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.     

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.     

Comparison of wheat albumin inhibitors of α-amylase and trypsin

Wheat albumins were extracted from whole wheat flour with 150 mM sodium chloride solution and precipitated between 0·4 and 1·8 M ammonium sulphate. The albumin precipitate was separated by gel filtration on Sephadex G100 into five peaks. Three peaks (II, III, and IV), whose MWs were 60 000, 24 000 and 12 500 daltons respectively, were active toward several insect α-amylases, whereas only peak III inhibited human saliva and pancreatic α-amylases. Peaks III and IV also inhibited trypsin. In each active peak, we found several α-amylase inhibitors slightly different in their electrophoretic mobilities in a Tris—glycine buffer system (pH 8·5), whereas only one major trypsin inhibitor was present in peaks III and IV. In contrast to α-amylase inhibitors that were all anodic, trypsin inhibitors migrated to the cathode under our experimental conditions. From a quantitative standpoint, wheat albumins that inhibit trypsin are negligible, whereas about 2/3 of the total albumin inhibits amylases from different origins. All inhibitor components of peak III were active toward both insect and mammalian α-amylases. Moreover, they reversibly dissociated in the presence of 6 M guanidine hydrochloride giving two similar subunits.     

Increased levels of albumin in bronchial washing fluid of patients with bronchial carcinoma. Could albumin be considered as a tumor marker?

BACKGROUND: The aim of this study was to evaluate the significance of albumin in bronchial washing fluid (BWF) and its relationship to three tumor markers (CEA, CA 19-9 and NSE). METHODS: Serum and BWF samples were collected in a group of 60 patients. Albumin and tumor markers in the BWF and serum of three groups: a control group (CG), a chronic bronchitis group (CBG) and a lung cancer group (CaG), were analyzed in a prospective cross-sectional study. The diagnostic yields of the tests in each environment (serum and BWF) were evaluated by using as cutoff points the values of the corresponding 90th percentile of CG and CBG taken together. RESULTS: A significant difference in albumin level (p < 0.001) was noted in the BWF of patients with cancer compared with the other two groups. In addition, a significant difference in CEA level (p < 0.001) was observed in the serum of cancer patients compared with the other two groups. The cutoff values for CEA in serum and albumin in BWF were 2.20 ng/mL and 2.00 g/dL, respectively. The areas under the corresponding ROC curves were 93% and 97%. Combination of CEA-serum and albumin-BWF by logistic regression analysis increased their diagnostic value. CONCLUSION: Measurement of albumin levels in BWF could be a useful additional diagnostic tool to differentiate malignant from non-malignant lung diseases. Moreover, the combined measurement of CEA in serum and albumin in BWF could be of aid in the follow-up of lung cancer patients.     

Sulfenic acid—A key intermediate in albumin thiol oxidation

The single thiol of human serum albumin (HSA-SH) is the predominant plasma thiol. Both circulating albumin and pharmaceutical preparations are heterogeneous regarding the thiol redox status, as revealed by anion-exchange–hydrophobic interaction chromatography. Sulfenic acid (HSA-SOH) is an intermediate in HSA-SH oxidation processes that was detected through different techniques including mass spectrometry. Recently, quantitative data led to the determination of rate constants. The preferred fate of HSA-SOH is the formation of mixed disulfides. Alternatively, HSA-SOH can be further oxidized to sulfinic and sulfonic acids. Oxidized forms increase under disease conditions, underscoring the importance of HSA-SH as a plasma scavenger of intravascular oxidants. We here provide a critical review of the oxidation of HSA-SH in the context of the intravascular compartment, with emphasis in the methodological approaches of mass spectrometry and chromatography for the analysis of albumin thiol redox states.     

Undermethylation at the 5′ end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma cells in culture

We have measured methylation of the albumin gene in clones of rat hepatoma cells that vary quantitatively in their rates of synthesis of albumin and in variant and hybrid cells that produce no albumin. Although the albumin gene is undermethylated for its entire length in rat liver, only the 5′ end is ever undermethylated in hepatoma cells. Moreover, undermethylation of the 5′ end of the gene appears to be necessary for stable expression of the albumin gene in hepatoma cells. Since undermethylation of this region is found in some variant cells that fail to produce albumin, it is not a sufficient condition for albumin gene expression. Despite the excellent correlation between undermethylation of the 5′ end of the albumin gene and its stable expression, the results argue against the possibility that the methylated state of such genes during development determines whether they will or will not be expressed.     

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.     

