warfarin and dansylsarcosine

The half-life of fatty acid-conjugated antidiabetic drugs are prolonged through binding to albumin, but this may not occur in diabetic patients with nephropathy complicated with hypoalbuminemia. We previously showed that human serum albumin (HSA) dimerized at the protein's Cys34 by 1,6-bis(maleimido)hexane has longer half-life than the monomer under high permeability conditions. The aim of this study was to investigate the superior ability of this HSA dimer as a plasma-retaining agent for fatty acid conjugated antidiabetic drugs.. The diabetic nephropathy rat model was prepared by administering a single injection of streptozotocin (STZ) intravenously, and the pharmacokinetic properties of HSA monomer and dimer were evaluated. Site-specific fluorescent probe displacement experiments were performed using warfarin and dansylsarcosine as site I and site II specific fluorescent probes, respectively.. The half-life of the HSA dimer in STZ-induced diabetic nephropathy model rats was 1.5 times longer than the HSA monomer. The fluorescent probe displacement experiment results for HSA monomer and dimer were similar, where fatty acid-conjugated antidiabetic drugs displaced dansylsarcosine but not warfarin in a concentration-dependent manner.. The HSA dimer shows potential for use as a plasma-retaining agent for antidiabetic drugs due to its favourable pharmacokinetic properties.

Topics: Animals; Binding Sites; Dansyl Compounds; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Dimerization; Drug Carriers; Fatty Acids; Fluorescent Dyes; Half-Life; Humans; Hypoglycemic Agents; Male; Maleimides; Permeability; Plasma; Protein Binding; Rats; Rats, Sprague-Dawley; Sarcosine; Serum Albumin; Warfarin

Human serum albumin nonenzymatically condenses with glucose to form stable Amadori adducts that are increased with the hyperglycemia of diabetes. The present study evaluated the influence of fatty acids, which are major endogenous ligands, on albumin glycation and of glycation on albumin conformation and exogenous ligand binding. Physiologic concentrations of palmitate, oleate, and linoleate reduced the ability of albumin to form glucose adducts, whereas glycation decreased intrinsic fluorescence, lowered the affinity for dansylsarcosine, and diminished the fatty acid-induced increase in limiting fluorescence of protein-bound warfarin that was observed with nonglycated albumin. The findings indicate that fatty acids impede the ability of albumin to undergo Amadori glucose modification and induce conformational changes affecting exogenous ligand binding, and that nonenzymatic glycation of albumin induces alterations in structural and functional properties that may have import in lipid transport and atherogenesis.

Topics: Dansyl Compounds; Fatty Acids, Unsaturated; Fluorescence; Glycosylation; Humans; Linoleic Acid; Oleic Acid; Palmitic Acid; Sarcosine; Serum Albumin; Warfarin

High-throughput screening of combinatorial libraries has evolved from studying large diverse libraries to analyzing small, structurally similar, focused libraries. This paradigm shift has generated a need for rapid screening technologies to screen both diverse and focused libraries in a simple, efficient, and inexpensive manner. We have proactively addressed these needs by developing a high-throughput, solution-based method combining size exclusion (SEC), two-dimensional liquid chromatography (2-D LC), and mass spectrometry (MS) for determining the relative binding of drug candidates in small, focused medicinal libraries against human serum albumin (HSA). Two types of libraries were used to evaluate the performance of the system. The first consisted of five diverse ligands with a wide range of hydrophobicities and whose association constants to HSA cover 3 orders of magnitude. A beta-lactam library composed of structurally similar compounds was used to further confirm the validity of the methodology. The ability to distinguish site-specific interactions of drugs competing for individual domains of the HSA receptor is also demonstrated. Comparison of chromatographic profiles of the library components before and after incubation with the receptor using multiple reaction monitoring allowed a ranking of the ligands according to their relative binding affinities. The observed rankings correlate closely with literature values of the association constants between the respective ligands and HSA. This simple, rugged methodology can screen a wide spectrum of chemical entities from combinatorial mixtures in less than 6 min.

