Effect of bilirubin on the activity and thermokinetic characteristics of brain (synaptosomal) membrane NO synthase

NO synthase activity was found in the plasma (synaptosomal) membrane particles isolated from the homogenate of adult rat brain (without cerebellum) under conditions preventing the protease attack and formation of reactive oxygen species. The NO synthase discovered exhibited some properties of a neuronal constitutive integral membrane enzyme and was inhibited by N-nitro-L-arginine. NO synthase activity decreased when bilirubin entered the synaptosomal membrane in vitro. Bilirubin caused the shift of the transition temperature in the temperature dependence of NO synthase activity in Arrhenius plots. The incorporation of bilirubin into synaptosomal membranes resulted in an increase in the apparent activation energy for NO synthase within a temperature range of 10-30 degrees C. The membrane NO synthase was susceptible to the photodynamic effect of membrane-bound bilirubin molecules. Monomeric human serum albumin without organophilic ligands exerted a protective effect on NO synthase in bilirubin-containing membrane particles.     

The binding of bilirubin to albumin. A study using spin-labelled bilirubin

Binding between human serum albumin and a spin-labelled derivative of bilirubin was investigated by circular dichroism, fluorescence quenching, electron spin resonance and visible spectroscopy. The orders of magnitude of the binding constants obtained by flurorescence quenching and electron spin resonance spectroscopies were 10(7) and 10(3) 1 . mol-1, respectively. These data suggest that most spin-labelled bilirubin interacts with human serum albumin at the side not holding the spin-labelled side-arm. CD measurements showed the presence of at least two sites, associated with opposite Cotton effects. It is worthy of note that the Cotton sign of the first site is inverted with respect to the corresponding one of bilirubin. CD measurements on mixed systems (spin-labelled bilirubin/human serum albumin/bilirubin) were also performed. The decomposition of the ternary curves shows that the rotatory power of bilirubin bound to human serum albumin is higher in the ternary system than in the binary (bilirubin/human serum albumin). The corresponding CD measurements for the binding between spin-labelled bilirubin and bovine serum albumin are also reported and discussed.     

Effect of Bilirubin on the Membrane Potential of Rat Brain Synaptosomes

The effect of the neurotoxic pigment bilirubin on the membrane potential of rat brain synaptosomes was studied by using the tetraphenylphosphonium ion (TTP+) technique. Bilirubin induces a rapid depolarization of synaptosomes, as reflected by an efflux of previously accumulated [3H]TTP+. This phenomenon persisted when the membrane potential across either the plasma membrane of the synaptosome or the inner membrane of the entrapped mitochondria was selectively depressed, thus indicating that both components of the synaptosomal membrane potential were affected by bilirubin. Bovine serum albumin, used at a albumin/bilirubin molar ratio of 1:1, had the capacity to completely prevent and reverse the effect of bilirubin. This fact demonstrates that the bilirubin-induced TPP+ release from synaptosomes is a reversible process that requires the presence of bilirubin interacting with the synaptosomal membranes. These results, together with the inhibition by bilirubin of [3H]TPP+ and [2-14C]acetate uptake by synaptosomal plasma membrane vesicles isolated from rat brain, suggest that bilirubin depresses the membrane potential across the synaptosomal plasma membrane by a mechanism involving alterations in ion permeability. This effect could be of relevance in the pathogenesis of bilirubin encephalopathy.     

The Biological Effects of Bilirubin Photoisomers

Although phototherapy was introduced as early as 1950’s, the potential biological effects of bilirubin photoisomers (PI) generated during phototherapy remain unclear. The aim of our study was to isolate bilirubin PI in their pure forms and to assess their biological effects in vitro. The three major bilirubin PI (ZE- and EZ-bilirubin and Z-lumirubin) were prepared by photo-irradiation of unconjugated bilirubin. The individual photoproducts were chromatographically separated (TLC, HPLC), and their identities verified by mass spectrometry. The role of Z-lumirubin (the principle bilirubin PI) on the dissociation of bilirubin from albumin was tested by several methods: peroxidase, fluorescence quenching, and circular dichroism. The biological effects of major bilirubin PI (cell viability, expression of selected genes, cell cycle progression) were tested on the SH-SY5Y human neuroblastoma cell line. Lumirubin was found to have a binding site on human serum albumin, in the subdomain IB (or at a close distance to it); and thus, different from that of bilirubin. Its binding constant to albumin was much lower when compared with bilirubin, and lumirubin did not affect the level of unbound bilirubin (Bf). Compared to unconjugated bilirubin, bilirubin PI did not have any effect on either SH-SY5Y cell viability, the expression of genes involved in bilirubin metabolism or cell cycle progression, nor in modulation of the cell cycle phase. The principle bilirubin PI do not interfere with bilirubin albumin binding, and do not exert any toxic effect on human neuroblastoma cells.     

