Serum albumin and lipoproteins as the quinidine binding molecules in normal human sera.

To substantiate the binding of quinidine in human sera and predict variations of binding dissociation constants and number of binding sites were determined for separate serum proteins. Human sera were fractionated by gel filtration and ultracentrifugation, and binding was evaluated by equilibrium dialysis at pH 7·30 at 20° and 37° in a Krebs-Ringer phosphate buffer. Quinidine was bound to all serum lipoproteins and to serum albumin. The binding was influenced by the buffer composition. In sodium phosphate buffer there were two separate binding sites for quinidine on LDL and HDL, while there was only one detectable binding site on VLDL and HDL in a Krebs-Ringer phosphate buffer. On LDL also there appeared to be one binding site but it exhibited a positive cooperative binding effect at lower concentrations of quinidine. This effect was assumed to be caused by inorganic ions of the Krebs-Ringer phosphate buffer. At a therapeutic level of quinidine in normal human serum the concentration of quinidine bound to serum proteins was 1·062 × 10^?5 M. Calculated from the evaluated binding parameters VLDL contributed with 0·101 × 10^?5 M of this binding, LDL with 0·143 × 10^?5 M, HDL with 0·083 × 10^?5 M and albumin with 0·699 × 10^?5 M.     

The influence of salts and differences in protein isolation procedure on the binding of quinidine to human serum albumin

The influence of buffer and two different albumin preparations on the albumin-quinidine interaction was investigated. Human serum albumin was prepared by either alcohol fractionation or ultracentrifugation with subsequent gel filtration. The interaction between albumin and quinidine was determined by equilibrium dialysis. It was inhibited by halide ions and consequently different binding parameters were found to be valid for the complex in sodium phosphate and in Krebs-Ringer phosphate buffer. The influence of Ca²⁺, Mg²⁺ and SO^2?₄ in physiological amounts was negligible. The albumin obtained by alcohol fractionation possessed one binding site for quinidine, while the albumin isolated by ultracentrifugation with subsequent gel filtration possessed two binding sites when tested in a Krebs-Ringer phosphate buffer. In sodium phosphate buffer both albumin preparations had two independent binding sites, and showed essentially identical binding parameters.     

Binding of [3H]mianserin to bovine serum albumin,human serum albumin and α1-acid glycoprotein

Scatchard plots which were curvilinear with negative slopes were obtained when the binding of [³H]mianserin to bovine serum albumin (BSA), human serum albumin (HSA), defatted human serum albumin (D-HSA) and α₁-acid glycoprotein (α₁-AGP) was studied with equilibrium dialysis with constant protein concentrations and various ligand concentrations. Binding parameters were estimated graphically and with a non-linear least-squares computer program, assuming two classes of independent binding sites. α₁-AGP had the highest binding affinity (K) and binding capacity (nK). The binding parameters, n and K were not independent of protein concentration when the BSA concentration was varied. Linear atypical Scatchard plots with positive slopes were obtained when the protein concentration was varied for BSA, HSA and D-HSA, at a fixed ligand concentration.     

Binding of phenytoin, L-tryptophan and O-methyl red to albumin. Unexpected effect of albumin concentration on the binding of phenytoin and L-tryptophan

The binding of phenyl in and o-methyl red to HSA and the binding of L-tryptophan to BSA has been studied by equilibrium dialysis at 37°, pH 7.4. The data for the binding of o-methyl red to HSA studied by either variation of the o-methyl red concentration or variation of the albumin concentration gave identical Scatchard plots. Scatchard plots of the data obtained for phenytoin and l-tryptophan, at constant ligand concentration, but with a range of albumin concentrations, were unusual and had a positive slope. Values for the apparent association constant (k) and number of binding sites (n) could not be obtained from these plots, but it was apparent that n and/or k decrease as albumin concentration increases.     

Further studies of binding of bromosulphthalein sodium by human serum albumin: Effects of albumin concentration and buffer composition

1. Binding isotherms of equilibrium solution concentration of bromosulphthalein (BSP) determined on the number of moles of BSP bound per mole of human serum albumin (HSA) in 310 ideal milliosmolar pH 7.4, Krebs-Henseleit and Krebs improved mammalian Ringer number 1 buffers at 37 degrees C were determined using continuous diafiltration. The albumin concentration range was from about 10 to 30 g/litre.2. The results indicate a competition between HSA polymerization and HSA binding BSP, confirming in more physiological conditions, the findings of Crawford, Jones, Thompson & Wells (1972) with pH 7.4 phosphate buffer.3. The results in Krebs-Henseleit buffer were markedly different from those in Krebs mammalian Ringer buffer and it is suggested that the differences in ionic composition influence the HSA conformation and so affect the competition between HSA polymerization and HSA binding BSP.     

