Binding Study of the Fluorescence Probe l-Anilino-8-naphthalenesulfonate to Human Plasma and Human and Bovine Serum Albumin Using Potentiometric Titration

The binding of l-anilino-8-naphthalenesulfonate (ANS) to bovine serum albumin (BSA), human serum albumin (HSA), and human plasma has been studied by potentiometric titration utilizing a laboratory constructed ion selective electrode (ISE) of ANS. Three classes of ANS binding sites were found on BSA, HSA, and plasma at 25 and 37°C. Computer analysis of the data resulted in estimates for the association constants, number of binding sites (HSA, BSA), and binding capacity of each class. The association constants for the first class of binding sites at 25°C were found to be 7.53 (±0.59) × 10⁵, 2.70 (±0.20) × 10⁵, and 2.64 (±0.26) × 10⁵ M ^–l for BSA, HSA, and plasma, respectively. Lower values for the association constants of all binding classes were estimated at the higher temperature (37°C). The binding capacity for ANS decreased in the order BSA, plasma, HSA.     

Binding of the organophosphates parathion and paraoxon to bovine and human serum albumin

Binding of parathion and paraoxon to bovine serum albumin (BSA) and human serum albumin (HSA) was studied by using equilibrium dialysis. The concentration of unbound organophosphate was determined from its anticholinesterase activity.Binding of parathion to BSA was shown to be reversible. The organophosphates interact with only one type of binding sites in BSA and HSA. The affinity constants at pH 7.2 and 4° C for the interaction of BSA or HSA and parathion were found to be 2.7×10⁶ and 1.5×10⁶ M^–1, respectively. The affinity constants for the interaction of the serum albumins and paraoxon were considerably lower, 6.0×10³ and 1.6×10⁴ M^–1, respectively. Lowering the pH from 7.2 to 4.8 did not significantly affect the binding parameters. The great difference of affinity of the serum albumins to parathion and paraoxon is discussed with respect to the fate of parathion in the body.     

Drug-protein binding kinetics in patients with Type I diabetes

Summary Sera from 17 patients with Type I diabetes and 19 healthy volunteers have been examined to evaluate whether the kinetics of the binding of drugs to Site II of serum albumin is altered in diabetes. Stopped-flow measurements showed that the association velocity and the affinity constants of the fluorescent marker dansylsarcosine were significantly lower in diabetics (160 s^–1 and 2.0 × 10⁵ l·mol^–1) than in non-diabetics (196s^–1 and 4.0 × 10⁵ l·mol^–1). The dissociation velocity was not different [20.3 s^–1 vs. 19.4 s^–1]. Although patients with a reduced albumin concentration were excluded the diabetics had significantly lower concentrations than the healthy volunteers. There was a significant correlation between decreased glycosylation of albumin and increased association velocity. The dissociation velocity constants were correlated with the molar concentration ratio of free fatty acids/human serum albumin. Thus, the extent of glycosylation and the amount of fatty acids bound per mole albumin can both affect the kinetics of drug binding to Site II. The lower affinity in patients with Type I diabetes is due to the increased in the glycoalbumin concentration.     

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%.     

Binding of 3H-desmethylphalloin to isolated plasma membranes from rat liver

Summary ³H-desmethylphalloin (DMPh), a phallotoxin chemically very similar to phalloidin, binds specifically to rat liver plasma membranes in vitro. Already after 30 sec at 37°C, >90% of maximal binding had taken place. At low ³H-DMPh concentrations, isolated plasma membranes bind 20 times more of the cyclopeptide per mg protein as compared with microsomes or mitochondria. Scatchard plot argues for the presence of two different binding sites in the plasma membranes. The dissociation constants of them at 37°C are 2.2·10^–8 moles/l and 1.2·10^–6 moles/l, respectively. ³H-DMPh bound on membranes could be exchanged by nonlabeled phalloidin but not by antamanide or tetraethylammonium ions.     