Kinetics of bilirubin oxidase and modeling of an immobilized bilirubin oxidase reactor for bilirubin detoxification

The unbound bilirubin concentration and the enzymatic rate of bilirubin degradation by bilirubin oxidase in bilirubin-serum albumin solutions have been investigated experimentally and theoretically. A stoichiometric bilirubin-serum albumin binding analysis shows that the unbound bilirubin concentration depends only on the molar ratio of the total bilirubin concentration to the total serum albumin concentration. From the theoretical analysis and the measured unbound bilirubin concentrations, serum albumin may be modelled as a molecule having two binding sites, primary and secondary, with stoichiometric equilibrium constants of K(1) = 6 x 10(7)M(-1) and K(2) = 4.5 x 10(6)M(-1), respectively. The rate of total bilirubin degradation in bilirubin-serum albumin mixtures is zero order. An immobilized bilirubin oxidase reactor model, which shows good agreement with experimental bilirubin conversions, is presented. At a flow rate of 1 mL/min with a 8-mL reactor volume, a 50% bilirubin conversion per pass was observed with an inlet bilirubin concentration of 350muM and a serum albumin concentration of 500muM.     

Effect of bilirubin and serum albumin on the Na,K-ATPase activity in the synaptosomal membrane

The absorption spectrum of visible light, characteristic of the free bilirubin being in the aqueous medium, with a single maximum at 440 nm and with the shoulder in the region of 410-420 nm is transformed into the spectrum with two maxima in the region of 460 and 500 nm, respectively, when the pigment is bound in vitro by the synaptosomal membrane. There are two types of sites for bilirubin binding in the membrane particles, differing in the values of constants of association (Ka = 0.6 . 10(5) and approximately 2.02 . 10(5) M-1, respectively) and in the values of the maximum binding of bilidiene (5.0 and 7.0 nmoles/mg of membrane proteins, respectively). The binding of bilirubin by the synaptosomal membrane leads to a decrease in the specific activity of the membrane Na+,K+-ATPase. The enzyme activity is further decreasing when suspension of the membrane particles is exposed to the blue light (lambda max = 450-460 nm) in the presence of bilirubin. The addition of the serum albumin into the incubation medium potentiates the inhibition effect of bilirubin, when the suspension of membrane particles is lighted in the presence of bilirubin. The alkalization of the medium up to pH 7.8 (from pH 7.2) removes this potentiation effect of the addition of serum albumin.     

Mechanism of hepatocellular uptake of albumin-bound bilirubin

We previously demonstrated that unconjugated bilirubin spontaneously diffuses through phospholipid bilayers at a rate which exceeds albumin dissociation, suggesting that solvation from albumin represents the rate-limiting step in hepatic bilirubin clearance. To further examine this hypothesis, we studied the uptake of bovine serum albumin (BSA)-bound bilirubin by cultured hepatoblastoma (HepG2) cells. Uptake of bilirubin was saturable, with a K(m) and V(max) of 4.2+/-0.5 microM (+/-S.E.M.) and 469+/-41 pmol min(-1) mg(-1) at 25 degrees C. Substantial bilirubin uptake also was observed at 4 degrees C (K(m)=7.0+/-0.8 microM, V(max)=282+/-26 pmol min(-1) mg(-1)), supporting a diffusional transport mechanism. Consistent with reported solvation rates, the cellular uptake of bilirubin bound to human serum albumin was more rapid than for BSA-bound bilirubin, indicative of dissociation-limited uptake. Counterintuitively, an inverse correlation between pH and the rate of bilirubin flip-flop was observed, due to pH effects on the rate of dissociation of bilirubin from albumin and from the membrane bilayer. The identification of an inflection point at pH 8.1 is indicative of a pK(a) value for bilirubin in this range. Taken together, our data suggest that hepatocellular uptake of bilirubin is dissociation-limited and occurs principally by a mechanism involving spontaneous transmembrane diffusion.     

