Showing papers on "Human serum albumin published in 1996"

Chapter 33. Plasma Protein Binding of Drugs

Abstract: Publisher Summary The role of plasma protein binding is recognized as an important factor in drug disposition and efficacy. This chapter discusses the structures of drug-binding proteins, particularly human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG), the relationship between ligand structure and protein binding and the effect of plasma protein binding on drug disposition and pharmacological activity. Human serum albumin (HSA) binds a wide variety of endogenous and exogenous ligands with association constants typically in the range of 10 4 to 10 6 M –1 . As the most abundant protein in plasma, it plays an important role in the maintenance of blood pH and colloidal osmotic pressure and accounts for most of the thiol content of plasma. Numerous competition and spectroscopic studies, using various ligands, have identified two sites (I and II) most relevant for drug binding. The interaction of various nonsteroidal anti-inflammatory drugs (NSAIDs) with HSA has been studied extensively. The interaction of other drug classes with HSA has also been investigated. Covalent modification of plasma proteins can be produced by direct attachment of an electrophilic moiety of the parent molecule to nucleophilic protein residues or by metabolic activation of the parent to a more reactive species. These reactions are important as their potential to form immunogenic adducts, with the possibility of producing idiosyncratic hypersensitivity reactions. Microdialysis is a potent technique to measure the extent of binding in vivo or in vitro in plasma. high-performance liquid chromatography (HPLC) methods, utilizing protein stationary phases, offer the potential for rapid screening, especially for those molecules bound to albumin. The blood brain barrier (BBB) effectively restricts the access of polar, hydrophilic drugs into the central nervous system (CNS), thus establishing a significant drug delivery impediment.

Fluorescence behavior of tryptophan residues of bovine and human serum albumins in ionic surfactant solutions: A comparative study of the two and one tryptophan(s) of bovine and human albumins

Abstract: The fluorescence behavior of two tryptophans (Trp-134, Trp-213) in bovine serum albumin (BSA) and a single tryptophan (Trp-214) in human serum albumin (HSA) was examined. The maximum emission wavelength (λmax) was 340.0 nm for both proteins. In a solution of sodium dodecyl sulfate (SDS), the λmax of BSA abruptly shifted to 332 nm at 1 mM SDS and then reversed to 334 nm at 3 mM SDS. The λmax of HSA gradually shifted to 330 nm below 3 mM SDS, although it returned to 338 nm at 10 mM SDS. In contrast to this, in a solution of dodecyltrimethylammonium bromide, the λmax positions of BSA and HSA gradually shifted to 334.0 and 331.5 nm, respectively. Differences in the fluorescence behavior of the proteins are attributed to the fact that Trp-134 exists only in BSA, with the assumption that Trp-213 of BSA behaves the same as Trp-214 of HSA. The Trp-134 behavior appears to relate to the disruption of the helical structure in the SDS solution.

Serum albumin is a specific inhibitor of apoptosis in human endothelial cells

Abstract: Excess blood vessels are removed by apoptosis of endothelial cells, however, the signals responsible for this have not been defined. Apoptosis of cultured human umbilical vein endothelial cells is induced by deprivation of serum or adhesion. In this paper, apoptosis in human umbilical vein and microvascular endothelium was induced by deprivation of serum and or adhesion. Apoptosis was confirmed on the basis of morphology, ultrastructure and internucleosomal cleavage of DNA. Loss of endothelial adhesion was found to be an early event in cultured endothelial cell apoptosis and was exploited to quantitate apoptosis. The effect of: bovine serum albumin; human serum albumin; recombinant human albumin; dithiothreitol reduced human and bovine albumin; CNBr treated human and bovine albumin as well as ovalbumin upon endothelial apoptosis was determined. Native bovine and human albumin as well as recombinant human material inhibited apoptosis at physiological concentrations with identical dose response curves in both umbilical vein and microvascular cells. Dithiothreitol treatment destroyed all protective activity while bovine but not human albumin was partially inactivated by CNBr treatment. The unrelated protein ovalbumin was not protective. Albumin did not inhibit apoptosis if cells were also deprived of adhesion. The data suggest that albumin is a specific inhibitor of human endothelial apoptosis but does not protect cells also deprived of adhesion. Reduced supply of albumin to endothelium in poorly perfused blood vessels may provide a mechanism for the removal of excess blood vessels in remodelling tissues. Also, the failure of albumin to protect endothelial cells deprived of adhesion from apoptosis may reflect the need to remove potentially micro-embolic cells detached due to trauma.

