Affinity For Albumin Research Articles

In Vitro Study of Ketoprofen and Indapamide Used in Multidrug Therapy: 1H-NMR Analysis

ABSTRACT Interaction of ketoprofen (KP, a nonsteroidal anti-inflammatory drug) and indapamide (IDP, a thiazide-related diuretic) with human serum albumin (HSA) in low-affinity binding sites (LAS) and competition between these medicines used in multidrug therapy have been studied by proton nuclear magnetic resonance (1H-NMR) spectroscopy. The analysis of chemical shifts, Δσ (ppm), of drug proton resonances and the values of the association constant, Ka [M−1], were used to estimate the effect of IDP and KP on the KP–HSA and IDP–HSA complexes, respectively. The results showed the changes in the affinity of human serum albumin toward the drug with different mechanisms of action (KP and IDP) and the competition in the binding to HSA.

Effects of green-, yellow- and red-emitting CdTe QDs on the interaction between 7-hydroxyflavone/quercetin with human serum albumin in vitro

In this study, 7-hydroxyflavone and quercetin were studied for their affinities for human serum albumin (HSA) in the presence and absence of quantum dots (QDs). Three typical CdTe QDs with maximum emissions of 535 nm (green-emitting, G-QDs), 598 nm (yellow-emitting, Y-QDs) and 654 nm (red-emitting, R-QDs) were tested. The fluorescence intensities of HSA decreased remarkably with addition of QDs. Quercetin resulted in obvious blue-shifts of the maximum λ em of HSA from 340.8 to 332.6 nm and 7-hydroxyflavone hardly changed the maximum λ em of HSA in the absence of QDs. The extents of shifts of the maximum λ em of HSA induced by 7-hydroxyflavone or quercetin in the presence of QDs were larger than that in the absence of QDs. When 8.00 µmol/L of 7-hydroxyflavone was added, the fluorescence decreased by 68.1%, 65.7% and 71.8% in the presence of G-QDs, R-QDs and Y-GDs, respectively, from the original G-QDs, R-QDs and Y-GDs that resulted in 42%, 43% and 55%, respectively. When 8.00 µmol/L of quercetin was tested, the fluorescence decreased by 72.4%, 69.5% and 75.6% in the presence of G-QDs, R-QDs and Y-GDs, respectively. G- and R-QDs hardly affected the affinities of 7-hydroxyflavone and quercetin for HSA. However, Y-QDs decreased the affinities of 7-hydroxyflavone and quercetin for HSA by about 13.61% and 6.8%, respectively.

Exogenous albumin inhibits sorafenib-induced cytotoxicity in human cancer cell lines

Sorafenib is an orally administered multikinase inhibitor that exhibits anti-angiogenic and anti-tumor activity. Sorafenib is also known to bind to protein (>99.5%), suggesting protein binding may be involved in sorafenib pharmacokinetic variability. Albumin is a major drug-binding protein. In this study, we examined the effect of albumin on sorafenib-induced cytotoxicity using two in vitro culture cell lines, human hepatoma Huh-7 cells and androgen-independent prostate cancer PC-3 cells. The cells were cultured and incubated, and cytotoxicity was assessed. Results were confirmed by western blotting. The presence of exogenous albumin markedly blocked the sorafenib-induced cytotoxicity in the two cell lines. Albumin concentration, the change of pharmacological signal transduction as Raf-B, vascular endothelial growth factor (VEGF), and phosphorylation of MEK1/2 or ERK1/2 were found to be decreased by sorafenib. Co-incubation of warfarin, a representative coumarin anticoagulant and potent binding activity, with albumin enhanced the cytotoxic effects by sorafenib. These mechanisms depend on the high binding proper ties of sorafenib and exogenous albumin. Furthermore, we investigated the effects of endo genous albumin expression on sorafenib-induced cytotoxicity using the siRNA knockdown system or transfected expression vector assay. However, the cytotoxic effects by sorafenib showed little change either with the knockdown or overexpression of albumin. Our results suggest that particular care should be taken with albuminemia or the combined use of drugs with a high affinity for albumin, such as warfarin, and sorafenib in the treatment of cancer patients. Our findings may be useful to the cancer therapeutic strategy by sorafenib.