A dynamic model for bilirubin binding to human serum albumin

Site-directed mutagenesis of human serum albumin was used to study the role of various amino acid residues in bilirubin binding. A comparison of thermodynamic, proteolytic, and x-ray crystallographic data from previous studies allowed a small number of amino acid residues in subdomain 2A to be selected as targets for substitution. The following recombinant human serum albumin species were synthesized in the yeast species Pichia pastoris: K195M, K199M, F211V, W214L, R218M, R222M, H242V, R257M, and wild type human serum albumin. The affinity of bilirubin was measured by two independent methods and found to be similar for all human serum albumin species. Examination of the absorption and circular dichroism spectra of bilirubin bound to its high affinity site revealed dramatic differences between the conformations of bilirubin bound to the above human serum albumin species. The absorption and circular dichroism spectra of bilirubin bound to the above human serum albumin species in aqueous solutions saturated with chloroform were also examined. The effect of certain amino acid substitutions on the conformation of bound bilirubin was altered by the addition of chloroform. In total, the present study suggests a dynamic, unusually flexible high affinity binding site for bilirubin on human serum albumin.     

Effect of the binding of bilirubin to either the first class or the second class of binding sites of the human serum albumin molecule on its photochemical reaction.

The kinetics of the photochemical changes of bilirubin were studied at a constant concentration of bilirubin bound either to the first class or to the second class of binding sites of the human serum albumin molecule. The more the bilirubin binds to the first class of binding sites in the human serum albumin molecule, the more readily geometric photoequilibrium to give (ZE)-bilirubin takes place. The more the bilirubin binds to the second class of binding sites or allosterically transformed binding sites induced by added SDS, the more readily structural photoisomerization, i.e. the formation of (EZ)-cyclobilirubin, takes place. When the serum bilirubin concentration is at low, safe, values bilirubin binds exclusively to the first class of binding sites and serves as an antioxidant [Onishi, Yamakawa & Ogawa (1971) Perinatology 1, 373-379]; at these concentrations human serum albumin protects bilirubin from irreversible photodegradation by only allowing readily reversible geometric photoisomerization. As the serum bilirubin concentration increases to high, and potentially dangerous, values, bilirubin binds to the second class of binding sites, and under these conditions human serum albumin seems to promote the photocyclization of bilirubin. During irradiation human serum albumin seems to act by retaining low, useful, concentrations of bilirubin while facilitating irreversible photoisomerization of excess bilirubin.     

Comparison of kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin with that bound to rat serum albumin.

It has been stated by McDonagh, Palma & Lightner [(1982) J. Am. Chem. Soc. 104, 6867-6871] that complexing of bilirubin with serum albumin has a marked species-dependent influence on bilirubin photoisomerization in vitro and in vivo. Therefore the kinetics for the quantitatively important reaction: (Formula: see text) of the photochemical interconversion between bilirubin and its photoisomers bound to human or rat serum albumin in aqueous solution, assayed by h.p.l.c., was used to elucidate the observed species-dependent difference. The relative rate constants for bilirubin bound to human serum albumin, except for k4, the rate of interconversion from (ZZ)-bilirubin into (EZ)-bilirubin, proved to be considerably larger than those for bilirubin bound to rat serum albumin. In accordance with these rate constants, the formation of photoisomers of bilirubin bound to human serum albumin, except for (EZ)-bilirubin, is very rapid and much greater than that for bilirubin bound to rat serum albumin.     

The role of albumin in the hepatic transport of bilirubin: studies in mutant analbuminemic rats

Bilirubin and other cholephilic organic anions are bound to albumin in the circulation; their hepatic uptake involves a carrier-mediated process. To investigate the possible role of serum albumin in the transhepatic transport of a cholephilic ligand, plasma clearance of radioactive bilirubin and its biliary excretion as well as its interaction with plasma proteins were compared between normal and mutant analbuminemic rats (NAR). With a tracer amount of 3H-labeled bilirubin, its plasma clearance and biliary excretion were comparable in both animal groups. However, the plasma clearance of a loading dose of the ligand was significantly increased and its biliary recovery was low in NAR as compared with normal animals. In accord with these findings in vivo, gel permeation chromatographic analysis revealed that the bilirubin binding capacity of serum proteins was significantly lower in NAR than in control animals. When bilirubin was administered to NAR as a mixture with equimolar albumin, its plasma disappearance was considerably decreased and its biliary recovery was increased. Similar effects were observed when albumin was replaced by an equimolar amount of glutathione S-transferases (ligandins). These observations indicate that, although ligand-protein interaction in the circulation is important for directing bilirubin to the plasma membranes of the hepatocyte, this mechanism is not specific for albumin.     