Binding of δ9-tetrahydrocannabinol and diazepam to human serum albumin

Cannabis is the most commonly used illicit drug worldwide. Cannabis users also appear to use other psychoactive drugs more frequently than noncannabis users. Here, Δ9-tetrahydrocannabinol (THC) and diazepam binding to human serum albumin (HSA) and HSA-heme is reported. THC binds to two different binding sites of HSA (K(d1) ≤ 10(-7) M and K(d2) = 10(-3)M) without affecting diazepam binding (K(d) = 1.2 × 10(-5) M). THC binding to the high-affinity site accounts for the low free fraction of the drug in plasma. Moreover, THC increases the affinity of heme for HSA. Accordingly, the affinity of THC for HSA-heme is higher than that for HSA. THC could bind to FA2 and FA7 sites, as substantiated by docking simulations; nevertheless, the observed allosteric effect(s) suggests that the primary binding site of THC is the FA2 cleft that positively modulates heme affinity. Possibly, the HSA conformational transition(s) induced by THC binding could account for drug delivery to the liver through receptor- mediated endocytosis.     

Pollutant-induced modulation in conformation and β-lactamase activity of human serum albumin

Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.     

Impaired drug-binding capacities of in vitro and in vivo glycated albumin

Albumin, the major circulating protein in blood, can undergo increased glycation in diabetes. One of the main properties of this plasma protein is its strong affinity to bind many therapeutic drugs, including warfarin and ketoprofen. In this study, we investigated whether or not there were any significant changes related to in vitro or in vivo glycation in the structural properties and the binding of human albumin to both therapeutic drugs. Structural parameters, including redox state and ketoamine contents of in vitro and in vivo glycated purified albumins, were investigated in parallel with their affinity for warfarin and ketoprofen. High-performance liquid chromatography was used to determine the free drug concentrations and dissociation constants according to the Scatchard method. An alternative method based on fluorescence spectroscopy was also used to assess drug-binding properties. Oxidation and glycation levels were found to be enhanced in albumin purified from diabetic patients or glycated with glucose or methylglyoxal, after determination of their ketoamine, free thiol, amino group and carbonyl contents. In parallel, significant impairments in the binding affinity of in vitro and in vivo glycated albumin, as indicated by the higher dissociation constant values and confirmed by higher free drug fractions, were observed. To a lesser extent, this alteration also significantly affected diabetic albumin affinity, indicated by a lower static quenching in fluorescence spectroscopy. This work provides useful information supporting in vivo diabetic albumin could be the best model of glycation for monitoring diabetic physiopathology and should be valuable to know if glycation of albumin could contribute to variability in drugs response during diabetes.     

Characteristics of a nickel–albumin binding assay for assessment of myocardial ischaemia

Background: The aim of this study was to describe a method to measure ischaemia-induced alterations of the binding capacity of serum albumin to exogenous nickel.

Methods: We measured the levels of cardiac troponin I (cTnI), serum albumin, ischaemia-modified albumin (IMA) measured by a cobalt–albumin binding assay (CABA), and a nickel–albumin binding assay (NABA) in the following groups: myocardial infarction (n?=?32) and non-ischaemic chest pain (n?=?64).

Results: IMA, cTnI and NABA levels were higher in the myocardial infarction group. NABA presented a higher ability to discriminate myocardial ischaemia than CABA.

Conclusions: Patients with myocardial infarction have reduced nickel binding to human serum albumin, and NABA may have an important role as an early marker of myocardial ischaemia.     

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.     

The effects of dexamethasone on α-fetoprotein and albumin synthesis in cultured hepatoma 7777 cells

Dexamethasone inhibits -fetoprotein (AFP) synthesis, and stimulates albumin synthesis, in cultured hepatoma 7777 cells. These changes are due to a decrease in AFT-mRNA, and an increase in albumin-mRNA, in cells.     

Ischemia-modified albumin as a marker of vascular dysfunction and subclinical atherosclerosis in β-thalassemia major

Background: Ischemia-modified albumin (IMA) is an altered type of serum albumin that forms under conditions of oxidative stress and an independent predictor of major adverse cardiovascular events.

Objectives: To measure the levels of IMA in 45 children and adolescents with β-thalassemia major (β-TM) compared with 30 healthy controls and assess its relation to lipid peroxidation, vascular complications and subclinical atherosclerosis.

Methods: β-TM patients without symptoms of heart disease were studied focusing on transfusion history, chelation therapy, serum ferritin, malondialdehyde (MDA) and IMA levels. Echocardiography was performed and carotid intima media thickness (CIMT) was assessed.

Results: IMA and MDA levels were significantly higher in β-TM patients compared with controls (p?Conclusion: Our results highlight the role of oxidative stress in the pathophysiology of vascular complications in thalassemia. IMA could be useful for screening of β-TM patients at risk of cardiopulmonary complications and atherosclerosis because its alteration occurs in early subclinical disease.     

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.