Molecular and practical aspects of the enzymatic properties of human serum albumin and of albumin–ligand complexes

Background

Human serum albumin and some of its ligand complexes possess enzymatic properties which are useful both in vivo and in vitro.

Scope of review

This review summarizes present knowledge about molecular aspects, practical applications and potentials of these properties.

Major conclusions

The most pronounced activities of the protein are different types of hydrolysis. Key examples are esterase-like activities involving Tyr411 or Lys199 and the thioesterase activity of Cys34. In the first case, hydrolysis involves water and both products are released, whereas in the latter cases one of the products is set free, and the other stays covalently bound to the protein. However, the modified Cys34 can be converted back to its reduced form by another compound/enzymatic system. Among the other activities are glucuronidase, phosphatase and amidase as well as isomerase and dehydration properties. The protein has great impact on the metabolism of, for example, eicosanoids and xenobiotics. Albumin with a metal ion-containing complex is capable of facilitating reactions involving reactive oxygen and nitrogen species.

General significance

Albumin is useful in detoxification reactions, for activating prodrugs, and for binding and activating drug conjugates. The protein can be used to construct smart nanotubes with enzymatic properties useful for biomedical applications. Binding of organic compounds with a metal ion often results in metalloenzymes or can be used for nanoparticle formation. Because any compound acting as cofactor and/or the protein can be modified, enzymes can be constructed which are not naturally found and therefore can increase, often stereospecifically, the number of catalytic reactions. This article is part of a Special Issue entitled Serum Albumin.     

Precursor–product relationship between intrahepatic albumin and plasma albumin

Rats were injected with [(3)H]leucine, and at various times thereafter labelled albumin was isolated by electrophoresis from their livers and blood plasma. The specific radioactivity of each protein was determined by spectrophotometry and liquid-scintillation spectrometry. Intrahepatic albumin was shown to be identical with plasma albumin by its electrophoretic mobility and antigenicity. It was found that intrahepatic albumin was the direct precursor of plasma albumin. Comparison of their specific radioactivities showed that intrahepatic albumin attained a higher specific radioactivity before plasma albumin. When plasma albumin reached its maximum specific radioactivity, that of intrahepatic albumin had decreased to a similar value. Thereafter, the specific radioactivity of intrahepatic albumin remained lower than that of plasma albumin.     

Is albumin gradient or fluid to serum albumin ratio better than the pleural fluid lactate dehydroginase in the diagnostic of separation of pleural effusion?

Background  

To determine the accuracy of serum-effusion albumin gradient (SEAG) and pleural fluid to serum albumin ratio (ALBR) in the diagnostic separation of pleural effusion into transudate and exudate and to compare SEAG and ALBR with pleural fluid LDH (FLDH) the most widely used test.     

Biotin-decorated anti-cancer nucleotide theranostic conjugate of human serum albumin: Where the seed meets the soil?

Human serum albumin is playing an increasing role as a drug carrier in clinical settings. Biotin molecules are often used as suitable tags in targeted anti-tumor drug delivery systems. We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anti-cancer fluorinated nucleotide conjugated with a biotinylated dual-labeled albumin. Interestingly, in vitro and in vivo study revealed stronger anti-tumor activity of the non-tagged theranostic conjugate than that of the biotin-tagged conjugate, which can be explained by decreased binding of the biotin-tagged conjugate to cellular receptors. Our study sheds light on the importance of site-specific albumin modification for the design of albumin-based drugs with desirable pharmaceutical properties.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

Modulation of the albumin–paraoxon interaction sites by fatty acids: Analysis by the molecular modeling methods

It is known that albumin can break the ester bonds in organophosphorus compounds (OPs). Amino acids responsible for esterase and pseudoesterase activity of albumin towards OPs are still not determined. It is assumed that Sudlow’s site I with residue Tyr150 exhibits the “true” esterase activity; and Sudlow’s site II containing residue Tyr411, a pseudoesterase one. Binding of fatty acids to albumin affects the efficiency of its interaction with xenobiotics; however, the effect of fatty acids on the interaction of albumin with OPs was not studied. The purpose of this work was to study the interaction of OPs with potential sites of albumin enzymatic activity and to examine the effect of fatty acids on the efficiency of such interaction using the molecular modeling methods by the example of paraoxon, a known inhibitor of acetylcholinesterase, and oleic acid. The structures of the protein complexes with paraoxon and oleic acid were determined by the molecular docking procedure; the conformational changes were calculated by the molecular dynamics method. It has been shown that sorption of oleic acid in one of the fatty acid-binding sites leads to the conformational changes in Sudlow’s sites I and II due to a “reversal” of the side chains of Arg410 and Arg257 residues by 90°. It has been found that this change in geometry reduces the affinity of Sudlow’s site I and increases the Sudlow’s site II affinity to paraoxon. The amino acid residue Ser193, which was previously identified as a site of possible albumin esterase activity, is not able to bind paraoxon efficiently. It is assumed that its activity can be affected by the interaction of the oleic acid molecules with other fatty acid-binding sites. It is hypothesized that the lesser toxicity of paraoxon compared to soman may be associated not only with its lower inhibitory activity against cholinesterases, but also with the increased affinity of paraoxon to albumin. It was concluded that albumin may serve as an alternative means of OP detoxification.     