Topics: Acecainide; beta-Lactams; Chromatography, Gel; Chromatography, Liquid; Combinatorial Chemistry Techniques; Dansyl Compounds; Humans; Imipramine; Indomethacin; Pharmaceutical Preparations; Protein Binding; Quinidine; Reproducibility of Results; Sarcosine; Serum Albumin; Tandem Mass Spectrometry; Tryptophan; Warfarin

Two approaches were utilized to increase the throughput of pulsed ultrafiltration assays of ligand binding to human serum albumin, reducing the volume of the ultrafiltration chamber and combining pulsed ultrafiltration with high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-MS). Affinity constants for binding of ligands to human serum albumin were determined using pulsed ultrafiltration with ultraviolet absorbance detection. The first affinity constants (Ka1) were measured for the binding of dansylsarcosine, dansylamide, 7-anilinocoumarin-4-acetic acid and warfarin, and were determined to be 1.8 x 105, 5 x 104, 8 x 104, and 2.0 x 105 M-1, respectively. The throughput of pulsed ultrafiltration analyses was tripled compared to previous pulsed ultrafiltration measurements by reducing the volume of the chamber. In addition, the use of LC-MS with pulsed ultrafiltration permitted the simultaneous comparison and rank ordering of ligand mixtures for binding to serum albumin. For example, the throughput of these pulsed ultrafiltration measurements was tripled by analyzing three ligands as a mixture.

Topics: Chromatography, Liquid; Dansyl Compounds; Humans; Ligands; Mass Spectrometry; Sarcosine; Serum Albumin; Ultrafiltration; Warfarin

The structure of human serum albumin (HSA) in the pressure-induced denatured state was investigated by fluorescence spectroscopy. HSA undergoes a conformational change in the pressure range from 0.1 MPa to 400 MPa, at 25 degrees C. Several ligands bind to specific sites in HSA, and the fluorescence spectra of these ligands were used to study the conformational state of this protein. The warfarin-binding site (site I) and the dansylsarcosine-binding site (site II), are located in subdomains II and III, respectively. The fluorescence spectra of these probes reflected the structural changes in each of these subdomains. Dansylsarcosine completely dissociated from its binding site in domain III above 300 MPa, but substantial affinity of warfarin remained in this pressure range. Similar results were obtained for the urea-induced denaturation of HSA; although dansylsarcosine completely dissociated at urea concentration above 6 M, warfarin remained bound to site I in domain II at these concentrations. These results suggest that the structure of domain III is unfolded both in the initial stages of both pressure- and urea-induced denaturation of HSA. HSA possesses a single tryptophan residue (Trp-214) in domain II, and fluorescence from this residue reflects structural changes in this domain. In the urea-induced denatured state of HSA, a red-shift in the wavelength of maximum fluorescence occurred over urea concentrations ranging from 4 M to 6 M. This shift indicated that a structural change in domain II occurred simultaneously with the unfolding of domain III in this concentration range. On other hand, the shift in the wavelength of maximum fluorescence of Trp-214 was comparatively small in the pressure range from 0.1 MPa to 400 MPa indicating that the environment of Trp-214 was not affected. These results indicate that preferential unfolding of domain III occurs in the pressure-induced denatured state of HSA.

Topics: Bilirubin; Circular Dichroism; Dansyl Compounds; Fluorescent Dyes; Humans; Pressure; Protein Conformation; Protein Denaturation; Sarcosine; Serum Albumin; Spectrometry, Fluorescence; Urea; Warfarin