Impaired drug-binding capacities of in vitro and in vivo glycated albumin

Albumin, the major circulating protein in blood, can undergo increased glycation in diabetes. One of the main properties of this plasma protein is its strong affinity to bind many therapeutic drugs, including warfarin and ketoprofen. In this study, we investigated whether or not there were any significant changes related to invitro or invivo glycation in the structural properties and the binding of human albumin to both therapeutic drugs. Structural parameters, including redox state and ketoamine contents of invitro and invivo glycated purified albumins, were investigated in parallel with their affinity for warfarin and ketoprofen. High-performance liquid chromatography was used to determine the free drug concentrations and dissociation constants according to the Scatchard method. An alternative method based on fluorescence spectroscopy was also used to assess drug-binding properties. Oxidation and glycation levels were found to be enhanced in albumin purified from diabetic patients or glycated with glucose or methylglyoxal, after determination of their ketoamine, free thiol, amino group and carbonyl contents. In parallel, significant impairments in the binding affinity of invitro and invivo glycated albumin, as indicated by the higher dissociation constant values and confirmed by higher free drug fractions, were observed. To a lesser extent, this alteration also significantly affected diabetic albumin affinity, indicated by a lower static quenching in fluorescence spectroscopy. This work provides useful information supporting invivo diabetic albumin could be the best model of glycation for monitoring diabetic physiopathology and should be valuable to know if glycation of albumin could contribute to variability in drugs response during diabetes.

Nickel(II)-Schiff base complex recognizing domain II of bovine and human serum albumin: Spectroscopic and docking studies

It has been spectroscopically monitored that a mononuclear nickel(II)-Schiff base complex {[NiL]·CH3OH=NSC} exhibits greater binding affinity for bovine serum albumin (BSA) than that of its human counterpart (HSA). Moreover the modes of binding of NSC with the two serum albumins also differ significantly. Docking studies predict a relatively rare type of ‘superficial binding’ of NSC at domain IIB of HSA with certain mobility whereas for BSA such phenomena has not been detected. The mobile nature of NSC at domain IIB of HSA has been well correlated with the spectroscopic results. It is to be noted that thermodynamic parameters for the NSC interaction also differ for the two serum albumins. Occurrence of energy transfer between the donor (Trp of BSA and HSA) and acceptor (NSC) has been obtained by means of Förster resonance energy transfer (FRET). The protein stability on NSC binding has also been experimented by the GuHCl-induced protein unfolding studies. Interestingly it has been found that NSC–HSA interaction enhances the protein stability whereas NSC–BSA binding has no such impact. Such observations are indicative of the fact that the conformation of NSC is responsible in recognizing the two serum albumins and selectively enhancing protein stability.

The bile steroid chenodeoxycholate is a potent antagonist at NMDA and GABAA receptors

The bile steroids (BS) cholic acid and chenodeoxycholic acid are produced in hepatocytes and in the brain. Nothing is known about neuronal actions of BS. Deficiency in a 27-hydroxylase enzyme coincides with reduced production of chenodeoxycholic acid (CDCA) and a relative increase in cholic acid in an inherited lipid storage disease, cerebrotendinous xanthomatosis, characterized by neurological dysfunctions, which can be treated by dietary CDCA. We have examined the modulation of hypothalamic network activity by nine common BS. Cholate and CDCA significantly reduced the firing of hypothalamic neurons and synchronized network activity with CDCA being nearly 10 times more potent. The synthetic BS dehydrocholate synchronized the activity without affecting the firing rate. Gabazine, a GABAA receptor antagonist, occluded synchronization by BS. Whole-cell patch clamp recordings revealed a block of NMDA- and GABAA-receptors by BS. Potencies of nine common BS differed between NMDA and GABAA receptors, however in both cases they correlated with BS affinities for albumin but not with their lipophilicity, supporting a direct action at ligand gated ion channels. GABAergic synaptic currents displayed a faster decay under BS. Our data provide new insight into extrahepatic functions of BS revealing their neuroactive potential.

