Inhibition of drug binding to human serum albumin by cholecystographic agents

The binding of two cholecystographic agents to human serum albumin (HSA) was evaluated by means of two different complementary methodologies. In particular, the inhibition of drug HSA binding caused by iopanoic- and iophenoxic-acid was investigated by circular dichroism (CD) and resonant mirror (RM) optical biosensor techniques. The CD study allowed to obtain information both on the cholecystographic agent binding site and on the effect of the binding on the protein conformation. Iopanoic acid (IOP), a drug potentially useful for thyrotoxic disorders, resulted a direct competitor for ligands that selectively bind to site II, in agreement to literature data. No definite evidence was obtained for the highest affinity binding site of iophenoxic acid (IOPH), however, this diagnostic tool markedly affected the binding of ligands to the most characterized high affinity sites on HSA, namely sites I, II and III. Binding parameters were obtained by optical biosensor analysis: K(D) values were 3.6 x 10(-7) and 2.8 x 10(-8) M for IOP and IOPH, respectively.     

Palmitate and stearate binding to human serum albumin

Multiple binding equilibria of two apparently insoluble ligands, palmitate and stearate, to defatted human serum albumin were studied in a 66 mM sodium phosphate buffer (pH 7.4) at 37 °C, by determination of dialytic exchange rates of ligands among identical equilibrium solutions. The experimental data were analysed by a computerised curve fitting procedure using equilibrium equations for multiple binding of ligands, containing relative binding constants, valid whether the ligands are truly insoluble or are slightly soluble and irrespective of aggregation in aqueous solution. A best-fit set of relative binding constants was found, and subsequently 30 sets of acceptable constants for each set of data in order to evaluate the variation. The data were first fitted by the relative Scatchard's equation, then by the relative, stoichiometric equation. Scatchard's equation is deduced on the presumption that cooperativity is absent while the stoichiometric equation is valid even when cooperativity is present. It was found with palmitate as well as with stearate that the two equations fitted the data equally well, and it was concluded that the observations were compatible with absence of cooperativity. The relative Scatchard binding constants were converted to relative, stoichiometric constants and it was found that the variations of the latter were slight. © Munksgaard 1997.     

Binding of teicoplanin to human serum albumin

Summary The interaction between the main components of the new glycopeptide antibiotic teicoplanin, A2–2, A2–3, A2–4, A2–5 and A3–1, and human serum albumin has been studied in vitro by equilibrium dialysis (pH 7.4, 37°C).From Scatchard analysis of the data, the calculated association constants (Ka) were: A2–2, 2.47×10⁴, A2–3, 2.86×10⁴, A2–4, 2.95×10⁴ and A2–5, 3.87×10⁴ mol·l^–1. The number of binding sites per albumin molecule ranged between 1.23 to 1.31. A3–1 had a lower affinity with a Ka of about 5×10³ mol·l^–1.Extrapolated to the in vivo situation, the data suggested that about 90–95% of A2 components will be bound to serum albumin, and about 68–72% of A3–1.The in vitro findings were confirmed by a pharmacokinetic study in volunteers given [¹⁴C] teicoplanin i.v., in whom the fraction of teicoplanin bound to serum protein ranged between 87.6 and 90.8%.     

Site- and orientation-specific binding of hematoporphyrin monomethyl ether to human serum albumin revealed by single spectral kinetic parameter

Interaction of a drug molecule with human serum albumin (HSA) is usually studied by fluorescence responses of the ligand or/and the single tryptophan residue (Trp-214) of the protein, but qualitative spectral information may lead to multiple conclusions. In this work, we report a study on the interaction of hematoporphyrin monomethyl ether (HMME) with human serum albumin (HSA), using the environment-sensitive spectra of HMME and reaction-induced fluorescence response of Trp-214. Particularly, the single kinetic parameter, the linear slope, was derived from the concentration-dependent absorbance or fluorescence of HMME in a certain solvent. A quantitative change in the slope at [HMME]/[HSA] = 1:1 clearly demonstrated a specific binding of HMME to site I. The microenvironment in site I may be comparable to that in DMSO solvent, because of the similarity of the slope. Linear correlation of the fluorescence to the absorbance of HMME in site I indicates that the energy transfer is not responsible for Trp-214 fluorescence quenching but an electron transfer may be possible. In addition, much higher rate observed for the binding of HMME or 2-taurine-substituted HB (THB) with HSA than that of hypocrellin B was due to the electrostatic attraction under physiological condition.     