Structural and functional differences between fetal and adult serum albumin

The binding of bilirubin with adult of fetal human serum albumin has been studied by steady-state fluorescence emission spectroscopy. The 1:1 complex between bilirubin and the two albumin samples shows very similar fluorescence properties, as well as essentially identical accessibility of the protein-bound bilirubin to fluorescence quenchers added to the aqueous medium. The intramolecular distance between bilirubin and the single tryptophyl residue can be estimated to be 2.4 +/- 0.2 nm for both proteins by singlet-singlet energy transfer. These findings suggest that fetal and adult human serum albumin have a very similar three-dimensional structure; the different binding capacity for bilirubin displayed by the two proteins is likely to be the consequence of small differences in the physico-chemical properties of some amino acid residues close to the bilirubin binding site, as indicated by pH-titration experiments of the intrinsic albumin fluorescence.     

Multiple binding of bilirubin to human serum albumin and cobinding with laurate

Numerical analysis of multiple binding of two ligands to one carrier has been accomplished, using the principle of several sets of acceptable binding constants, with bilirubin-laurate-albumin as an example. Binding of bilirubin to defatted human serum albumin was investigated by a spectroscopic method, based upon a difference of light absorption spectrum for free and bound bilirubin. The observations were supplemented with previous data from an independent technique, measurement of oxidation rates of free bilirubin with hydrogen peroxide and peroxidase. A continuous isotherm was obtained, showing binding of at least 4 mol bilirubin per mole albumin with the following stoichiometric binding constants, 1.11 X 10(8), 1.7 X 10(7), 8 X 10(5), and 4 X 10(4) M-1 at pH 8.2, ionic strength 0.15 M, 25 degrees C. The binding is anticooperative at all steps. A saturation level was not reached. Cobinding of bilirubin and laurate was studied, with up to 2 mol of each ligand per mole albumin, using the peroxidase method for determination of free equilibrium concentrations of bilirubin, and a dialysis rate technique for free laurate. The findings could be described in terms of a stoichiometric model. Heterotropic cooperativity was present among the first bilirubin and the first and second laurate molecules. More than two molecules of either ligand can be bound at the same time.     

A novel method to determine total and free serum bilirubin.

The determination of total (unconjugated) and free serum bilirubin concentrations using a novel and sensitive method based on static fluorescence quenching of daneyl bovine serum albumin was developed. The method allowed the use of a sample of 5 μl or less to determine total bilirubin over a range of 10–200 μg/ml with good recovery (94.9 ± 2.2%). For the determination of total bilirubin, a denaturation medium containing 8 m urea, 10 mm dithloerythreitol, and 0.1 m Tris was employed to eliminate interference by human serum albumin itself. The method was also tested with patients' sera containing negligible conjugated bilirubin in order to compare it to a commonly used “diazo” method. The correlation between the two methods gave a practically linear relation (γ = 0.99). The effects of a number of potentially interfering substances were tested and the results showed the test was specific for bilirubin. Concentrations of free bilirubin were determined without adding a denaturation agent. The experimental values were in agreement with those calculated theoretically using the isotherm of a single binding site and an association constant of human serum albumin to bilirubin of 1.5 × 10⁸m^?1.     

Effect of bilirubin and its photodegradation products on the respiration of mitochondria isolated from rat liver

In 0.05--0.1 mmol.l-1 concentration, bilirubin inhibits ADP-activated respiration of isolated liver mitochondria; it has no effect on respiration in the absence of ADP. Bilirubin-induced inhibition of respiration is not abolished by serum albumin, but bilirubin bound to serum albumin and the photodegradation products of bilirubin have no inhibitory effect.     

Affinity labeling of the primary bilirubin binding site of human serum albumin.

A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site.     