Results of molecular docking as descriptors to predict human serum albumin binding affinity

Pharmacokinetic properties of a compound are important in drug discovery and development. These properties are most often estimated from the structural properties of a compound with a structural–activity relationship (QSAR) approach. Rapid advances in molecular pharmacology have characterized a number of important proteins that shape the pharmacokinetic profile of a compound. Previous studies have shown that molecular docking, which is capable of analyzing compound–protein interactions, could be applied to make a categorical estimation of a pharmacokinetic property. The present study focused on the binding affinity of human serum albumin (HSA) as an example to show that docking descriptors might also be useful to estimate the exact value of a pharmacokinetic property. A previously reported dataset containing 94 compounds with log K HSA values was analyzed. A support vector regression model based on the docking descriptors was able to approximate the observed log K HSA in the training and validation dataset with an R 2 = 0.79. This accuracy was comparable to known QSAR models based on compound descriptors. In this case study, it was shown that an account of protein flexibility is essential to calculate informative docking descriptors for use in the quantitative estimation of log K HSA.

Serum Albumin Binding of Structurally Diverse Neutral Organic Compounds: Data and Models

Binding to serum albumin has a strong influence on freely dissolved, unbound concentrations of chemicals in vivo and in vitro. For neutral organic solutes, previous studies have suggested a log-log correlation between the albumin-water partition coefficient and the octanol-water partition coefficient (K(ow)) and postulated highly nonspecific binding that is mechanistically analogous to dissolution into solvents. These relationships and concepts were further explored in this study. Bovine serum albumin (BSA)-water partition coefficients (K(BSA/w)) were measured for 83 structurally diverse neutral organic chemicals in consistent experimental conditions. The correlation between log K(BSA/w) and log K(ow) was moderate, with R(2) = 0.76 and SD = 0.43. The log K(BSA/w) of low-polarity compounds including a series of chlorobenzenes and polycyclic aromatic hydrocarbons increased with log K(ow) linearly up to log K(ow) = 4-5, but then the linear relationship apparently broke off, and the increase became gradual. The fitting of polyparameter linear free energy relationship models with five solute descriptors was just comparable to that of the log K(ow) model (R(2) = 0.78-0.79, SD = 0.41-0.42); the relatively high SD obtained suggests that solvent dissolution models are not capable of modeling albumin binding accurately. A size limitation of the binding site(s) of albumin is suggested as a possible reason for the high SD. An equilibrium distribution model indicates that serum albumin generally has high contributions to the binding in the serum of polar compounds and relatively small low-polarity compounds, whereas albumin binding for large low-polarity compounds is outcompeted by the strong partitioning into lipids due to low relative affinity of albumin for these compounds.

Evaluation of the real-time protein adsorption kinetics on albumin-binding surfaces by dynamic in situ spectroscopic ellipsometry

Functionalized surfaces with an affinity for albumin over competing serum proteins were prepared by immobilizing linear peptides or a small chemical ligand (SCL) with albumin-binding properties on silanized silicon surfaces. The real time adsorption of human serum albumin from single- and multi-component systems was monitored by dynamic ellipsometry. The experimentally obtained time-dependent adsorption data were analyzed by two models: a) a pseudo-first-order model and b) a biphasic kinetic model that accounted for the formation of tightly and loosely bound complexes. The biphasic kinetic model better fit the experimental data, and the binding constants were determined by non-linear regression analyses. The net forward rate constant for the tightly bound complex formation was distinctively higher for surfaces functionalized with peptides (~ 0.014 min − 1 ) when compared to surfaces functionalized with SCL (~ 10 − 3 min − 1 ).

Engineering Bispecificity into a Single Albumin-Binding Domain

Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed.