Interaction of Benzothiadiazides with Human Serum Albumin Studied by Dialysis and Spectroscopic Methods

The interaction of a series of benzothiadiazides with human serum albumin (HSA) was investigated by equilibrium dialysis (ED) and spectroscopic methods including circular dichroism (CD). The primary binding site of benzothiadiazides was designated site II, the diazepam site on the HSA molecule, as indicated by displacement experiments using different site-selective probes. Tyrosine and lysine amino acid residues were probably involved in the binding site of these compounds to HSA. Both electrostatic and hydrophobic interactions were found to play a role in the binding of these compounds to HSA. Among the compounds tested, chlorothiazide had the highest affinity (K₁ = 5.5 × 10⁴M^–1, K₂ = 5.8 × 10³ M^–1).The primary binding affinity of the compounds for HSA was of the order: chlorothiazide > cyclopenthiazide > polythiazide > ethiazide > trichlormethiazide = methyclothiazde > hydrochlorothiazide. Binding was insensitive to the N-B transition of HSA. The binding site is proposed to consist of a cationic site on the surface of the HSA molecule with a hydrophobic crevice to accommodate the aromatic ring of the compounds. Positions 3 and 7 of the benzothiadiazide molecule is thought to affect the binding affinity to HSA.     

Plasma protein binding and interaction studies with piroxicam

Summary The binding of non-steroidal antirheumatic drug piroxicam to human serum albumin, human plasma and serum has been studied by equilibrium dialysis at 22°C, pH 7.4. The binding data were analyzed according to Scatchard model. The values of binding parameters obtained for human serum albumin are quite similar to those obtained for human plasma and serum (N ₁=0.3, K ₁=3.0· 10⁵ l/mol; N ₂=7, K ₂=3.5 · 10³ l/mol). We suggest that piroxicam interacts with the albumin fraction in human plasma proteins.The displacement of piroxicam (in the therapeutical concentration of 4.5 · 10⁵ mol/l) from the binding to human serum albumin and human plasma has been studied. The concentration of albumin and albumin fraction in plasma was 2.9·10^–4 mol/l. The displacement substances were drugs — diazepam, warfarin and salicylic acid, and endogenous substances-bilirubin and palmitic acid. Only in the presence of salicylic acid in high clinical concentration (14.5·10^–4 mol/l) and palmitic acid in the molar ratio to albumin 5:1, free piroxicam substantially increased, which may be of clinical significance. Other studied substances displaced piroxicam only in high concentrations exceeding the therapeutical and physiological range.The evidence was found for the similarity of piroxicam and warfarin high-affinity binding site.     

The protein binding of methotrexate by the serum of normal subjects

Summary The protein binding of methotrexate by serum from eight normal volunteers was assessed by continuous ultrafiltration at pH 7.4 and 37°C. Methotrexate concentrations were measured by radioimmunoassay and the data analysed by the method of Scatchard. The major binding protein was albumin which bound 87.3% of the drug in serum. Analysis of the Scatchard plots indicated two distinct groups of binding sites. Class I was found to have 0.16±0.05 (S D) binding sites with an intrinsic association constant of 71.15±35.98 (S D)×10⁴ M^–1: Class II had 2.01±0.93 (S D) binding sites and an affinity of 0.18±0.15×10⁴ M^–1. No great change in the percentage of methotrexate bound occurred until the total concentration of the drug exceeded 50 µMol 1^–1.     

Modification of human serum albumin binding of methotrexate by folinic acid and certain drugs used in cancer chemotherapy

Summary The binding of methotrexate (MTX) and citrovorum factor (CF) to human serum albumin (HSA) was investigated. The affinity constant for MTX was 820 M^–1, with 2 binding sites, and for CF 2340 M^–1, with 1.5 binding sites. MTX and CF, which are used together in high dose therapy, compete for HSA binding. Competition for HSA binding between MTX and adriamycin, bleomycin and cyclophosphamide, drugs often used in association with MTX in cancer chemotherapy, was also demonstrated. The clinical importance of such competition depends on the drug/protein concentration ratio which is extremely variable.     