Mammalian albumins have two main structurally selective ligand binding sites. Site I binds drugs such as azapropazone, phenylbutazone and warfarin; whereas benzodiazepines, some dansyl amino acids, such as dansylsarcosine, and short chain fatty acids like octanoic acid interact with site II. However, it is not known if non-mammalian albumins have similar binding loci. In this study, drug binding sites on chicken albumin were investigated using site selective fluorescent probes (warfarin and dansylsarcosine) and p-nitrophenyl acetate (NPA); the hydrolysis of which is selectively inhibited by site II ligands. Azapropazone and phenylbutazone decreased the binding of warfarin and dansylsarcosine to a similar extent. Diazepam and octanoic acid also inhibited binding of the two fluorescent probes in a non-selective manner. However, the fluorescence intensity of the warfarin-chicken albumin complex decreased when the pH was increased from 6.0-9.0; but by contrast, the fluorescence of bound dansylsarcosine remained unchanged. Furthermore, the hydrolysis of NPA was selectively inhibited by dansylsarcosine, diazepam and octanoic acid (ligands selective for site II on mammalian albumins), but not by site I selective ligands such as azapropazone and warfarin. Overall, the results suggest that chicken albumin, like mammalian albumins, has discrete binding sites for warfarin and dansylsarcosine.

Topics: Albumins; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anticoagulants; Apazone; Binding Sites; Chickens; Dansyl Compounds; Humans; Hydrogen-Ion Concentration; Hydrolysis; Ligands; Nitrophenols; Sarcosine; Species Specificity; Warfarin

The interaction of sulphamethazine (SMZ) with pig plasma proteins and albumin was studied by ultrafiltration and equilibrium dialysis. Binding to pig plasma proteins was monophasic (affinity approximately 9.0 mol/L x 10(3)) and the main binding protein was albumin. At 37 degrees C and pH 7.4, the affinity of SMZ for albumin was about 8.0 mol/L x 10(3) and the number of binding sites was estimated as 1.4. Increasing the temperature from 4 to 45 degrees C resulted in a seven-fold decrease in affinity, and increasing pH from 6.0 to 8.0 enhanced affinity for pig albumin ten-fold. The free energy of binding (-delta G) and enthalpy change (-delta H) were around 5.5 and 5.1 Kcal/mol, respectively. The total entropy change (delta S) was small and positive, around 2 cal/mol/degree K. Studies with the fluorescent probes warfarin and dansylsarcosine, suggest that these bind to separate sites on porcine albumin. SMZ displaced both probes and inhibited the deacetylation of p-nitrophenyl acetate by pig albumin. We conclude that: (1) binding of SMZ to pig plasma proteins and albumin is weak; (2) the interaction with albumin is exothermic and enthalpy driven, and (3) pig albumin, like other mammalian albumins, appears to possess discrete binding sites for warfarin and dansylsarcosine. SMZ interacts with both these loci.

Topics: Analysis of Variance; Animals; Anti-Infective Agents; Binding Sites; Blood Proteins; Dansyl Compounds; Fluorescent Dyes; Hydrogen-Ion Concentration; Male; Protein Binding; Sarcosine; Serum Albumin; Sulfamethazine; Swine; Thermodynamics; Warfarin

The interaction of uremic toxins including indole-3-acetic acid (IA), indoxyl sulfate (IS), hippuric acid (HA) and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) with human serum albumin (HSA) has been investigated by three methods of fluorescent probe displacement, ultrafiltration and equilibrium dialysis. The binding parameter of CMPF was found to have the strongest affinity (10(7)M-1) among all the uremic toxins studied. Competitive experiment based on the method of Kragh-Hansen suggested that IA, IS and HA bind to site II, whereas CMPF binds to site I. The present limited data indicated that the four uremic toxins caused inhibition to any endo- or exogenous substances on HSA.