Probing the binding of morin to human serum albumin by optical spectroscopy

Morin [2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one], a member of flavonols, is an important bioactive compound by interacting with nucleic acids, enzymes and protein. Its binding to human serum albumin was investigated by fluorescence quenching, fluorescence anisotropy, and UV–vis absorbance under the simulative physiological condition. Fluorescence quenching data show that the interaction of morin with HSA forms a non-fluorescent complex with the binding constants of 1.394 × 10⁵, 1.489 × 10⁵, 1.609 × 10⁵ and 1.717 × 10⁵ M⁻¹ at 292, 298, 303 and 310 K, respectively. The thermodynamics parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be 8.97 kJ mol⁻¹ and 129.15 J mol⁻¹ K⁻¹ via van’t Hoff equation. From the spectroscopic results and thermodynamics parameters, it is observed that van der Waals and hydrogen bonds are predominant intermolecular forces when forming the complex. The distance r = 4.25 nm between donor (Trp214) and accepter (morin) was estimated based on the Förster theory of non-radiative energy transfer. The red shift of UV–vis absorbance shows that morin is bound to several amino acids on the hydrophobic pocket of HSA. Moreover, the competitive probes, such as warfarin and ibuprofen (site I and II probes, respectively), reveal that the binding location of morin to HSA in the site I of the hydrophobic pocket, which corresponds to the results of UV–vis absorbance, while morin also binds other lower affinity binding sites on HSA from the fluorescence anisotropy spectroscopy.     

Tocainide analogues binding to human serum albumin: A HPLAC and circular dichroism study

A series of synthesised tocainide analogues were characterized for their human serum albumin (HSA) binding, using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). The synthesis and physico-chemical characterization of compounds 7a–7d is reported here. For the HPLAC investigation HSA was covalently immobilized to the silica matrix of the HPLC column, using an anchoring procedure, which allows the binding properties of the protein to be maintained. The HSA-based column was used for getting information on the high affinity binding sites of the tocainide analogues to HSA. According to the displacement chromatography approach, the retentions of the analytes were determined in the absence and in the presence of increasing concentrations of competitors known to bind to specific binding sites on the protein. The same system, drug/protein, was investigated in solution by CD.     

Fatty acid and drug binding to a low-affinity component of human serum albumin,purified by affinity chromatography

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component.     

Reversible binding of antidiabetic drugs, repaglinide and gliclazide, with human serum albumin

Mechanism of interaction of antidiabetic drugs, repaglinide and gliclazide, to human serum albumin has been studied using fluorescence spectroscopic technique. Repaglinide had much higher affinity for human serum albumin when compared with gliclazide. The order of association constants was 10(5) for both the drugs. The size, hydrophobicity and flexibility of the drug molecules play a major role in explaining the binding behaviour of these drugs. Hydrophobic interactions are predominantly involved in the binding. However, drugs do not share common sites with 1-anilinonaphthalene-8-sulphonate on the human serum albumin molecule. Both tyrosine and tryptophan residues participate in the interaction. Repaglinide and gliclazide are bound to site II on the human serum albumin molecule, and the aromatic ring of 411Tyr appears to be involved in binding within site II. Although they do not bind at site I, their binding at site II may cause conformational changes thereby affecting the binding of other ligands to site I. Site-specificity can be useful in predicting the competitive displacement of these drugs by other co-administered drugs, resulting in fluctuations of the blood glucose levels in diabetic patients. Stern-Volmer analysis of quenching data indicated that the tryptophan residues are not fully accessible to the drugs and predominantly dynamic quenching mechanism is involved in the binding.     

人血白蛋白临床应用进展

目的了解人血白蛋白的临床应用现状。方法对近期国内外相关文献进行分析和评价。结果与结论人血白蛋白临床用途广,其主要适应证包括出血性休克、外伤性休克、烧伤、成人呼吸窘迫综合征、肝硬化伴有腹水及水肿、恶性肿瘤等,但在某些治疗领域的使用还是存在着争议。     

The effects of glycation on the binding of human serum albumin to warfarin and l-tryptophan

Diabetes leads to elevated levels of glucose in blood which, in turn, can lead to the non-enzymatic glycation of serum proteins such as human serum albumin (HSA). It has been suggested that this increase in glycation can alter the ability of HSA to bind to drugs and other small solutes. This study used high-performance affinity chromatography (HPAC) to see if there is any significant change related to glycation in the binding of HSA to warfarin and l-tryptophan, which are often used as probe compounds for Sudlow sites I and II of HSA in drug binding studies with this protein. It was found through frontal analysis studies that both of these compounds gave a good fit to a single-site binding model with glycated HSA under the conditions used in this study. There was no significant change in the association equilibrium constants or specific activities for warfarin with HSA at pH 7.4 and 37 °C under glycation conditions that were representative of those expected in pre-diabetes or diabetes, but a 4.7- to 5.8-fold increase in binding affinity for l-tryptophan with glycated HSA was observed. These results indicate that warfarin and l-tryptophan can be successively used as site-selective probes for glycated HSA; however, changes in the affinity of l-tryptophan may need to be considered in such an application. These results should be valuable in future competition studies using these compounds as probes to examine the interactions of other drugs and solutes with Sudlow sites I and II and to determine how changes in HSA glycation can affect the serum protein binding of various pharmaceutical agents during diabetes.     

