenic properties studied. The electrophoretic mobility increased proportionally to the degree of substitution. In the same manner the formation of high molecular weight aggregates increased. A loss of BGG and HSA specific determinants was observed too. The physicochemical and antigenic properties of these antigen-suppressive agent-conjugates with low and medium coupling degree (to MP26-BGG and MP20-HSA) were similar to the unmodified antigens. The affinity of rabbit anti-DNP-antibodies decreased from K0 = 1,4 - 10(6) M-1 of the unmodified antibodies to 6,5 - 10(5), 3,6 - 10(5) and 3,4 - 10(5) M-1 of the antibodies conjugated with 15, 21 and 29 MP-groups per antibody molecule. The precipitation capacity of the MP-conjugated antibodies decreased to 89, 85 and 61% for the same coupling ratios. These results suggest that the upper limit of the coupling ratio of the antibodies is 20 to 1. 相似文献
7.
Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments. 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin). 相似文献
8.
9.
The binding of triton X-100 to bovine serum albumin has been shown to exhibit positive cooperativity. Subsequent equilibrium dialysis studies indicate that the binding of Triton X-100 to sheep serum albumin likewise shows positive cooperativity, the first two stepwise equilibrium constants being K1 = 1.24 X 10(4) M-1 and K2 = 1.62 X 10(4) M-1. However, the mechanism for Triton X-100 binding to human serum albumin differs in that the binding isotherm indicates the binding sites are independent and identical. In the latter case the binding is described by the Scatchard model with an equilibrium constant of K = 7.2 X 10(3) M-1. The studies were conducted at 16 degrees C in pH 7.0, I = 0.05 phosphate buffer. 相似文献
10.
Binding of a spin-labelled photoallergen to human serum albumin 总被引:1,自引:0,他引:1
The binding site for 3,3',4',5-tetrachlorosalicylanilide (T4CS), a potent photoallergen, on human serum albumin (HSA) was studied by electron spin resonance spectroscopy using a spin-labelled analogue 3,5-dichlorosalicylamido-4-(2,2,6,6-tetramethylpiperidine 1-oxyl) (DCS-TEMPO) of T4CS in the absence of ultraviolet irradiation. DCS-TEMPO bound non-covalently (K = 5.8 X 10(6) M-1) to one major binding site on HSA. This binding site could be blocked by the photochemical binding of T4CS to the protein. Limited tryptic digestion of HSA or chemical modification of its single tryptophan residue with 2-hydroxy-5-nitrobenzyl bromide was found to reduce the binding constant of the T4CS/DCS-TEMPO-binding site. These observations are in good agreement with earlier conclusions on the nature of the T4CS-binding site and suggest a location for this site close to the single tryptophan residue of the HSA molecule. 相似文献
11.
The binding of cicletanine to human serum, isolated proteins and red blood cells was studied in vitro by equilibrium dialysis. Our results show this drug is highly bound to serum (97.3%) at therapeutic levels. No saturation to the binding sites was seen. Human serum albumin was shown to mainly responsible for this binding (93.5%) with a saturable process characterized by one binding site with a moderate affinity (K = 75800 M-1) and a non saturable process with a low total affinity (nK = 6400 M-1). Like many basic lipophilic drugs, cicletanine showed a saturable binding to alpha-1-acid glycoprotein with one site and a moderate affinity (K = 38,800 M-1). Its binding to lipoproteins and red blood cells was weak and non saturable. Over the range of therapeutic concentrations, the unbound fraction in blood remains constant (3.6%). Moreover, interactions were studied using bilirubin and non esterified fatty acids at pathological concentrations and these endogenous compounds did not alter cicletanine binding human serum or to human serum albumin likewise cicletanine shared the diazepam-site on HSA but no inhibition could take place between cicletanine and the drugs sharing the same binding site in serum at therapeutic levels. 相似文献
12.