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

Abstract: The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (K A) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of K A. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM–1 s–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate.

Serum albumin is a significant intermediate in cholesterol transfer between cells and lipoproteins

Abstract: The function of albumin in the movement of cholesterol into and out of non-cholesterol-loaded fibroblasts has been investigated. Cholesterol efflux from cholesterol labeled normal human skin fibroblasts to fatty acid-free human serum albumin (HSA) is biphasic with a rapid first phase that plateaus at about 15 min followed by a nearly linear phase up to 90 min, the longest incubation in this study. Saturation of efflux is observed at about 10 mg of albumin/mL. Efflux is specific to albumin since other molecules, such as ovalbumin or gelatin, do not induce efflux. The ability of HSA to induce cellular cholesterol efflux is low compared to reconstituted discoidal lipoprotein A-I (LpA-I). HSA at 2 mg/mL produces a rate of cholesterol efflux similar to that of LpA-I at 45 micrograms of protein/mL; however, these concentrations are within the physiological range for both HSA and apolipoprotein A-I (apoA-I). The efflux to the medium containing both LpA-I and HSA is greater than that to each of them alone but does not show complete additivity, indicating a competition between HSA and LpA-I. The HSA-mediated cholesterol movement is bidirectional as demonstrated by the transfer of cholesterol from HSA-(3H)- cholesterol complexes to fibroblasts; moreover, the HSA-mediated transfer is much faster than that from cholesterol-containing LpA-I (0.8 versus 0.2 pmol (micrograms of cell protein)-1 (90 min)-1. However, the presence of either low-density lipoprotein (LDL) or LpA-I in the incubation medium significantly inhibits the transfer of cholesterol from HSA-(3H)-cholesterol complexes to fibroblasts, thus allowing the bidirectional transfer of cholesterol between HSA and cells to possibly operate as a net efflux. In conclusion, albumin plays a significant role in cholesterol transfer between cells and lipoproteins.

Two distinguishable fluorescent modes of 1-anilino-8-naphthalenesulfonate bound to human albumin.

Abstract: We study the interaction of 1-anilino-8-naphthalenesulfonate (ANS) with human (HSA) and bovine serum albumin (BSA) by phase and modulation fluorescence spectroscopy. We determined that both HSA and BSA show one or two distinguishable fluorescent sites, depending of the ANS/serum albumin ratio. At above a 1∶1 ANS/HSA molar ratio, the steady-state emission spectra for ANS can be resolved in two components: component 1, emitting with a lifetime (τ1) of 16 ns and a λ1max of 478 nm, with a quantum yield (фf1) of 0.67, and component 2, with a lifetime (τ2) of 2–4 ns and a λ2max of 483 nm, with an average quantum yield (фf2) of about 0.11. Considering these findings, the binding analysis is fitted with a model of two independent sites. Site 1 has an association constantKas1=0.87×106M−1 and a capacity of 1.04 mol of ANS/mol of HSA, and site 2 aKas2=0.079×106M−1 and a capacity of 2.34 mol of ANS/mol of HSA. Analysis of fluorescence lifetime distributions shows that the rigidity of the fluorophore environment at site 1 changes when site 2 is occupied. These findings suggest an interconnection between the two sites and that ligands can stabilize the protein's globular structure. To assess the identity of the ANS binding sites we used diazepam as a marker of the site located at the IIIA HSA subdomain and aspirin as a marker of sites located at the IIIA and IIA HSA subdomains. Both ligands displace ANS only from site 1, suggesting that it corresponds to the binding site located at the IIIA sub-domain of the protein. We determined that theKas values for diazepam and aspirin are 0.113× 106 and 0.021×106M−1 respectively.