Role of the medium acidity in the complex formation of pyropheophorbide a with albumin and lipoproteins

The spectral characteristics of the photosensitizer pyropheophorbide a (PPP) complexes with its carriers, that is, serum albumin and low density lipoproteins, were investigated in aqueous solutions at pH 7.4 and 5.0. The acidic pH had no effect on the quantitative parameters of PPP binding to lipoproteins but reduces its affinity for albumin. Differential role of acidification in the binding of PPP to biomacromolecules should be considered in the design of PPP-based drugs given that pH is frequently lowered in the sites of the disease.

Unbound Bilirubin does not Increase during Ibuprofen Treatment of Patent Ductus Arteriosus in Preterm Infants

To determine whether ibuprofen displaces bilirubin from albumin in preterm infants. A total of 34 preterm neonates (<32 weeks gestation) treated by ibuprofen (10-5-5 mg/kg) were included in this prospective open-label study. Total bilirubin (TB), unbound bilirubin (UB), and ibuprofen concentrations were measured before, 1 hour, and 6 hours after the first dose; before and 1 hour after the second dose; and 72 hours after the beginning of treatment. The infants were screened by auditory brainstem responses and by neurologic examination at term. At baseline, TB, UB, apparent binding affinity of albumin (Ka), and albumin concentrations were 6.0±1.6 mg/dL, 1.9±2.2 μg/dL, 14.1±5.8 L·μmol(-1), and 28.7±2.3 g/L, respectively. Ibuprofen treatment had no effect on TB, UB, or Ka values. No correlation between UB or Ka and ibuprofen concentrations was found. No neurologic symptoms or significant modifications of auditory brainstem responses were observed at term. Ibuprofen (10-5-5 mg/kg) did not displace bilirubin in preterm infants with a baseline TB concentration <8.8 mg/dL.

Molecular structure-affinity relationship of dietary flavonoids for bovine serum albumin

The relationship between the molecular structure of dietary flavonoids and their affinities for bovine serum albumin (BSA) were fully investigated by fluorescence titration analysis. The binding process with BSA was significantly affected by the molecular structure of flavonoids under study. The methylation of hydroxyl group in flavonoids enhanced their binding affinities for BSA by 1 to 794 times. Hydroxylation on rings A, B and C also strongly influenced the affinity for BSA. The glycosylation weakened the affinities for BSA by 1-2 orders of magnitude depending on the conjugation site and the class of sugar moiety. The hydrogenation of the C2=C3 double bond slightly increased the binding affinity. The galloylated catechins and pyrogallol-type catechins exhibited higher binding affinities for BSA than non-galloylated and catechol-type catechins, respectively. The affinities for BSA increased with increasing partition coefficients and decreased with increasing hydrogen bond donor and acceptor numbers of flavonoids, which suggested that the binding interaction was mainly caused by hydrophobic forces.

Protein delivery based on uncoated and chitosan-coated mesoporous silicon microparticles

Mesoporous silicon is a biocompatible, biodegradable material that is receiving increased attention for pharmaceutical applications due to its extensive specific surface. This feature enables to load a variety of drugs in mesoporous silicon devices by simple adsorption-based procedures. In this work, we have addressed the fabrication and characterization of two new mesoporous silicon devices prepared by electrochemistry and intended for protein delivery, namely: (i) mesoporous silicon microparticles and (ii) chitosan-coated mesoporous silicon microparticles. Both carriers were investigated for their capacity to load a therapeutic protein (insulin) and a model antigen (bovine serum albumin) by adsorption. Our results show that mesoporous silicon microparticles prepared by electrochemical methods present moderate affinity for insulin and high affinity for albumin. However, mesoporous silicon presents an extensive capacity to load both proteins, leading to systems were protein could represent the major mass fraction of the formulation. The possibility to form a chitosan coating on the microparticles surface was confirmed both qualitatively by atomic force microscopy and quantitatively by a colorimetric method. Mesoporous silicon microparticles with mean pore size of 35 nm released the loaded insulin quickly, but not instantaneously. This profile could be slowed to a certain extent by the chitosan coating modification. With their high protein loading, their capacity to provide a controlled release of insulin over a period of 60–90 min, and the potential mucoadhesive effect of the chitosan coating, these composite devices comprise several features that render them interesting candidates as transmucosal protein delivery systems.