Using the antigens testosterone-17 beta-hemisuccinate and testosterone-3-(o-carboxymethyl) oxime, each coupled to bovine serum albumin, we have produced 44 monoclonal antibodies to testosterone. Of the 17 monoclonal antibodies raised against the 17 beta-linked antigen 8 showed extremely low affinity for testosterone (Ka less than or equal to 8 X 10(7) M-1) and none had an affinity greater than 5 X 10(9) M-1. Of the 27 monoclonal antibodies raised against the 3-linked antigen 2 had affinities less than 8 X 10(7) M, 7 had affinities greater than 5 X 10(9) M-1 and one had an affinity (Ka = 9 X 10(10) M-1) greater than that of a high affinity rabbit antiserum (Ka = 6 X 10(10) M-1). The affinity constant (Ka = 5 X 10(9) M-1) measured in the serum of the mouse whose spleen gave rise to the greatest number of high affinity antibodies, was significantly higher than those measured in the sera of the remaining mice (Ka = 0.7 - 3 X 10(8) M-1). The cross-reactions of the monoclonal antibodies varied widely but none showed an overall improvement in specificity when compared with the corresponding rabbit antisera. Results suggest that as well as the structure of the steroid antigen careful selection of the spleen donor facilitates the development of monoclonal antibodies with good binding characteristics. 相似文献
13.
Jiang YL 《Bioorganic & medicinal chemistry》2008,16(12):6406-6414
As one of the natural polyphenols, resveratrol possesses hydroxyl substituted trans-stilbene structure and exerts impact on health by inhibiting multiple human enzymes, such as cyclooxygenase, F1 ATPase, and tyrosinase. Resveratrol has to be bound by human serum albumin (HSA) to keep a high concentration in serum, since its solubility is low in water. To improve water solubility and bioavailability, two resveratrol aliphatic acids and their esters have been designed and synthesized. The solubilities of the resveratrol and its derivatives have been measured using a standard procedure. The two aliphatic acids showed better solubilities in pure water and phosphate buffer (pH 7). The binding affinities of resveratrol derivatives for HSA were also measured, and the drug-protein interaction mechanism was investigated using fluorescence, UV-vis, and NMR spectroscopies. Interestingly, resveratrol hexanoic acid (5) was found to be a much better ligand (K(a)=(6.70+/-0.10)x10(6) M(-1)) for HSA than resveratrol (K(a)=(1.64+/-0.07)x10(5) M(-1)), and there was 41-fold improvement for the binding affinity. It was the first time that the increase of fluorescence of resveratrol moiety was observed during the binding to HSA, suggesting that 5 should be bound tightly by HSA. The UV-vis absorption spectroscopy revealed a maximum absorption shift from 318 to 311 nm with decreasing intensity by 20% upon complexation, suggesting that the pi-pi conjugation of the stilbene structure was impaired during the binding. Although HSA was reported to have only one binding site for resveratrol, the Job's and molar ratio plots suggested that HSA should bind two molecules of 5. NMR study suggested that phenyl group (B ring) in the center of the molecule of 5 should be involved in the pi-pi stacking interactions with HSA aromatic amino acid residues. Molecular geometry calculation of 5 with Spartan software showed that the stilbene structure had two conformers, orthogonal and planar ones. The former (E=-1.432 KJ/mol) was more stable than the latter (E=-0.128 KJ/mol), suggesting that the former should be the conformer of 5 in the complexation with HSA. 相似文献
14.
Bovine serum albumin: characterization of a fatty acid binding site on the N-terminal peptic fragment using a new spin-label 总被引:1,自引:0,他引:1
J C Hsia L T Wong C T Tan S S Er S Kharouba E Balaskas D O Tinker R C Feldhoff 《Biochemistry》1984,23(25):5930-5932
A new spin-label, 4-(L-glutamo)-4'-[(1-oxy-2,2,5,5-tetramethyl-3L-pyrrolidinyl )amino]-3, 3'-dinitrodiphenyl sulfone, is shown to bind to one high-affinity binding site on bovine serum albumin (K = 5 X 10(4) M-1, n = 1). Analysis of the binding of the spin-label to the amino-terminal half (peptic fragment PB) and the carboxy-terminal half (peptic fragment PA) of BSA, and their complex (PA-PB), indicates that the spin-label binds to a long-chain fatty acid binding site located on PB. The usefulness of the novel specificity of the spin-label in characterizing this binding site is discussed. 相似文献
15.