Chemical modification of earthworm fibrinolytic enzyme with human serum albumin fragment and characterization of the protease as a therapeutic enzyme

Abstract: The strongest fibrinolytic protease (F-III-2) in the six enzyme proteins purified from earthworm, Lumbricus rubellus [N. Nakajima et al., Biosci. Biotech. Biochem., 57, 1726-1730 (1993)] has been modified chemically with fragmented human serum albumin (mol. wt., 10,000-30,000). The modified enzyme lost the antigenicity of the native enzyme and reacted with the antisera against human serum albumin, the human serum albumin fragments, and the conjugate with the native enzyme to form precipitation lines, which fused with each other. The conjugate was significantly more resistant to inactivation by protease inhibitors in rat plasma. The enzyme was a non-hemorrhagic protein and did not induce platelet aggregation. The enzyme kept potent proteolytic activity for fibrin and fibrinogen than that of human plasmin. The enzyme easily solubilized actual fibrin clots (thrombi) of whole blood induced by thrombin in a rat's vena cava. The continuous fibrinolysis for fibrin suspension in an enzyme reactor system using the modified enzyme immobilized to oxirane-activated acrylic beads has been achieved without any inactivation of the activity at least for more than 1 month. The N-terminal amino acid sequence of the protein was also investigated and the sequence showed local similarity to those of the serine proteases such as plasmin and chymotrypsin.

Comparative serum protein binding of anthracycline derivatives.

Abstract: The binding of doxorubicin, iododoxorubicin, daunorubicin, epirubicin, pirarubicin, zorubicin, aclarubicin, and mitoxantrone to 600 μM human serum albumin and 50 μM alpha1-acid glycoprotein was studied by ultrafiltration at 37°C and pH 7.4. Anthracycline concentrations (total and free) were determined by high-performance liquid chromatography (HPLC) with fluorometric detection. Binding to albumin (600 μM) varied from 61% (daunorubicin) to 94% (iododoxorubicin). The binding to alpha1-acid glycoprotein (50 μM) was more variable, ranging from 31% (epirubicin) to 64% (zorubicin), and was essentially related to the hydrophobicity of the derivatives. Simulations showed that the total serum binding varied over a broad range from 71% (doxorubicin) to 96% (iododoxorubicin).

Covalent binding between bucillamine derivatives and human serum albumin

Abstract: Purpose. To clarify the mechanism of covalent binding between human serum albumin (HSA) and drugs containing thiol groups, we studied the interactions between HSA and bucillamine (BA) and its derivatives. Methods. To determine the concentration of HSA-drug conjugate, we used columns of N-methylpyridium polymer cross-linked with ethylene glycol dimethacrylate (4VP-Me), and analyzed the reaction between HSA and B A derivatives kinetically. Following pseudo first-order reaction kinetics, the rate constants of reduction of non-mercaptoalbumin (HNA) to mercaptoalbumin (HMA) (ka) and formation of HSA-drug conjugate (kc) were determined. Results. Formation of HSA-drug conjugate was observed only for drugs containing one thiol group. In compound IV, the plots of ka and kc against pH were found to be linear. The HSA-drug conjugate was affected by various factors such as pKa, pH, temparture and the microenviroment of Cys34. The increases in ka and kc. against pH were mainly due to the increase in mercaptide ion concentration. Further, fatty acid affected the microenviroment of Cys34, which increased HSA-drug formation. Conclusions. Cys34 located in a crevice on the surface of the protein plays an important role on the formation of HSA-drug conjugate. These results may be useful for elucidating the reaction mechanisms between various proteins and thiol compounds.

Mechanism of stereoselective serum binding of ketoprofen after hemodialysis.