Adsorption of bilirubin with polylysine carrying chitosan-coated nylon affinity membranes

Microporous polyamide membranes were activated by bisoxirane and subsequently bound with chitosan (CS) to amplify reactive groups. Then polylysine (PLL) as ligand was immobilized onto the CS-coated nylon membranes. The contents of CS and PLL of PLL-attached membranes were 93.2 and 90.4 mg/g nylon membrane, respectively. Such PLL-attached membranes were used to adsorb bilirubin from the bilirubin-phosphate solution and bilirubin-albumin solution. The adsorption mechanism of bilirubin and the effects of temperature, initial concentration of bilirubin, albumin concentration and ionic strength on adsorption were investigated by batch experiments. The results showed that the adsorption capacity increased with increasing the temperature while decreased with increasing the NaCl concentration and albumin concentration, and the adsorption isotherm fitted the Freundlich model well. The result of dynamic experiment showed PLL-attached membranes can well remove the bilirubin from the bilirubin-albumin solution.     

The displacement of albumin bound bilirubin by free fatty acids in vivo

Variations in bilirubinaemia in response to alterations of the free fatty acid level was studied in conscious and unstressed Gunn rats. When only one molecule of bilirubin is bound to albumin (without bilirubin overload), no displacement of bilirubin is observed, even if the protein binds as much as 6 molecules of free fatty acids. After overloading with exogenous unconjugated bilirubin, the second site of fixation of bilirubin on albumin is partly occupied; in this situation, a displacement is observed, but only when more than 3.5 molecules of free fatty acids are simultaneously bound to the protein. In vivo, free fatty acids do not spontaneously reach such levels as those responsable for the observed displacement of bilirubin. In the ranges of bilirubin and free fatty acids concentrations likely to be encountered clinically, free fatty acids might not represent an effective agent of displacement for bilirubin, as it is commonly thought.     

Circular dichroism of bilirubin-amine association complexes: insights into bilirubin-albumin binding

Bichromophoric (4Z, 15Z)-bilirubin-IX alpha, the yellow-orange cytotoxic pigment of jaundice, adopts either of two intramolecularly hydrogen-bonded enantiomeric conformations that are in dynamic equilibrium in solution. The addition of optically active amines induces the pigment solutions to exhibit intense bisignate circular dichroism in the region of the bilirubin long wavelength uv-visible absorption band. The most intense circular dichroism Cotton effects, (delta epsilon) approximately equal to 130, are induced by beta-arylamines and are comparable to those exhibited by bilirubin complexes with serum albumin and other proteins. Like serum albumin and other proteins, the optically active base acts as a chiral complexation agent to induce an asymmetric transformation of bilirubin, whose induced bisignate circular dichroism Cotton effect is characteristic of exciton splitting of the component pyrromethenone chromophores. The amines thus serve as chiral templates for molecular recognition, and the complementary action of the amine complexation sites provides insight into the binding forces important in protein-bilirubin heteroassociation.     

Circular dichroism and hydrodynamic measurements of ternary protein--two ligand systems: serum albumin-bilirubin-drug or alcohol.

The effects of various ligands on bilirubin-serum albumin complexes in aqueous solution were investigated at pH 7.4 and 27 °C by circular dichroism (CD) measurements. The ligands included various penicillins, benzoic acid derivatives, and various lower aliphatic alcohols, using a molar excess of charcoal-treated human or bovine serum albumin with respect to bilirubin. In all cases investigated, significant changes in the visible-range CD spectra of the bilirubin-serum albumin complexes occurred within a certain range of added ligand concentrations. For several such ligand systems, analogous CD effects could be measured on both diluted and undiluted human blood serum or plasma. For part of the isolated albumin-ligand systems, significant dissociation of the bilirubin from the albumin was demonstrated by electrophoretic and analytical ultracentrifugation measurements, while other systems did not reveal measurable dissociation under the conditions used, indicating the formation of a ternary complex. A scheme of equilibria among all complex components is proposed, which includes both dissociation of the bilirubin and ternary complex formation in which the bilirubin conformation appears to be modified. At least two different sets of binding sites (competitive and noncompetitive) for added ligands are assumed. Values of apparent parameters describing the formation of ternary complexes from the bilirubin-albumin complex are estimated for a number of systems. Some relationships between the chemical structure of a ligand and its effect on the bilirubin-serum albumin complex are deduced. The relevance of the results obtained for the isolated protein-ligand complexes with respect to in vivo conditions is evaluated.