Cannabis is the most commonly used illicit drug worldwide. Cannabis users also appear to use other psychoactive drugs more frequently than noncannabis users. Here, Δ9-tetrahydrocannabinol (THC) and diazepam binding to human serum albumin (HSA) and HSA-heme is reported. THC binds to two different binding sites of HSA (K(d1) ≤ 10(-7) M and K(d2) = 10(-3)M) without affecting diazepam binding (K(d) = 1.2 × 10(-5) M). THC binding to the high-affinity site accounts for the low free fraction of the drug in plasma. Moreover, THC increases the affinity of heme for HSA. Accordingly, the affinity of THC for HSA-heme is higher than that for HSA. THC could bind to FA2 and FA7 sites, as substantiated by docking simulations; nevertheless, the observed allosteric effect(s) suggests that the primary binding site of THC is the FA2 cleft that positively modulates heme affinity. Possibly, the HSA conformational transition(s) induced by THC binding could account for drug delivery to the liver through receptor- mediated endocytosis. 相似文献
16.
A rapid and efficient method for the synthesis of various poly‐substituted benzo[b][1,8]naphthyridines in high yield has been developed via the Friedländer condensation of 2‐aminoquinoline‐3‐carbaldehyde 1 with various alicyclic ketones in a base catalyst (aq. potassium hydroxide). A series of benzo[b][1,8]naphthyridines branched with various side‐chains and substituents were prepared with the aim of being investigated as a fluorescent agents. Electronic absorption and fluorescence properties of some representative benzonaphthyridines (3d, 5b and 21f) in homogeneous organic solvents, dioxane–water binary mixtures and in the microheterogeneous media (sodium dodecyl sulphate (SDS), cetyl trimethyl ammonium bromide (CTAB) and Triton‐X100 micelles) have been examined. A linear correlation between solvent polarity and fluorescence properties was observed. Further, the interaction of these benzonaphthyridines (3d, 5b and 21f) with human serum albumin (HSA) and bovine serum albumin (BSA) in phosphate buffer have been examined by UV‐vis absorption and fluorescence spectroscopy. The fluorescence intensity of 3d, 5b and 21f increases with the increasing HSA and BSA concentration. These benzonaphthyridines also quench the 345 nm fluorescence of BSA in phosphate buffer (λex 280 nm). These compounds have potential for use as neutral and hydrophobic fluorescence probes for examining the microenvironments in proteins, polymers, micelles, etc. Copyright © 2011 John Wiley & Sons, Ltd. 相似文献
17.
Analysis of the tryptophan fluorescence emission spectra of caldesmon and its 34 kDa C-terminal fragment indicates that all tryptophan residues are located on the surface of the molecule, accessible to solvent. All three tryptophan residues of the 34 kDa fragment and four of the five tryptophan residues of intact protein are accessible to free water, whereas one located in the N-terminal region of molecule is accessible only to bound water molecules. The temperature dependence of the fluorescence parameters indicates higher thermal stability of the 34 kDa fragment than the whole caldesmon molecule. The interaction of the 34 kDa fragment of caldesmon (like that of the intact molecule) with calmodulin is accompanied by a blue shift of the fluorescence emission maximum and an increase in the relative quantum yield. Computer-calculated binding constants show that the binding of calmodulin to the 34 kDa fragment (K = 2.5 x 10(5) M-1) is of two orders of magnitude weaker than that to intact caldesmon (K = 1.4 x 10(7) M-1). The interaction with tropomyosin results in a blue shift of the spectrum of the 34 kDa fragment, yet there is no effect on the spectrum of intact caldesmon. Binding constants of tropomyosin to caldesmon (K = 3.8 x 10(5) M-1) and its 34 kDa fragment (K = 2.3 x 10(5) M-1) are similar. Binding of calmodulin to caldesmon and to the 34 kDa fragment affects their interaction with tropomyosin. 相似文献
18.