Gallopamil binding to human serum proteins

Summary We have determined the extent and variability in the binding of gallopamil to human serum proteins. Binding was determined by equilibrium dialysis in healthy volunteer serum, human serum albumin (45 g·l^–1), and alpha-1 acid glycoprotein (AAG) (600 mg·l^–1) at a pH of 7.4.Nonlinear regression analysis of gallopamil binding over a wide range of concentrations (10^–9 to 10^–4 M) in healthy volunteer serum suggested two classes of binding sites (k_ass.1=4.7×10⁵M^–1, k_ass.2=4.1×10⁴M^–1). These values were in close agreement with those obtained from binding to AAG and human serum albumin.Gallopamil free fraction over the concentration range of 10 to 100 ng·ml^–1 was independent of concentration. The free fraction in 20 volunteers was 0.075 at a concentration of 10 ng·ml^–1. Gallopamil free fractions were also determined in human serum albumin, to which various concentrations of AAG were added. Bound/free ratios correlated with AAG.As we changed the pH of the serum from 7.0 to 8.0, the free fraction changed from 0.1 to 0.05.Verapamil, lignocaine, procainamide, propranolol, 40H-propranolol, MEGX, and NAPA all caused an increase in the free fraction of gallopamil in serum. However, tocanide, quinidine, diltiazem, GX, norverapamil, D620, D617, and desacetyl diltiazem had no effect on gallopamil binding.Therefore, the data strongly suggest AAG as the high affinity, low capacity binding site and albumin as the low affinity, high capacity binding site for gallopamil, variability in gallopamil binding can be explained by alterations in AAG concentrations, pH, and the presence of other drugs and their metabolites.Presented in part at the 88th Annual Meeting of the American Society for Clinical Pharmacology and Therapeutics, March 1987, Orlando, Florida, USA     

Stopped-flow studies on drug-protein binding

Summary Binding curves obtained by the stopped-flow method for the association of warfarin and human serum albumin (HSA) at pH 6.0 and 9.0 have been analysed with digital- and analog computers. The first association product (WHSA') at pH 6–9 does not contribute to the observed fluorescence enhancement during warfarin-HSA complex formation. A similar consecutive relaxation process leads to a more stable warfarin-HSA complex, with HSA in the neutral (N)-form (pH 6) and base (B)-form (pH 9). This rearrangement can be measured by stopped-flow (k ₂=31 s^–1 at pH 6; k₂=63 s^–1 at pH 9). At pH 6 a further concentration dependent relaxation process has been observed indicating that the complex of warfarin with the N-form of HSA gets partially converted into its B-form with a half-time for this NB transition in the range of 0.2–0.4 s. A drug such as warfarin can act as effector molecule for conformational changes of the HSA tertiary and quaternary structure during the formation of a high affinity complex.     

Stability of warfarin solutions for drug-protein binding measurements: spectroscopic and chromatographic studies

Warfarin is commonly used in drug-protein binding studies as a displacement marker for Sudlow site I on the protein human serum albumin (HSA). This study examined the stability of aqueous warfarin solutions prepared for such experiments. This was investigated using NMR spectroscopy and affinity chromatography. It was found by 1H NMR that warfarin underwent a slow first-order conversion in aqueous solution. The rate of this reaction increased with temperature, giving rate constants at pH 7.4 of 0.0086 h(-1) at 25 degrees C and 0.041 h(-1) at 37 degrees C. It was concluded from further 1H and 13C NMR studies, along with molecular modeling, that this process involved the conversion of the minor cyclic hemiketal form of warfarin to its major cyclic hemiketal. This reaction had a small but measurable effect on the binding of R- and S-warfarin to HSA, as demonstrated by HPLC using an immobilized HSA affinity column. From this work, general guidelines were developed concerning the usable lifetimes for warfarin that is prepared in aqueous solutions for studies of drug-protein binding.     