Expression patterns of mRNAs for α-fetoprotein and albumin in the developing rat: the ontogenesis of hepatocyte heterogeneity

Summary In developing and normal adult rat liver the expression patterns of the mRNAs for -fetoprotein (AFP) and albumin (ALB) were analysed byin situ hybridization using specific³⁵S-labelled complementary DNA probes. In the developing liver AFP and ALB mRNA are found from embryonic day (ED) 11 and 12, respectively, onward. At ED 20 the first signs of a zonal distribution of these mRNAs across the liver lobule can be observed, AFP mRNA concentration being higher in the pericentral area and ALB mRNA concentration higher in the periportal area. This distribution pattern of reciprocal, overlapping gradients of mRNA can be clearly recognized in the neonatal period. In the adult liver AFP mRNA can no longer be detected and similar to the neonatal situation, ALB mRNA is expressed across the entire porto-central distance decreasing in concentration going from the portal to the central area.Transient extra-hepatic expression of AFP mRNA is found in the embryonic heart and in the epithelial lining of intestine and lung furthermore, AFP and ALB mRNA are found to be transiently expressed in the developing renal tubules. Similar expression patterns have been observed for other liver-characteristic mRNAs (Moormanet al., 1990), suggesting that common regulatory factors are operative during development.     

Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme–albumin

Human serum heme–albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA–heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K ₀ = (3.9 ± 0.4) × 10⁻⁴ and (1.3 ± 0.1) × 10⁻⁵ M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K _h = (4.3 ± 0.4) × 10⁻³ and (1.2 ± 0.1) × 10⁻⁴ M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H ₀ = (1.9 ± 0.2) × 10⁻⁸ M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H _d = (2.1 ± 0.2) × 10⁻⁷ M]. In the absence and presence of CO₂, the values of the second-order rate constant (l _on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 10⁵ and 4.3 × 10⁵ M⁻¹ s⁻¹, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO₂. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow’s site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow’s site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.     

Ligand binding strategies of human serum albumin: How can the cargo be utilized?

Human serum albumin (HSA), being the most abundant carrier protein in blood and a modern day clinical tool for drug delivery, attracts high attention among biologists. Hence, its unfolding/refolding strategies and exogenous/endogenous ligand binding preference are of immense use in therapeutics and clinical biochemistry. Among its fellow proteins albumin is known to carry almost every small molecule. Thus, it is a potential contender for being a molecular cargo/or nanovehicle for clinical, biophysical and industrial purposes. Nonetheless, its structure and function are largely regulated by various chemical and physical factors to accommodate HSA to its functional purpose. This multifunctional protein also possesses enzymatic properties which may be used to convert prodrugs to active therapeutics. This review aims to highlight current overview on the binding strategies of protein to various ligands that may be expected to lead to significant clinical applications. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Spectral diagnostics of the interaction between pyridoxine hydrochloride and bovine serum albumin in vitro

The interaction between pyridoxine hydrochloride (VB6) and bovine serum albumin (BSA) were studied by spectroscopic methods including fluorescence spectroscopy and UV-visible absorption spectra. The quenching mechanism of fluorescence of BSA by VB6 was discussed. The number of binding sites n and observed binding constant K(b) was measured by fluorescence quenching method. The thermodynamic parameters DeltaH(theta), DeltaG(theta), DeltaS(theta) at different temperatures were calculated and the results indicate the binding reaction is mainly entropy-driven and hydrophobic interaction played major role in the reaction. The distance r between donor (BSA) and acceptor (VB6) was obtained according to FOrster theory of non-radiation energy transfer. Synchronous fluorescence and three-dimensional fluorescence spectra were used to investigate the structural change of BSA molecules with addition of VB6, the result indicates that the secondary structure of BSA molecules is changed in the presence of VB6.     