Study of curcumin and genistein interactions with human serum albumin

Curcumin, the yellow pigment from the rhizoma of Curcuma longa, is a widely studied polyphenolic compound which has a variety of biological activity as anti-inflammatory and antioxidative agent. Genistein one of the flavonoids found in soybean and chickpeas inhibits DNA strand breaks acting as a direct scavenger of reactive oxygen species. Human serum albumin (HSA) with high affinity binding sites is a major transporter for delivering several endogenous compounds and drugs in vivo. The aim of this study was to examine the interactions of curcumin and genistein with human serum albumin at physiological conditions, using constant protein concentration and various pigment contents. FTIR, UV–Visible, CD and fluorescence spectroscopic methods were used to analyse drug binding mode, the binding constant and the effects of pigment complexation on HSA stability and conformation. Structural analysis showed that curcumin and genistein bind HSA via polypeptide polar groups with overall binding constants of K_curcumin = 5.5 (±0.8) × 10⁴ M⁻¹ and K_genistein = 2.4 (±0.40) × 10⁴ M⁻¹. The number of bound pigment (n) is 1.33 for curcumin and 1.49 for genistein. The HSA conformation was altered by pigment complexation with reduction of α-helix and increase of random coil and turn structures suggesting a partial protein unfolding.     

Erythromycin binding to human serum

Erythromycin binding to human serum was measured under conditions of binding equilibrium. The binding is sensitive to pH changes, decreasing at acid pH. Over a great range of serum dilution, the bound fraction is semilogarithmically related to serum concentration. Binding is shown to be completely reversible. With increasing erythromycin concentration a specific part of binding is saturable and specifically displaceable by erythromycin in excess, whereas a nonspecific part linearly increases with total concentration. Erythromycin is specifically bound to a single class of noninteracting binding sites with an apparent dissociation constant K_d=5.9 μM (38°C). The kinetic and thermodynamic parameters at 25° are: K_d = 8.4 μM, ΔH° = +4.4 × 10³ cal per mole, ΔG° = ?6.9 × 10³ cal per mole, ΔS°= +38 e.u.     

Variable binding of propranolol in human serum.

Human serum proteins were fractionated by ultracentrifugation and gel filtration. Binding of propranolol was determined by equilibrium dialysis. Propranolol was distributed to lipoproteins independent of drug concentration. Two groups of propranolol binding sites were found to be present in the protein preparation containing albumin, α₁-acid glycoprotein, transferrin and prealbumin. The first binding site with a dissociation constant of 7.5 × 10^?7 was present in number equivalent to concentration of α₁-acid glycoprotein. The propranolol binding to serum samples from 21 healthy males expressed as binding ratio B/F and per cent binding ranged from 7.5 to 19.2 and 88.2 to 95.0 respectively. The binding ratio was correlated to concentration of α₁-acid glycoprotein (r = 0.85, P < 0.001), but not to concentrations of albumin and lipoproteins. The results indicate that α₁-acid glycoprotein is the main propranolol binding protein in human serum.     

Factors influencing the protein binding of azosemide using an equilibrium dialysis technique

Various factors most likely to influence the plasma protein binding of azosemide to 4% human serum albumin (HSA) were evaluated using equilibrium dialysis at the initial azosemide concentration of 10 μg mL^?1. It took approximately 8h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% dextran (the ‘buffer’) using a Spectra/Por 2 membrane (molecular weight cut-off 12000–14000) in a water bath shaker kept at 37°C and a rate of 50 oscillations min^?1. Azosemide was fairly stable both in 4% HSA and in the ‘buffer’ for up to 24h. The binding of azosemide to 4% HSA was constant (95.5 ± 0.142%) at azosemide concentrations ranging from 5 to 100 μg mL^?1. However, the extent of binding was dependent on HSA concentration: the values were 88.4, 91.0, 92.2, 94.2, 94.9, 94.9, and 94.9% at albumin concentrations of 0.5, 1, 2, 3, 4, 5, and 6% respectively. The binding was also dependent on incubation temperature; the binding values were 97.0, 94.9, and 94.9% when incubated at 6, 28, and 37°C, respectively. The binding of azosemide was also influenced by buffers containing various chloride ion concentrations and buffer pHs. The binding values were 95.3, 94.9, and 93.6% for the chloride ion concentrations of 0, 0.249, and 0.546%, respectively, and the unbound values were 6.8, 5.1, 3.8, 3.4, and 3.3% for buffer pHs of 5.8, 6.4, 7.0, 7.4, and 8.0, respectively. The binding of azosemide was independent of the quantity of heparin (up to 40 UmL^?1), AAG (up to 0.16%), sodium azide (NaN₃, up to 5%), its metabolite, Ml (up to 10 μg mL^?1), and anticoagulants (EDTA and citrate).     