Binding of a metabolite of triflusal (2-hydroxy-4-trifluoromethylbenzoic acid) to serum proteins in rat and man

Summary 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) is the main active metabolite of the platelet anti-aggregant drug triflusal. Its binding to plasma proteins of rats and healthy volunteers in vitro and in vivo has been studied.Rats were given a single oral dose of 50 mg·kg^–1 triflusal and the healthy volunteers received 300 mg as a single oral dose or a multiple dose regimen of 600 mg every 24 h and 300 mg every 8 h, both for 13 days. Protein-free HTB was obtained by ultrafiltration. Unbound and total HTB concentrations were determined by HPLC.HTB was primarily bound to albumin in plasma. The Scatchard plots suggested two types of binding sites for HTB on the albumin molecule. In rats, the binding constants (K=intrinsic affinity constant, n=number of binding sites) were K₁=1.4×10⁵ l·mol^–1, n₁=1.23, and K₂=4.1×10³ l·mol^–1 and n₂=3.77. The mean plasma concentration in rats after oral administration was 185 (37) g·ml^–1 (protein-free HTB: 2.44 (0.77)%). The binding constants in human plasma were K₁=4.7×10⁵ l·mol^–1, n₁=1.93, K₂=4.3 l·mol^–1 and n₂=4.28.The plasma HTB concentration in man (n=8) was 35 g·ml^–1 (C_max) after a single oral dose of triflusal 300 mg, 172.96 g·ml^–1 (C_max·ss) during the multiple dosage regimen of 300 mg every 8 h, and 131 g·ml^–1 (C_max·ss) during the multiple oral dose regimen of 600 mg every 24 h. Unbound HTB ranged from 0.27 to 0.43%, depending on dose. HTB had high affinity for plasma albumin, which was not saturable after therapeutic doses. It showed linear elimination.     

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     

Molecular Mechanisms of the Adsorption of a Model Protein (Human Serum Albumin) on Poly(Methylidene Malonate 2.1.2) Nanoparticles

Purpose. To understand the molecular mechanisms involved in protein–methylidene malonate 2.1.2 polymer interactions. Methods. To assess the importance of electrostatic forces in polymer-protein interactions use was made of HAS and its derivatives, which were anionized by succinylation and aconitylation. Surface plasmon resonance measurements, using the three HSA molecules as immobilized ligands and polymer nanoparticles as analytes in the liquid phase, allowed the determination of initial kinetic constants and affinity constants at equilibrium at two different temperatures. Results. Saturation of binding for the three proteins occurred at approximately 900 protein molecules/nanoparticle. The apparent affinity decreased with increasing electronegativity of the proteins. Surface plasmon resonance measurement of proteins, covalently linked to the chip matrix, showed a high affinity for the nanoparticles (K_A 10¹⁰ M^-1) and confirmed the moderate decrease of affinity with increasing electronegativity of the modified albumins. Measurements at 25 and 37°C showed no significant increase in the albumin-nanoparticle interactions. Dissociation of the proteins from the nanoparticles could only be realized with chaotropic salt solutions. Conclusions. These results suggest the molecular forces initiating the protein-nanoparticle interactions are mainly of electrostatic nature followed by stabilization by hydrophobic forces. The high affinity confirms the nanoparticles as excellent carriers for protein delivery.     

Protein binding of 2-chloro 2′-deoxyadenosine (cladribine) in healthy subjects and in patients with leukaemia

The plasma protein binding of 2-chloro-2-deoxyadenosine (CdA) at 37°;C was studied by ultrafiltration in 5 healthy volunteers, in 11 patients with haematological malignancies and in purified protein preparations. In the patients, the binding of CdA to plasma proteins was 25.0% and in healthy subjects it was 21.1%. In a solution of human serum albumin (40 g·1^–1), 24.3% CdA was bound, but less than 5% was bound in a solution of ₁-acid-glycoprotein (0.7 g·1^–1). No dependence of binding on the concentration of CdA was found within a range 25–1000 nmol·1^–1.In conclusion, due to its limited binding to plasma proteins, any change in the binding of CdA is unlikely to have a major influence on its pharmacological effect.     