Elucidating the molecular interaction of serum albumin with nizatidine and the role of β-cyclodextrin: multi-spectroscopic and computational approach

Abstract

Nizatidine is a histamine H2 receptor antagonist which act by inhibiting the production of stomach acid, thereby, finds its application in treating various diseases related to the gastrointestinal tract. Studying albumin–drug interaction is important for understanding the pharmacokinetics and pharmacodynamics of therapeutic candidates. In the present work, the interaction of nizatidine with BSA was investigated by employing multi-spectroscopic and computational studies. The formation of BSA–nizatidine complex was characterised by UV-visible and fluorescence based-spectroscopic studies. Steady-state fluorescence demonstrated the static mode of quenching of BSA by nizatidine. The interaction was spontaneous and nizatidine binds to BSA with a stoichiometry of 1:1. Forster resonance energy transfer calculations revealed that there was a high possibility of energy transfer between nizatidine and BSA. The resultant secondary structural change in BSA on the addition of nizatidine was studied by circular dichroism spectroscopy. Moreover, synchronous and three-dimensional fluorescence spectroscopy was used to determine the conformational changes occurred in the structure of albumin on the binding of nizatidine. Competitive-site marker experiments suggested that nizatidine binds in the Sudlow site II of BSA. Additionally, the effect of β-cyclodextrin as an inclusion compound on the interaction was studied. Furthermore, molecular modelling and simulation studies were performed to corroborate the results obtained above.

Communicated by Ramaswamy H. Sarma     

Biophysical insights into the binding characteristics of bovine serum albumin with dipyridamole and the influence of molecular interaction with β cyclodextrin

Abstract

The binding characteristic of anti-platelet drug dipyridamole has been investigated with a transport protein, serum albumin. A multi-spectroscopic approach has been employed, and the results were well supported by in silico molecular docking and simulation studies. The fluorescence quenching of serum albumin at three different temperatures revealed that the mechanism involved is static and the binding constant of the interaction was found to be of the order of 10⁴ M^?1. The reaction was found to be spontaneous and involved hydrophobic interactions. Synchronous, 3D fluorescence and CD spectroscopy indicated a change in conformation of bovine serum albumin (BSA) on interaction with DP. Using site-selective markers, the binding site of DP was found to be in subdomain IB. Molecular docking studies further corroborated these results. Molecular dynamic (MD) simulations showed lower RMSD values on interaction, suggesting the existence of a stable complex between DP and BSA. Furthermore, since β-Cyclodextrin (βCD) is used to improve the solubility of DP in ophthalmic solutions, therefore, the effect of (βCD) on the interaction of BSA and DP was also studied, and it was found that in the presence of βCD, the binding constant for BSA-DP interaction decreased. The present study is an attempt to characterize the transport of DP and to improve its bioavailability, consequently helping in dosage design to achieve optimum therapeutic levels.

Communicated by Ramaswamy H. Sarma     

Fractionation of nuclear and cytoplasmic ribonucleic acids from rat tissues on the methylated albumin–kieselguhr column

1. The conformation of RNA was found to affect its behaviour on methylated albumin-kieselguhr chromatography. The less regular the secondary structure of RNA, the more tightly it binds to the methylated albumin-kieselguhr column. 2. The presence of various denaturing agents (such as urea or perchlorate) in the medium while RNA was adsorbed on the column increased the resolving power of the technique as exemplified by the separation of rat liver rRNA into two distinct peaks. A special procedure for selective adsorption of the cytoplasmic DNA-like RNA on the preparative scale has been developed. Polyribosomal mRNA (rapidly labelled RNA formed in the presence of small doses of actinomycin D) can also be adsorbed selectively by the column. 3. A type of tissue specificity was detected in nuclear RNA from rat liver, kidney, thymus and spleen by using a modified salt and temperature gradient for the chromatographic fractionation (Lichtenstein, Piker & Shapot, 1967; Shapot, Lichtenstein & Piker, 1967). It was also found that cytoplasmic RNA from the different rat tissues contained no tenaciously bound fraction at all, whereas it constituted about 50% of the nuclear RNA. The problem of the possible biological function of the tenaciously bound fraction is discussed.     

Direct demonstration of the highly reactive tyrosine residue of human serum albumin located in fragment 299–585

The reactivity of tyrosine residues in human serum albumin (HSA) was investigated by the nitration of the albumin with tetranitromethane. It could clearly be demonstrated that one tyrosine residue reacts faster and with about 20-fold higher reactivity than all others. This highly reactive tyrosine residue is located in fragment 299–585 of HSA and is supposed to be Tyr 411. This tyrosine residue is specifically involved in the indole and benzodiazepine binding site of HSA. Since some other amino acid residues, located in fragment 124–298, are also involved in the indole and benzodiazepine binding site, it is concluded that this important binding site of HSA depends on the tertiary structure of the albumin.     