Topics: Binding Sites; Dansyl Compounds; Furans; Hippurates; Humans; Indoleacetic Acids; Indoles; Propionates; Sarcosine; Serum Albumin; Uremia; Warfarin

The fluorescent probes warfarin and dansylsarcosine are known to selectively interact with binding sites I and II, respectively, on human albumin. This paper investigates whether similar binding sites exist on bovine, dog, horse, sheep and rat albumins. Binding sites on albumins were studied by: (1) displacement of warfarin and dansylsarcosine by site I (phenylbutazone) and site II (diazepam) selective ligands; (2) the effects of non-esterified fatty acids (carbon chain lengths: C5-C20) and changes in pH (6-9) on the fluorescence of warfarin and dansylsarcosine; and (3) the ability of site selective ligands to inhibit hydrolysis of 4-nitrophenyl acetate. For bovine, dog, horse, human and sheep albumins the fluorescence of bound warfarin and dansylsarcosine was selectively decreased by phenylbutazone and diazepam, respectively. For these albumins medium chain fatty acids (C1-C12) reduced the fluorescence of dansylsarcosine (maximum inhibition with C9) whereas long chain acids (C12-C20) enhanced the fluorescence of warfarin (maximum increases with C12). In addition, changes in pH from 6 to 9 increased the fluorescence of warfarin and although site I ligands (warfarin/phenylbutazone) had no pronounced effects on 4-nitrophenyl acetate hydrolysis, site II ligands (dansylsarcosine/diazepam) significantly inhibited this reaction. Rat albumin behaved differently from the other albumins studied in that the C12-C20 fatty acids and changes in pH did not enhance the fluorescence of warfarin. Moreover, the differential effects of site I and site II ligands on the fluorescence of warfarin/dansylsarcosine and hydrolysis of 4-nitrophenyl acetate were less apparent with rat albumin. The results suggest bovine, dog, horse and sheep albumins have binding sites for warfarin and dansylsarcosine with similar properties to sites I and II on human albumin. By contrast, the warfarin binding site and to a lesser degree the dansylsarcosine site, of rat albumin have different characteristics from these sites on the other albumins studied.

Topics: Albumins; Animals; Binding Sites; Binding, Competitive; Cattle; Dansyl Compounds; Diazepam; Dogs; Fatty Acids, Nonesterified; Fluorescence; Horses; Humans; Hydrogen-Ion Concentration; Kinetics; Nitrophenols; Phenylbutazone; Rats; Sarcosine; Sheep; Warfarin

The sites to which valproic acid and its main unsaturated metabolites (2-en-2-propyl pentanoic acid and 4-en-2-propyl pentanoic acid) bind to on human albumin were investigated by (1) measuring their ability to displace the fluorescent probes warfarin and dansylsarcosine and (2) by assessing the extent to which they inhibited the hydrolysis of 4-nitrophenyl acetate. Valproate and its metabolites displaced both warfarin and dansylsarcosine, and they also inhibited the hydrolysis of 4-nitrophenyl acetate. The order of potency for inhibition of both binding and hydrolysis was: 2-en-2-propyl pentanoic acid greater than 4-en-2-propyl pentanoic acid greater than or equal to valproate. It is concluded that valproic acid and its unsaturated metabolites can displace ligands from the warfarin binding site (site I) and the benzodiazepine/indole binding site (site II), but the primary interaction is with site II. Furthermore, the introduction of a double bond into the carbon backbone of valproate increases affinity for albumin at both sites.

Topics: Binding Sites; Binding, Competitive; Dansyl Compounds; Diazepam; Fatty Acids; Fatty Acids, Unsaturated; Humans; In Vitro Techniques; Phenylbutazone; Protein Binding; Sarcosine; Serum Albumin; Structure-Activity Relationship; Valproic Acid; Warfarin

The binding of three fluorescent ligands (warfarin, dansylsarcosine and 1-anilino-8-naphthalene sulphonate) to human albumin was analysed using simultaneous non-linear least squares regression analysis. Both mock and actual fluorescence data were examined and the results indicated that reliable estimates of the binding parameters as well as the molar fluorescence of bound ligand could be obtained. The advantage of this method of analysis is that it makes full use of all the experimental data and it eliminates the need for the graphical procedures usually employed to estimate the molar fluorescence of bound ligand and its binding constants. This type of analysis can be extended to other systems where some physical property of the bound ligand varies with increasing protein concentration.