Stability of the New Anticancer Platinum Analogue 1,2-Diaminomethyl-Cyclobutane-Platinum(II)-Lactate (Lobaplatin; D19466) in Intravenous Solutions

The chemical stability of the new anticancer platinum analogue 1,2-diaminomethyl-cyclobutane-platinum(II)-lactate (D19466) in infusion media was studied in an accelerated stability testing experiment with a selective HPLC-UV method. Variables were time, temperature, light, concentration, and infusion mixture. Mean reaction rate constants of decomposition were, respectively, 0.9555 *10^–2, 2.127 *10^–2, and 4.221 *10^–2 hr^–1 at 37, 56, and 66°C at a concentration of 200 mg/L in normal saline. From the Arrhenius equation, shelf lives (5% loss) at 4, 22, 37, and 121°C were, respectively, calculated to be 41.6, 13.2, 5.7, and 0.15 hr. Mean reaction rate constant in 5% dextrose was 3.106 * 10^–2 hr^–1 (200 mg/L; 56°C) and differed from that in normal saline (P < 0.005). Mean reaction rate constant in Ringer lactate was 2.084 *10^–2 hr^–1 (200 mg/L; 56°C) (P > 0.05). There was no influence of normal daylight on the rate of decomposition. It is recommended to prepare D19466 infusions in normal saline. Chemical stability is then maximal 12 hr at room temperature or 24 hr at 4°C. No protection against normal daylight is required. Sterilization by heat is not possible.     

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.     

Albumin binding in uraemia: quantitative assessment of inhibition by endogenous ligands and carbamylation of albumin

Summary The binding capacity of human serum albumin (HSA) for small acidic molecules is known to be reduced in chronic renal failure (CRF). The contribution of competitive inhibition by accumulated endogenous ligands and of structural changes in HSA has now been evaluated. In a fluorimetric in vitro assay using HSA and two dansylated amino acids the inhibitory properties of various endogenous ligands were determined in concentration-effect studies. The effect of carbamylation of HSA on binding was also examined. The mode of inhibition, including binding parameters n and K_a, was determined. Finally, HSA binding in sera from controls and dialysis patients was compared in a modified assay.Thirty three substances were tested and were placed in 3 groups: strong inhibitors (IC₅₀ < 3*10^–5 mol · 1^–1, e. g. indolyl acids, furanoic acids), medium inhibitors (IC₅₀ > 3*10^–5, eg. vanillic acid), and no inhibition (e.g. urea, creatinine, guanidino compounds). Complete ( > 80 %) carbamylation of HSA reduced binding by 67 in a non-competitive mode. There was a significant reduction in the binding capacity of HSA from the dialysis patients ( 24 %), irrespective of medication.It is concluded that the uraemic binding defect of HSA is caused by competitive inhibition by the many physiological ligands accumulated in CRF and structural modifications of HSA. The assay presented proved useful for the rapid analysis of possible HSA binding inhibitors and for testing large groups of patients, e. g. comparison of dialysis treatments, and pharmacological binding studies.     

Human serum albumin interaction with oxaliplatin studied by capillary isoelectric focusing with the whole column imaging detection and spectroscopic method

Capillary isoelectric focusing (CIEF) with whole column imaging detection (WCID) was used to investigate drug-protein interactions. This study was designed to examine the interaction between the platinum-based anticancer drug, oxaliplatin, with human serum albumin (HSA) in aqueous solution at physiological pH with drug concentrations of 10 to 100 μM and a constant concentration of HSA (5.0 × 10⁻⁵ M). The reaction mixtures were incubated for 0, 0.5, 1, 12, 24, 48 and 72 h at 37 °C in a water bath. The CIEF results indicate that with increasing the drug concentration, the complex formation of protein adducts increased compared to low-drug concentrations and major structural changes were observed as the incubation time progressed. The altered CIEF profile demonstrated the possible conformation change due to the binding of the drug. Results also showed a significant protein's pI shift for higher HSA–oxaliplatin incubation ratios. Furthermore, spectroscopic evidence shows that oxaliplatin caused the fluorescence quenching of HSA by formation of HSA–oxaliplatin complex. Using the Stern–Volmer equation, the quenching constants were calculated in the linear range. The quenching rate constants K_q at three different temperatures indicating the presence of static quenching mechanism in the interactions of oxaliplatin with HSA. This paper describes the validity of the CIEF-WCID technique for the study of protein–drug interactions and provides useful information and insight into the interaction of anticancer drugs with HSA.