Application of headspace solid phase microextraction for study of noncovalent interaction of borneol with human serum albumin

Aim:

To investigate noncovalent interactions between borneol and human serum albumin (HSA) under near-physiological conditions.

Methods:

A 65-μm polydimethylsiloxane (PDMS) fiber was selected for sampling. The extraction temperature was kept at 37 °C, and the extraction time was optimized at 10 min. Borneol solutions of different concentrations were equilibrated in 600 μmol/L HSA and 67 mmol/L phosphate buffer solution (pH 7.4, 37 °C) for 24 h prior to solid phase microextraction (SPME) using headspace mode. The binding properties were obtained based on the calculation of extracted borneol amount using gas chromatography (GC) determination.

Results:

The headspace SPME extraction method avoided disturbance from the HSA binding matrix. The recovery showed good linearity for the borneol concentrations over the range of 0.4–16.3 μmol/L with a regression coefficient (R²) of 0.9998. The limit of detection and lower limit of quantitation were determined to be 0.01 μmol/L and 0.4 μmol/L, respectively. The binding constant and the percentage binding rate were estimated to be 2.4×10³(mol/L)^-1 and 59.5%, respectively.

Conclusion:

Headspace SPME coupled to GC is a simple, sensitive and rapid method for the study of borneol binding to HSA. The method may be applied in the determination of other protein binding properties in human plasma.     

The Binding of Thiopental to Serum Proteins Determined by Ultrafiltration and Equilibrium Dialysis

Abstract: An ultra filtration method is described by which it is possible to estimate the protein bound fraction of a drug at its original concentration in a serum sample. The determination is carried out at 37° and pH 7.4. For thiopental no difference was found between the values of protein binding whether they were determined by the ultrafiltration method or by a less time consuming equilibrium dialysis against an equal volume of phosphate buffered isotonic sodium chloride solution. The equilibrium dialysis was used to measure the concentration of bound and unbound thiopental molecules; and the binding parameters in a two class binding model were determined. No evidence was found for binding to other proteins than albumin. About one binding site belonging to the first class was found per 1000 albumin molecules whereas an average of about 5 secondary sites were found for each albumin molecule. The association constants for the primary class of binding sites were 3.4×10⁶M^?1 for albumin and 13.3×10⁶M^?1 for serum while the values for the secondary class were 2.2 and 1.2×10³M^?1 for albumin and serum respectively. The estimates of association constants and number of binding sites were based on experiments with total thiopental concentrations ranging from 0.4 to 80 μg/ml. In a phosphate buffered albumin solution with 2 g albumin per 100 ml, a pH increase from 5 to 8 caused an increase in protein binding from 36 to 76%. A Scatchard plot using data from experiments with increasing albumin concentrations resulted in a “plot” with positive slope. The use of a curve like this is discussed and it is concluded that the binding parameters for thiopental are influenced by the albumin concentration.     

Electrophoretic studies of clioquinol binding to human serum proteins.

The binding of radioisotope-labeled clioquinol to human serum proteins in vitro was studied by means of agarose gel electrophoresis. The technique gave a satisfactory separation of free and bound forms of clioquinol without a significant interaction between clioquinol and the supporting medium even at considerably high concentrations of the drug. The clioquinol binding proteins in serum were identified as albumin and lipo-proteins. Clioquinol was bound to albumin at an equimolar ratio with an apparent binding constant of 5.6 × 10⁴ M^?1. When the amount of clioquinol exceeded the binding capacity of albumin, lipoprotein classes of very low density, low density and high density were capable of serving as auxiliary carrier proteins of clioquinol.     

Factors influencing the protein binding of vancomycin.