Oxidation of bovine serum albumin initiated by the Fenton reaction—effect of EDTA,tert-butylhydroperoxide and tetrahydrofuran

Oxidation of bovine serum albumin (BSA) was investigated using different oxidants: The water-soluble azo-initiator 2,2′azo-bis-(2-amidinopropane) hydrochloride (AAPH), a combination of FeCl₃ and ascorbate or the Fenton oxidant consisting of FeCl₂, H₂O₂ and EDTA. In addition, the effects of exogenous compounds such as tert-butyl hydroperoxide (tBuOOH) or solvents such as tetrahydrofuran (THF), often used in model systems, was evaluated. The extent of protein damage was studied by measuring protein carbonyl groups and protein hydroperoxides. The interaction between Fenton oxidant and EDTA, THF or tBuOOH was further characterized using spin trapping electron spin resonance (ESR) spectroscopy. The results showed that the extent of protein oxidation depended on the oxidant used. The Fenton oxidant was the most reactive of the initiators tested. However, in the absence of EDTA, the Fenton system produced protein carbonyl groups on BSA equivalent to that obtained with the other oxidants, however, significantly more protein hydroperoxide was produced. Surprisingly, it was also found that addition of tBuOOH or THF to BSA reduced protein damage when the oxidation was initiated with the Fenton oxidant. ESR investigation showed that EDTA played a key role in the generation of free radicals. It was also revealed that in an EDTA containing system both tBuOOH and THF were able to react with radicals without inducing protein damage in effect protecting BSA from oxidative damage.     

Sandwich enzyme-linked immunosorbent assay for the quantification of human serum albumin fragment 408–423 in bodily fluids

Urinary levels of human serum albumin (hSA) fragment 408–423 have been proposed to represent an early marker for graft-versus-host disease (GvHD) and chronic kidney diseases. Here, we developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of hSA(408–423). The sandwich ELISA has a detection limit of 0.5 ng/ml and is highly specific for hSA(408–423) because it does not cross-react with other albumin fragments or the full-length precursor. This ELISA allows rapid and convenient quantification of hSA(408–423) in bodily fluids, further clarifying the prognostic and diagnostic value of this peptide in GvHD, kidney disease, and other disorders.     

Characterization of messenger-like ribonucleic acid from Saccharomyces cerevisiae by the use of chromatography on methylated albumin–kieselguhr

Chromatography on methylated albumin–kieselguhr of RNA from Saccharomyces cerevisiae was used to separate stable RNA from a tenaciously bound DNA-like RNA fraction. The tenaciously bound RNA, which was eluted with a dilute solution of sodium dodecyl sulphate, was characterized as messenger-like RNA by its sedimentation behaviour, nucleotide composition, lack of methylated bases and labelling kinetics. Chromatography of purified ribosomal RNA indicated a minor contamination of the tenaciously bound fraction with ribosomal RNA. On the other hand, a large portion of pulse-labelled polyribosomal RNA from protoplasts of Saccharomyces cerevisiae was tenaciously bound to the columns. The `chase' of isotopic label from the messenger-like RNA was found to be retarded during inhibition of protein synthesis both by cycloheximide and by starvation for a carbon source.     

Nanopore sensing of γ-cyclodextrin induced host-guest interaction to reverse the binding of perfluorooctanoic acid to human serum albumin

Perfluorooctanoic acid (PFOA) has been one of the most common perfluorochemicals, which are globally pervasive contaminants that are persistent, bioaccumulative, toxic, and have adverse impacts on human health. The highest concentration of PFOA occurs in the blood, where it strongly binds to human serum albumins (HSA). Thus, a method to reverse the HSA-PFOA binding is critical to help facilitate the faster elimination of PFOA from the body to minimize its toxicological effects. Inspired by the remediation effect of cyclodextrin (CD) to PFOA through host-guest interactions, herein, by elucidating inter-molecular interactions using a nanopore sensor, we demonstrated in vitro reversal of the binding of PFOA to HSA using γ-cyclodextrin (γ-CD). The competition behavior for the complexation of PFOA between HSA and γ-CD was discussed in combination with in situ nanopore current recording and nuclear magnetic resonance (NMR) characterization. The present work not only demonstrates the potential therapeutic application of γ-CD for PFOA removal from human blood, but also provides an emerging method for investigating interactions between organic compounds and proteins.