Albumin-binding domain conjugate for near-infrared fluorescence lymphatic imaging.

PurposeThe aim of this study was to develop and characterize a novel peptide imaging agent for noninvasive near-infrared fluorescence imaging of protein transport by the lymphatics. An imaging agent consisting of a cyclic albumin-binding domain (cABD) peptide, with sequence, Arg-Leu-Ile-Glu-Asp-Ile-Cys-Leu-Pro-Arg-Trp-Gly-Cys-Leu-Trp-Glu-Asp-Asp-Lys, was conjugated to a near-infrared fluorophore, IRDye800CW, allowing for enhanced vascular uptake, retention, and fluorescence imaging.ProcedureCharacterization of the cABD-IRDye800 peptide conjugate was performed using fluorescence spectroscopy to assess optical properties and SDS-PAGE and Biacore binding assays to determine binding affinity and specificity. Fluorescence imaging of normal C57BL/6 mice was conducted to monitor lymphatic uptake and retention.ResultscABD-IRDye800 exhibited approximately six times greater fluorescent yield and greater stability than indocyanine green, an agent previously used in humans to image lymphatic vasculature. The agent exhibited affinity for albumin with IC50 and Kd in the nanomolar range and demonstrated superior retention characteristics within mouse lymphatics when compared with IRDye800CW.ConclusionscABD-IRDye800 has utility for assessing lymphatic function in mouse models of human lymphatic disease and the potential for use in clinical diagnostic imaging of the lymphatic vasculature.Electronic supplementary materialThe online version of this article (doi:10.1007/s11307-011-0499-x) contains supplementary material, which is available to authorized users.

Quantification of Interactions between Serum Albumin and Endogenous Free Fatty Acids or Exogenous Chemicals by Stable Isotope-Coded Mass Spectrometry.

As primary endogenous ligands of serum albumin, free fatty acids exert versatile effects on the albumin conformation through cooperative or competitive interactions with exogenous chemicals. Based on equilibrium partition between n-hexane and aqueous phases, we have established three indexes, defined as R A, R V, and R T, for quantitative assessment of the intrinsic binding affinity, the affinitive variation induced by exogenous chemicals, and the topological dependence of albumin affinity, respectively. When albumin molecules in the aqueous phase are in native or denatured forms, or disturbed by exogenous chemicals, corresponding changes of free fatty acids in the n-hexane phase can be quantified by an iFFAM (isotope-coded free fatty acid methylation) approach. Free fatty acids from the control and the sample are differentially derived by d0- or d3-methanol and analyzed by gas chromatography-mass spectrometry. Changes of fatty acids can be revealed by peak ratios of d0- or d3-labeled fragment ions of fatty acid methyl esters.

Effect of CdTe QDs on the protein-drug interactions

Recently, investigations of biological toxicity of cadmium QDs and their toxic interaction with plasma proteins have attracted great interest. In this work, flavonoids were studied for the affinities for human serum albumin (HSA) in the presence and absence of CdTe G-QDs by fluorescence quenching method. CdTe G-QDs obviously enhanced the binding affinities of kaempferol, genistein and biochanin A by 3.78 to 154.88 times depending on the QDs concentration. However, the affinity of kaempferide for HSA was slightly weakened in the presence of G-QDs. The non-methylated flavonoids were more sensitive to G-QDs than their methylated forms. The affinities of kaempferide and kaempferol for HSA at first were slightly improved and then obviously decreased with increasing G-QDs concentration. For genistein, the affinities for HSA decreased with increasing G-QDs concentration. However, the G-QDs concentration showed no obvious effect on the affinity of biochanin A. The binding affinities of flavonoids for HSA improved with increasing QDs size.