Abstract: In vitro studies of serum protein binding were conducted to examine the influence of hemodialysis therapy on the stereoselectivity for ketoprofen (KP) in patients with renal dysfunction. In normal volunteers, only small differences in binding to undiluted serum were observed between the enantiomers of racemic KP (10 micrograms/mL). As compared with healthy volunteers, significantly impaired binding to undiluted serum was observed in uremic patients before they received dialysis. This reduced binding was not accompanied by alterations in stereoselectivity. At the end of dialysis, a similar extent of defective binding to undiluted serum was still observed, whereas interestingly, its stereoselectivity was significantly enhanced. These findings indicated that preferential inhibition for S-enantiomer serum binding was caused by changes that occurred during hemodialysis. Among various physiological parameters, only enantioselective inhibition of KP serum binding was correlated with heparin-induced increases in free fatty acids (FFA) levels and with unbound levels of uremic toxins with indole ring, indoxyl sulfate and indole acetate. To elucidate the effects of these accumulated endogenous substances in serum of patients, ultrafiltration was also used to study the binding of KP enantiomers to isolated human serum albumin under various conditions. After dialysis, mean albumin level and pH value in patients were slightly but significantly altered, but in vitro binding data indicated that these factors could clearly be ruled out as an explanation for the origin of the induced stereoselectivity. In contrast to palmitate, oleate and laurate markedly induced the stereoselective inhibition, which indicated that these fatty acids preferentially inhibited the binding of S-KP via a competitive and allosteric mechanism. Among the four uremic toxins, only indoxyl sulfate and indole acetate predominantly displaced the S-enantiomer rather than its antipode through competitive interaction. In addition, serum unbound levels of indole uremic toxins were correlated with elevated FFA levels. FFA displaced those indole uremic toxins in a manner similar to KP, which suggests that FFA also indirectly inhibits the S-enantiomer binding by increasing the unbound levels of indole uremic toxins. Thus it is likely that combined direct and cascade effects of transiently increased FFA and unbound indole uremic toxins contributed to the preferential elevation of the pharmacologically active S-KP fraction in uremic patients at the end of hemodialysis.

Extrinsic fluorescence probe study of Human Serum Albumin using Nile Red

Abstract: Nile red bound to human serum albumin (HSA) shows an order of magnitude increase in the probe's fluorescence intensity. Here, we report on the fluorescence characteristics of the probe-protein complex in Trizma buffer (pH 7.1), urea, guanidine hydrochloride, and AOT/isooctane/buffer reverse micelles using both steady—state and time-resolved fluorescence techniques. With a view to illustrating the use of extrinsic probe fluorescence spectroscopy in protein research, we demonstrate that protein unfolding can be observed through measurements of the probe's time-resolved anisotropy and steady-state fluorescence spectrum. Moreover, this shows that thermal unfolding is fundamentally different from using denaturant, with respect to changes in both the nanosecond diffusional rotation of the probe at intermediate stages and in the denatured protein's structure. Also, the large Stokes shift of Nile red allows the changes in the environment of the probe-protein complex in reverse micelles of varying waterpool size to be easily identified in the steady-state fluorescence. This was not seen in earlier work exploiting the intrinsic tryptophan fluorescence of HSA and further demonstrates the complementary information that extrinsic fluorescence probe studies can offer protein science. We discuss the complex acrylamide quenching characteristics of Nile red bound to HSA in terms of the possibility of at least two binding sites for the probe and the effect of acrylamide on the probe-protein structure at very high quencher concentrations.

Binding of bupivacaine to human serum proteins, isolated albumin and isolated alpha-1-acid glycoprotein. Differences between the two enantiomers are partly due to cooperativity.

Abstract: Binding parameters of R(+)- and S(-)-bupivacaine were determined for human serum proteins, human alpha-1-acid glycoprotein (AAG) and human serum albumin (HSA), using ultrafiltration. Binding parameters were estimated according to the Scatchard model of the law of mass action using nonlinear regression. A sigmoid (cooperativity) term was added when needed. Both enantiomers exhibited a two site binding profile for human serum and for a solution containing AAG and HSA at physiological concentrations. At concentrations lower than 40 microM (concentrations encountered in clinical situations), the low capacity, high affinity apparent site was predominant and S(-)-bupivacaine exhibited a higher free fraction than R(+)-bupivacaine. At concentrations higher than 60 microM, the opposite situation was observed and the S(-) enantiomer showed much higher binding to AAG than the R(+) enantiomer. Two cooperativity phenomena occurred. Negative cooperativity was observed when AAG and HSA were combined in the same solution. S(-) and R(+) enantiomers exhibited different behavior toward purified AAG and HSA due in part to complex allosteric cooperativity (positive or negative depending on the ligand/protein ratio). In conclusion, we observed stereoselective binding of bupivacaine to AAG and HSA. Moreover, cooperativity occurred, and the behavior of the two enantiomers showed marked differences in this respect.

Mode of interaction of loop diuretics with human serum albumin and characterization of binding site.

Abstract: Purpose. The purpose of this study was to investigate the binding mechanism of loop diuretics with HSA and to characterize the binding site on HSA.