Reversible binding of tolmetin,zomepirac, and their glucuronide conjugates to human serum albumin and plasma

Acyl glucuronides of drugs and bilirubin have been shown in the past decade to be reactive metabolites undergoing acyl migration and irreversible binding. The latter reaction has been hypothesized to be facilitated by or to proceed through the formation of a reversible complex. Furthermore, it has been suggested that the decreased binding seen in patients with compromised excretory function may be due to competition by elevated plasma concentrations of the glucuronides. In these reversible binding studies, we characterized the extent and the “site” of binding of tolmetin, zomepirac, their glucuronides and isomeric conjugates. We also examined the displacement between the parent drugs and their glucuronide conjugates using a rapid ultrafiltration method. Tolmetin exhibited three classes of binding sites with a primary association constant of 1.7×10⁶ M⁻¹ (K_dl=0.60 μM). The primary association constant of zomepirac (1.16×10⁶ M⁻¹, K_dl=0.86 μM) is similar to that of tolmetin. The β 1 and α/β3 glucuronides of both compounds bind to a lesser extent than their parent aglycones. The isomeric glucuronide conjugates of both compounds showed much stronger binding than the β/1 conjugates. Of the four glucuronides investigated, tolmetin glucuronide-α/β3 isomer was bound by fatty acid free human serum albumin with the highest affinity (4.6×10⁵ M⁻¹, K_d=2.22 μM). Protein binding of the parent drugs and conjugates were decreased significantly at pH 5.0. In displacement studies, except for salicylate and acetylsalicylate, drugs known to bind to Sites I and II as well as the digitoxin and tamoxifen binding sites had little inhibitory effect on the binding of tolmetin, zomepirac, and their glucuronide conjugates. Supported in part by Grant GM 36633 from the National Institute of General Medical Sciences.     

人血清白蛋白与硫代寡核苷酸的结合研究

目的研究二价阳离子对白蛋白与硫代寡核苷酸结合的影响以及结合硫代寡核苷酸后白蛋白构象的改变。方法应用表面等离子体共振、圆二色散以及荧光光谱法对白蛋白与硫代寡核苷酸的结合行为进行了表征。结果（1）随着pH值升高,结合能力降低;（2）锌离子能显著促进白蛋白与硫代寡核苷酸的结合, 镍离子也促进结合,但与锌离子相比作用稍弱;（3）与硫代寡核苷酸结合后,白蛋白结构发生变化,产生β-折叠构造。结论通过实验方法证明硫代寡核苷酸结合于白蛋白的正电荷密集区;表明白蛋白可能介导硫代寡核苷酸的胞吞行为。     

阿柔比星与人血白蛋白相互作用的特点

目的研究阿柔比星与人血清白蛋白的相互作用特点。方法用荧光猝灭光谱及同步荧光光谱技术测定在不同温度下阿柔比星对人血清白蛋白荧光的猝灭规律。结果阿柔比星对人血清白蛋白荧光呈规律性猝灭,17℃时的n为1.11,K为6.21×104,37℃时的n为1.12,K为6.41×104。阿柔比星使白蛋白的同步光谱中最大发射峰红移。结论阿柔比星与人血清白蛋白只有一个结合位点,表现为疏水作用,两者结合使白蛋白的极性略有增加。     

BTK-inhibitor drug covalent binding to lysine in human serum albumin using LC-MS/MS

Irreversible Bruton's tyrosine kinase (BTK) inhibitor drugs are designed to bind covalently to a free-thiol cysteine in the BTK protein active site. However, these reactive drugs bind to off-target proteins as well. In this study, seven BTK-inhibitor drugs containing acrylamide warheads were incubated with human serum albumin (HSA) and analyzed using an LC-MS/MS peptide mapping approach to determine the amino acid sites of drug covalent binding. Significant adduction at the free-thiol cysteine of HSA was only observed for two of the drugs. However, significant adduction was observed for at least four lysine residues. This is just a small percentage of the 59 total lysine residues in HSA. These four lysine residues are likely partially buried, accessible to the drugs, and exist at least partially in a neutral state. The levels of adduction observed in the in-vitro experimental conditions are only indicative of a relative propensity for adduction with the individual lysine residues of HSA, and are not in-vivo predictions. Widespread off-target lysine binding could impact clearance and bioavailability for irreversible inhibitor drugs. However, the extent of the impact on clearance may be limited in comparison to conjugation with glutathione.     