Various factors influencing the protein binding of vancomycin were examined using equilibrium dialysis method. Four per cent human serum albumin (HSA) and/or 0.08 per cent alpha-1-acid glycoprotein (AAG), dissolved in isotonic phosphate buffer, were dialyzed against isotonic phosphate buffer of pH 7.4 using Spectrapor 2 membrane. The protein binding of vancomycin to 0.08 per cent AAG was dependent on vancomycin concentrations; the values ranged from 21.1 per cent at the vancomycin concentration of 20 micrograms ml-1 to 5.30 per cent at 2400 micrograms ml-1. However, binding to 4 per cent HSA was relatively constant, 8.79 +/- 2.43 per cent over a vancomycin concentration range of 20-2400 micrograms ml-1. The values to 4 per cent HSA alone and 0.08 per cent AAG alone did not predict the greater binding of vancomycin in the presence of both proteins, especially at higher concentrations of vancomycin; the values to 4 per cent HSA with 0.08 per cent AAG were constant, 26.3 +/- 3.74 per cent, at the vancomycin concentration range of 20-2400 micrograms ml-1. This suggested an interaction between the proteins, which resulted in enhanced binding of vancomycin. The protein binding of vancomycin to 4 per cent HSA with 0.08 per cent AAG was not influenced by the different incubation temperatures (4 degrees, 22 degrees, and 37 degrees), quantities of heparin (up to 40 units ml-1) or AAG (up to 0.16 per cent), or buffers (isotonic phosphate buffer of pH 7.4, phosphate buffer of pH 7.4 and 0.9 per cent NaCl solution) at the vancomycin concentration of 80 micrograms ml-1. Vancomycin was found to be stable in human serum albumin or in isotonic phosphate buffer of pH 7.4.     

Spectroscopic and molecular docking studies for characterizing binding mechanism and conformational changes of human serum albumin upon interaction with Telmisartan

Human serum albumin (HSA), one of the most copious plasma proteins is responsible for binding and transportation of many exogenous and endogenous ligands including drugs. In this study, we intended to explore the extent and types of binding interaction present between HSA and the antihypertensive drug, telmisartan (TLM). The conformational changes in HSA due to this binding were also studied using different spectroscopic and molecular docking techniques. The spectral shifting and intensity variations upon interaction with TLM were studied using FT-IR spectroscopy. Binding constant and the change in absorption of HSA at its λ_max was analyzed using absorption spectroscopy. Eventually, the types and extent of binding interactions were confirmed using molecular docking technique. Results have shown that TLM significantly interacts with the binding site-1 of HSA utilizing strong hydrogen bonding with Glu292, and Lys195 residues. The UV-absorption intensities were found to be decreased serially as the drug concentration increased with a binding constant of 1.01 × 10³ M⁻¹. The secondary structure analysis using FT-IR spectroscopy also revealed a marked reduction in the α-helix (56%) component of HSA on interaction. This study gives critical insights into the interaction of TLM with HSA protein which eventually affects the concentration of TLM reaching the site of action and ultimately its therapeutic profile.     

Fluorescence spectroscopic study of serum albumin-bromadiolone interaction: fluorimetric determination of bromadiolone

Bromadiolone (BRD), a substituted 4-hydroxycoumarin derivative, is known to possess anti-coagulant activity with acute toxicity. In this paper, we report a study on the interaction of bromadiolone with the plasma proteins bovine serum albumin (BSA) and human serum albumin (HSA), using the intrinsic fluorescence emission properties of bromadiolone. Bromadiolone is weakly fluorescent in aqueous buffer medium, with an emission at 397 nm. Binding of bromadiolone with serum albumins (SA) leads to a marked enhancement in the fluorescence emission intensity and steady state fluorescence anisotropy (r_ss), accompanied by a blueshift of 10 nm. In the serum albumin–bromadiolone complex, selective excitation of tryptophan (Trp) residue results in emission from bromadiolone, thereby indicating a Förster type energy transfer from Trp to BRD. This quenching of Trp fluorescence by BRD was used to estimate the binding constant of the SA–BRD complex. The binding constants for BRD with BSA and HSA were 7.5 × 10⁴ and 3.7 × 10⁵ L mol⁻¹, respectively. Based on this, a new method involving SA as fluorescence-enhancing reagent for estimation of BRD in aqueous samples has been suggested. The detection limits of bromadiolone under the optimum conditions were 0.77 and 0.19 μg mL⁻¹ in presence of BSA and HSA, respectively.     