Spectroscopy studies of functionalized oxidized porous silicon surface for biosensing applications

In this paper we report detailed Fourier transform infra-red (FTIR) and Raman spectroscopic results obtained after the modification of a porous silica surface using different steps of functionalization and protein grafting. After the elaboration of porous silicon (PS) layers, with sufficient adjusted pore size and well defined porosity, we carried out complete thermal oxidation and we optimized the silanization step by varying the concentration of 3-aminopropyltriethoxysilane (APTES) molecules using different immersion and rinsing durations. Then we introduced a coupling agent, glutaraldehyde (GL) molecules, which has a great affinity for bovine serum albumin (BSA) molecule grafting. FTIR and Raman spectroscopic analyses present complementary spectra that allow us to get an idea about the chemical links and vibrational modes that appear during the functionalization process and after protein attachment. This proves that the biological molecules are covalently attached on the modified porous silica surface. Moreover, a modelling study of the reflectance spectra allows values of the volume fraction and refractive index variations to be estimated by assigning them with the number of APTES, glutaraldehyde and protein layers after each step. These results are well correlated to those obtained by the FTIR and Raman analyses. This work on porous silica single-layers is carried out in order to exploit it for the elaboration of functionalized optical waveguides to obtain a sensitive label-free optical biosensor.

ZnO-ZnS QDs interfacial heterostructure for drug and food delivery application: enhancement of the binding affinities of flavonoid aglycones to bovine serum albumin

Zero-dimensional nanostructures are green nanomaterials that have recently attracted increasing attention. However, very little information is available on whether or not these heterostructures affect drug transport in blood. In current work, flavonoid aglycones were studied for their affinities for bovine serum albumin (BSA) in the presence and absence of zinc oxide-zinc sulfide quantum dots (ZnO-ZnS QDs) in vitro. The fluorescence intensity of BSA decreased remarkably with increasing concentration of ZnO-ZnS QDs, resulting in an obvious red-shift of the maximum emission of BSA from 340 to 348 nm. The magnitudes of binding constants in the presence of QDs ranged from 104 to 106 L/mol, and the number of binding sites per BSA molecule (n) was determined as 1.12 ± 0.17. Although ZnO-ZnS QDs significantly increased the affinities for BSA of myricetin, luteolin, gallocatechin gallate, tectorigenin, and formononetin, they barely affected the binding affinities of flavone, (-)-epicatechin gallate, and quercetin. From the Clinical EditorSerum albumins are major transport proteins in blood that reversibly bind fatty acids, amino acids, drugs, and inorganic ions, which interactions have important effects on the distribution, free concentration, and metabolism of drugs in blood. In this research nine flavonoid aglycones were studied for their affinities for bovine serum albumin (BSA). Interestingly it was found that presence of ZnO-ZnS QDs significantly increased the affinities of BSA for several of these aglycones.

Chromatographic and Traditional Albumin Isotherms on Cellulose: A Model for Wound Protein Adsorption on Modified Cotton

Albumin is the most abundant protein found in healing wounds. Traditional and chromatographic protein isotherms of albumin binding on modified cotton fibers are useful in understanding albumin binding to cellulose wound dressings. An important consideration in the design of cellulosic wound dressings is adsorption and accumulation of proteins like albumin at the solid-liquid interface of the biological fluid and wound dressing fiber. To better understand the effect of fiber charge and molecular modifications in cellulose-containing fibers on the binding of serum albumin as observed in protease sequestrant dressings, albumin binding to modified cotton fibers was compared with traditional and chromatographic isotherms. Modified cotton including carboxymethylated, citrate-crosslinked, dialdehyde and phosphorylated cotton, which sequester elastase and collagenase, were compared for their albumin binding isotherms. Albumin isotherms on citrate-cellulose, cross-linked cotton demonstrated a two-fold increased binding affinity over untreated cotton. A comparison of albumin binding between traditional, solution isotherms and chromatographic isotherms on modified cellulose yielded similar equilibrium constants. Application of the binding affinity of albumin obtained in the in vitro protein isotherm to the in vivo wound dressing uptake of the protein is discussed. The chromatographic approach to assessment of albumin isotherms on modified cellulose offers a more rapid approach to evaluating protein binding on modified cellulose over traditional solution approaches.