Hydrolysis of carbaryl by human serum albumin

Human serum (HS) and human serum albumin (HSA) were able to hydrolyse the carbamate carbaryl. Carbarylase activity found in HSA was slightly activated by 1 mM Zn²⁺, Mn²⁺, Cd²⁺, Ni²⁺ and Na⁺ and by 0.01 mM Pb²⁺. The organophosphorus compounds paraoxon and O-hexyl O-2,5-dichlorophenyl phosphoramidate, caprylic acid, palmitic acid and the carboxyl ester p-nitrophenyl butyrate inhibited the hydrolysis of carbaryl by HSA, being in the last case a competitive inhibition. Using selective amino acid reagents, we concluded that Cys, Trp, Arg and Tyr seem to play important roles in the carbarylase activity of HSA. In addition, Tyr and Arg seem to be located in the active centre of the enzyme since carbaryl protected the activity from the inhibition. It was concluded that HSA hydrolyses carbaryl by a mechanism similar to that described for rabbit serum albumin based in transient carbamylation of a Tyr residue. The extrapolation of the hydrolysis rate to physiological albumin concentrations suggests that albumin might be playing a critical role in the detoxication of carbaryl.Abbreviations carbarylase carbaryl hydrolysing activity; - DCC N,N-Dicyclohexylcarbodiimide; - DEPC Diethyl pyrocarbonate; - DFP diisopropyl fluorophosphates; - DTNB 5,5-dithio-bis (2-nitrobenzoic acid); - HDCP O-hexyl O-2,5-dichlorophenyl phosphoramidate; - HS human serum;m - HSA human serum albumin; - NAI N-acetylimidazol; - NBS N-bromosuccinimide; - PNG phenylglyoxal; - p-NPB p-nitrophenyl butyrate     

Kinetics of drug binding to human serum albumin: allosteric and competitive inhibition at the benzodiazepine binding site by free fatty acids of various chain lengths

Summary The inhibition of dansylsarcosine (DS) binding at the benzodiazepine binding site of human serum albumin has been studied in the presence of saturated and unsaturated free fatty acids (FFA) of various chain lengths (C6–C20, C18:1, C18:2). In order to determine the mechanism of displacement, velocity constants for association (k ₂) and dissociation (k _–2) and binding constants (K _A and K _A) have been measured using the stopped-flow method.The inhibitory effect of FFA on DS binding kinetics at site II is dependent of their structure. With increasing amounts of FFA the association velocity constant of DS binding decreases from 520 s^–1 (fatty acid free albumin) by a factor of 3–10 and affinity decreases according to FFA chain length. Inhibition is strongest in the presence of caprylic, capric and lauric acid (C8–C12) i.e. with more than one mole FFA per mole albumin, DS association could no longer be measured. Short chain caproic and the long chain FFA C14–C20 showed only a less inhibitory effect since in the presence of a twofold excess k ₂ ranged between 100 and 200 s^–1. Dissociation velocity of DS from the benzodiazepine binding site could be measured in relationship to FFA chain length using ibuprofene, another drug binding at site II. Dissociation velocity constants k _–2 remained constant up to 2 moles FFA per mole albumin (k _–2 = 16–18 s^–1). A rise in k _–2 to 70 s^–1 was seen, however, when 2–4 moles capric, lauric, myristic and palmitic (C10–C16) acid were bound, whereas no change was observed when increasing concentrations of caproic, caprylic, stearic and arachic acid. The more compact unsaturated FFA oleic and linoleic acids did not inhibit DS binding to the same extent as their saturated homologue stearic acid. k ₂ and k _–2 values resembled those when comparable amounts of myristic acid were bound indicating that chain length is the relevant parameter.It can be concluded that two different types of FFA inhibition of binding at the benzodiazepine binding site exist. Short-chain FFA (C6–C8) may be specifically bound at binding site II thereby competitively displacing DS. Longchain FFA (C12–C20), however, occupy their binding sites far from the benzodiazepine binding site. Inhibition occurs via an allosteric mechanism. Capric acid is unique in showing as well as competitive as well as allosteric inhibition characteristics.Abbreviations k ₂ Dissociation velocity constant - k ₂ association velocity constant - K _A affinity constant - K _A affinity constant of the intermediate complex - DS dansylsarcosine - FFA free fatty acids - HSA human serum albumin This work has been presented in part at the Spring Meeting of the German Pharmacological Society in Mainz, March 1988 (Menke et al. 1988) Send offprint request to N. Rietbrock at the above address     