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.     

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.     

Interaction studies of a novel Co(II)-based potential chemotherapeutic agent with human serum albumin (HSA) employing biophysical techniques

Novel macrocyclic cobalt(II) complex C₃₆H₂₄N₈O₄S₄CoCl₂ with a butterfly topology was synthesized and characterized by spectroscopic (IR, ¹H, ¹³C NMR, EPR, UV-Vis, ESI-MS) and analytical methods. The complex exhibits distorted octahedral geometry around Co(II) metal ion, which was confirmed by EPR measurements with g_a, g_b, and g_c values (8.01, 2.20, and 1.66), respectively, and molar extinction coefficient ε = 58 M⁻¹cm⁻¹. The Interaction studies with human serum albumin (HSA) in phosphate buffer (0.1 M, pH 7.0) were studied by electronic absorption titration, fluorescence titration, circular dichroism, and cyclic voltammetry. Hyperchromism in fluorescence intensity indicates binding of complex with HSA near tryptophan residue in IIA subdomain leading to less polar microenvironment around tryptophan and more at tyrosine. The intrinsic binding constant K _b obtained from absorption spectral titrations was found to be 9.3 × 10⁴ M⁻¹, suggesting medium binding affinity of HSA with complex. CD spectrum indicates α-helical structure up to β-pleated secondary structure. CV data confirmed medium reversible binding with HSA. The binding of complex with HSA shows typical reversible mode of binding, which enables the delivery of drug candidate to the tissue enzymes and receptors in an efficient manner, and thereby affects the uptake of the drug.     

Cooperative interaction of warfarin and phenylbutazone with human serum albumin

Phenylbutazone is commonly thought to displace warfarin from human serum albumin (HSA) by direct competition, and the aim of the present study was to confirm or deny this mechanism using standard equilibrium dialysis. A number of titration curves for [¹⁴C]warfarin (4–1000 μm) in the presence of fixed concentrations of phenylbutazone (0–2000 μM) were obtained at 25° in 0.1 M, pH 7.0 phosphate buffer. The first two site dissociation constants (K₁ and K₂) for each of these curves were estimated according to Adair's equation. Comparison of K₁ with that predicted for a single site directly competitive model showed large discrepancies, indicating unacceptability of the model beyond experimental error. The data for the first warfarin site could be partly explained by a dual interaction mechanism where at lower phenylbutazone concentration (<100 μM) the interaction is negatively cooperative, and at higher levels (100–1000 μM) further displacement is caused by direct competition. Phenylbutazone also affects the second warfarin site resulting in an apparent positive cooperativity between the two warfarin sites. The results of this study suggest caution in assigning drugs to common binding sites on the basis of simple displacement data.     

Interaction of 3′ -O-caffeoyl d-quinic acid with human serum albumin

The interaction of chlorogenic acid (CGA) with human serum albumin (HSA) was studied from the viewpoint of thermodynamics and mechanism of binding at pH 6.0. The association constants (K_a) for the HSA-CGA interaction at 10, 25 and 40° C were 6.0 × 10⁴, 9.0 × 10³ and 2 × 10⁴ M^?1, resulting in AG of -6.21, -5.80, -6.32 kcal/mol, respectively. These high K_a -values showed that the interaction between CGA and HSA is strong, endothermic and entropically driven. Binding of chlorogenic acid induces conformational change in HSA as indicated by quenching of fluorescence emission intensity along with a red shift in the emission maxima from 338 to 350 mm. This suggested the involvement of the lone tryptophan residue in the region of binding. Far-ultraviolet circular dichroic data showed a decrease in the α-helical content of HSA from 56 to 50% upon binding of CGA. These data are also supported by the decrease in the apparent Tm of HSA by 4°C upon binding of CGA causing destabilization of the HSA molecule. The kinetics of the interaction involves a single step in the binding, and the kinetic curve attains equilibrium within 180 ± 5 s. Data on caffeic acid (CA) and quinic acid (QA), which are the hydrolysis products of the bidentate CGA molecule, indicate that CA interacts more strongly than CGA. CA binds with an association constant of 8 × 10⁴ M^?1and with a maximum number of binding sites of four. Microcalorimetric investigation of the interaction of these ligands with HSA suggests that the strength of binding follows the order CA?CGA?>QA with a single class of binding sites. The effect of temperature on the binding of CGA to HSA showed that the interaction is dominated by hydrophobic forces and hydrogen bonding.