The interaction of the coronary vasodilator dipyridamole with biological systems, protein and membranes has been studied through optical absorption and fluorescence spectroscopies. Using the analysis of the spectra and fluorescence intensity of dipyridamole (DIP) in solution, the interaction of this compound with the transport protein albumin (BSA) and with a model of cell membranes, namely micelles of lysophosphatidylcholine (L-PC), was investigated. Measurements were performed at pH 5.0 and pH 7.0 where the molecule of DIP is fully protonated and partially protonated, respectively. The quenching of fluorescence with nitroxide-stable radicals 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) as well as with acrylamide and iodide allowed the localization of the drug in the polar interface of micelles. Quenching by acrylamide and iodide in L-PC micelles demonstrated the effect of micelle protonation which increased the accessibility of iodide to the chromophore. An effective association constant was obtained both at pH 7.0 (7.5 x 10(3) M-1) and pH 5.0 (2.5 x 10(3) M-1) and a very good agreement with the proposed binding model was observed. The quantum yields of fluorescence data agree very well with the fluorescence lifetimes. The measurement of lifetimes was important to understand the kinetic data obtained from Stern-Volmer plots both of radical, acrylamide and iodide quenching of fluorescence. It was observed that, in the presence of micelles, the kq value increased for TEMPO while decreased for TEMPOL. This result, together with the vanishing solubility of DIP in saturated hydrocarbons and the preferential partition of TEMPO in micelles, suggested the localization of DIP in the polar micellar interface. This is also supported by the enhanced iodide quenching at pH 5.0, constancy of acrylamide quenching in the range of pH 7.0-5.0 and the partition of TEMPO and TEMPOL in SDS micelles. The association constant of DIP to BSA was also estimated both at pH 7.0 (2 x 10(4) M-1) and pH 5.0 (4 x 10(3) M-1). Quenching studies with nitroxide radicals, acrylamide and iodide also suggested the binding of the drug to a hydrophobic region of the protein. At pH 5.0, the protein undergo a conformational change which leads to a loosening of the overall structure so that the accessibility of the nitroxide radicals for DIP is increased at this pH. The differences in kq values at pH 7.0 and pH 5.0 suggested that at pH 7.0 the chromophore is protected in the protein site.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
19.
E Yavin 《Archives of biochemistry and biophysics》1984,230(1):129-137
The effects of human serum albumin (HSA) on the rate of dithionite reduction of iron(III)deuteroporphyrin (iron(III)Dp) have been investigated in order to further characterize the porphyrin binding site and the changes manifested in this site under various conditions. These studies were performed under pseudo-first-order conditions, and in the presence of carbon monoxide as a "trapping agent" for the reduced iron(II)porphyrin. The rate of reduction of the free iron(III)Dp in phosphate buffer at pH 7.4 follows second-order kinetics with a rate constant (4.2 X 10(9) M-1 s-1) suggestive of a diffusion-controlled process. A six-orders of magnitude decrease in the rate of reduction was observed with iron(III)Dp was complexed with HSA. This result is consistent with HSA-bound porphyrin being less accessible to the aqueous environment. Additional studies demonstrated that both pH and anions induce various alterations in the complex that are reflected in the rate of reduction of iron(III)porphyrin. 相似文献
20.
Binding equilibria for decanoate, octanoate, and hexanoate to defatted human serum albumin were investigated by dialysis exchange rate determinations in 66 mM sodium phosphate buffer, pH 7.4, 37 degrees C. The binding isotherms for decanoate and octanoate could not be fitted by the general binding equation. It was necessary to assume the presence of two albumin components, one with high affinity and one with low affinity, about 0.65 of the albumin having high binding affinity. The first stoichiometric binding constants for the high- and low-affinity albumin components were 1.1 X 10(7) and 1.4 X 10(5) M-1, respectively, for decanoate; 1.6 X 10(6) and 3.5 X 10(4) for octanoate; and 7.1 X 10(4) and 8.0 X 10(2) M-1 for hexanoate. The high-affinity albumin component binds 1 mol decanoate, 1 mol octanoate, or 2 mol hexanoate more than is bound to the low-affinity component. Chloride ions compete with the high-affinity binding of all three ligands. Albumin dimer, present in the commercial human serum albumin, has approximately the same binding properties as the monomer. Mercaptalbumin, isolated from the preparation, also consists of two proteins, with first stoichiometric binding constants 8.0 X 10(6) and 1.4 X 10(5) M-1 for decanoate, approximately 0.5 of the mercaptalbumin having high affinity. 相似文献