Evaluation of the binding effect of human serum albumin on the properties of granules

The main objective of this study was the application of a solution of human serum albumin as a granulating fluid. The properties of the granules formed were evaluated and compared with those when a conventional binder was applied in the same concentration. The powder mixture contained a soluble (mannitol) and an insoluble component (different types of cellulose). The protein solution applied exerted an appropriate aggregating effect if the system contained microcrystalline celluloses. Powdered cellulose was not suitable for the granulation with human serum albumin solution. As compared with the same concentration of the conventionally applied cellulose ethers as binder, the prepared granules exhibited a larger particle size, a significantly better compressibility, a higher breaking hardness and a favourable deformation process. These findings mainly reflect the good adhesive properties of the protein. The best compressibility and mechanical behaviour were attained on the application of the microcrystalline cellulose Vivapur type 105. This favourable behaviour may be connected with the wettability of cellulose. These results suggest that the formulation of tablets may be easier from an active agent in the serum that binds to albumin (e.g. interferon) since the amount of additives (binder) can be reduced.     

我院2013-2014年人血白蛋白临床使用规范性评价

目的了解我院人血白蛋白临床使用情况,评价其用药规范性。方法利用医院电子病历系统,回顾性抽查我院2013年1月至2014年12月使用人血白蛋白的1 009例住院患者,收集相关临床信息并统计。结果应用人血白蛋白的患者以中老年为主;用药原因以低蛋白血症、营养支持为主;用药前有28.15%的患者未检测血清白蛋白浓度;对比美国UHC指南、台湾人血白蛋白使用规定及中国人血白蛋白使用说明书,适应证符合率分别为15.36%、24.08%、63.83%。结论我院人血白蛋白使用广泛,但规范性较低,应加强管理。     

Reviewing the binding of a series of parabens to human serum albumin

To better understand the factors that contribute to the accumulation of unmetabolized parabens (p‐hydroxybenzoic acid esters) in breast cancer tissue, the binding of a series of parabens (methyl‐, ethyl‐, butyl‐, benzyl‐paraben) to human serum albumin (HSA) was investigated by fluorescence spectroscopy and also their ability to modify the binding parameters of albumin site markers. Emission spectra of HSA upon fluorescence excitation of Trp 214 residue at 295 nm were recorded at different molar ratios of PB/HSA and data were corrected for the inner‐filter effect. A significant inner‐filter effect was obtained for molar ratios of 2.0 and above. For lower molar ratios, a slight increase in fluorescence of HSA was detected. p‐Hydroxybenzoic acid, the main metabolite of parabens, did not modify the fluorescence of HSA whatever the molar ratio used. Binding parameters for compounds that are markers of site I, bilirubin and warfarin, were determined in the absence and presence of methyl, butyl and benzyl paraben at molar ratios of PB/HSA of 0, 1 and 2. No variation of the binding constants of these markers was observed. The results indicate that parabens weakly interact with HSA thus suggesting that they are in a free form in blood and therefore more available to reach tissues. Copyright © 2013 John Wiley & Sons, Ltd.     

Variation in the binding affinity of warfarin and phenprocoumon to human serum albumin in relation to surgery

Summary The binding equilibria of warfarin and phenprocoumon with defatted human serum albumin were studied by equilibrium dialysis in 33 mM sodium phosphate buffer, pH 7.4, 37°C. The binding isotherms for both ligands were consistent with binding to two similar and independent sites in the albumin molecule.The binding affinity of warfarin was markedly increased on adding palmitic acid up to palmitate 4 mol per mol albumin and then it decreased. The binding affinity of phenprocoumon varied similarly but to a lesser degree.Serum samples were obtained from 14 patients under-going knee joint surgery, six consecutive samples from each patient. The binding affinity of warfarin and phenprocoumon added in low concentrations to the serum samples was consistently less than to purified albumin. The binding affinity for warfarin increased slightly with increasing fatty acid concentrations during surgery, but the increase was much less than expected from the in vitro studies. The binding of phenprocoumon in the serum samples was not influenced by changing fatty acid concentration. The binding affinity for both drugs decreased markedly during the three days following surgery.