Topics: Albumins; Anilino Naphthalenesulfonates; Dansyl Compounds; Fluorescence; Fluorescent Dyes; Humans; Kinetics; Ligands; Linear Models; Models, Biological; Proteins; Regression Analysis; Sarcosine; Software; Warfarin

We have previously suggested that albumin in tubule fluid at concentrations found in the nephrotic syndrome (NS) binds furosemide, thereby diminishing diuretic effect. This mechanism may contribute to diuretic resistance in NS. If this hypothesis is correct, displacement of albumin from furosemide should restore diuretic response in tubule fluid containing albumin. To test this supposition, in vivo loop microperfusion was performed in rats using perfusates containing 6 microM furosemide in the presence or absence of 3.8 microM albumin, or furosemide and albumin to which 12 microM warfarin or 5.4 mM sulfisoxazole had been added. These drugs are inhibitors of albumin-furosemide binding in plasma. Albumin in the perfusate impaired furosemide effect on loop chloride reabsorption (1248 +/- 59 vs. 886 +/- 65 pEq/min; P less than 0.05). Addition of warfarin or sulfisoxazole to perfusate containing albumin normalized furosemide's effect. Neither drug affected furosemide response in the absence of albumin. Dansylsarcosine, a probe that binds albumin at a different site than furosemide, failed to normalize furosemide response in albumin perfusates. These data suggest that albumin in tubule fluid reduces diuretic response through a diminution in the free furosemide concentration. In as much as this mechanism contributes to diuretic resistance observed clinically in NS, displacement of furosemide from albumin binding sites may be a therapeutic strategy warranting study.

Topics: Albumins; Animals; Binding Sites; Binding, Competitive; Body Fluids; Dansyl Compounds; Drug Tolerance; Furosemide; Kidney Tubules, Distal; Male; Nephrotic Syndrome; Perfusion; Protein Binding; Rats; Rats, Inbred Strains; Sarcosine; Sulfisoxazole; Warfarin

Double-reciprocal plots of fluorescence intensity versus protein concentration are often used to obtain the intrinsic molar fluorescence (Fb) of ligands bound to acceptor molecules such as albumin. In this paper we show that these plots develop upward concave curvature as the concentration of albumin increases. Thus linear extrapolation of such plots cannot be employed to provide accurate values of Fb. It is suggested that a direct plot of fluorescence intensity versus log protein concentration should be employed to obtain Fb.

Topics: Albumins; Anilino Naphthalenesulfonates; Dansyl Compounds; Fluorescence; Protein Binding; Sarcosine; Warfarin

In the pH range 6-9, human serum albumin undergoes a conformational change termed the neutral-base (N-B) transition. Recently, it has been shown that the N-B transition causes enhanced binding at the warfarin-binding site (site I). The present study used fluorescence and equilibrium dialysis to investigate the effects of the N-B transition, chloride, calcium and fatty acids on the specific binding sites I and II on human serum albumin. The effect of the N-B transition of human serum albumin provides a further distinction between site I and II binding characteristics. The N-B transition of albumin caused a change in conformation at site I which resulted in increased binding of drugs and fluorescent probes at this site, whereas there was no effect on acidic drug binding at site II. These effects on site I and II are qualitatively similar to those induced by fatty acids (increased drug binding at site I and no change at site II). However, the effects of increasing pH and fatty acids were additive, showing that they were caused by two different conformational changes. The effect of Cl- on site I binding was pH-dependent and was abolished by the presence of fatty acid. Ca2+ reduced the fluorescence of site I probes but had no effect on a site II fluorescent probe. Effects of pH were also investigated with drugs not binding to site I or II. Increasing pH caused a decrease in binding to indomethacin, increases in binding of L-tryptophan, tolmetin and quinidine and no change in the binding of salicylic acid, diflunisal and phenytoin.

Topics: Binding Sites; Calcium; Chlorides; Dansyl Compounds; Humans; Hydrogen-Ion Concentration; Oleic Acid; Oleic Acids; Protein Binding; Protein Conformation; Sarcosine; Serum Albumin; Warfarin