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.     

Use of fluorescence enhancement technique to study bilirubin–albumin interaction

Bilirubin–albumin solution gave an emission spectrum in the wavelength range 500–600 nm with emission maxima at 528 nm when excited at 487 nm. The magnitude of fluorescence intensity increased on increasing bilirubin/albumin molar ratio. At three different albumin concentrations, namely, 1.0, 2.5 and 10.0 μM, there was an initial linear increase in fluorescence up to a molar ratio 1.0 in all cases beyond which it sloped off or decreased. This fluorescence enhancement was used to calculate the binding parameters of bilirubin–albumin interaction and the value of binding constant was found to be 1.72×10⁷ l/mol similar to the published values obtained with other methods. Different serum albumins, namely, human (HSA), goat (GSA), pig (PSA) and dog serum albumins (DSA) bound bilirubin with almost the same affinity when studied by the technique of fluorescence enhancement. Bilirubin–albumin interaction was also studied at different pH and ionic strengths. There was a decrease in bilirubin–albumin complex formation on either decreasing the pH from 9.0 to 7.0 or increasing the ionic strength from 0.15 to 1.0. These results suggest that the technique of fluorescence enhancement can be used successfully to study the bilirubin–albumin interaction.     

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.     

Interaction of bilirubin with the synaptosomal plasma membrane

The interaction of the neurotoxic pigment bilirubin with synaptosomal plasma membrane vesicles (SPMV) isolated from rat brain was investigated. The interaction seems to involve three steps: (a) a rapid formation of an electrostatic complex between bilirubin and polar lipid head groups; (b) a slow inclusion of the pigment into the hydrophobic core of the membrane; and (c) a SPMV-induced bilirubin aggregation, observed when membrane capacity for bilirubin is exceeded. The association constant of the initial complex increased markedly when pH was lowered below 7.4, particularly in SPMV isolated from newborn rats. A preferential binding of bilirubin to pure gangliosides and sphingomyelin was observed, thus suggesting a role for these lipids as first targets of the pigment in the synaptic membrane. The inclusion of bilirubin into the membranes was gradually enhanced when decreasing the pH or the age of the rats from which SPMV were isolated. In addition, membranes from 2-day-old rats have a higher capacity for bilirubin incorporation compared to those from adult rats. Experiments with reconstituted liposomes of varying protein and cholesterol contents suggest that the effect of age may be related to changes in synaptosomal membrane fluidity during development. Our results support the hypothesis that the interaction of bilirubin with the synaptic membrane plays an important role in the molecular mechanisms of bilirubin neurotoxicity.     

The orange-yellow pigment of heartwater-infected angora goats

Heartwater is a highly fatal disease of ruminants in South Africa, caused by the parasite Cowdria ruminantium. A consistent pigment, associated with serum albumin, could be readily extracted from heartwater exudates and serum samples using n-butanol. The pigment gave a characteristic absorption spectrum between 400 and 500 nm which was absent in control serum samples. RP-HPLC of the isolated pigment revealed two components with absorption maxima at 440 and 460 nm and mol. wts of 558 and 1239. The pigment closely resembled bilirubin and/or its conjugates when compared by HPLC.     

Interaction of bilirubin with brain capillaries and its toxicity

The interaction of bilirubin with isolated brain capillaries, and the effect of bilirubin on the uptake of 2-deoxyglucose by the capillaries were investigated with 1-month-old Sprague-Dawley rats. The binding of bilirubin to the brain capillaries was observed only at a molar ratio of bilirubin to bovine serum albumin higher than 1.0. An absorption spectrum with a microspectrophotometer of the bilirubin-capillary complex showed a broad absorption maximum from 425 to 440 nm with a shoulder near 490 nm, but no shoulder was observed in the case of the bilirubin emulsion. The bilirubin binding activity was dependent on pH and temperature of the medium, but was not affected by sulfhydryl blocking agents such as p-chloromercuribenzoate and N-ethylmaleimide. Bilirubin saturation kinetics gave an apparent K_m for bilirubin of 61.7 μM. Release of the bilirubin from the brain capillaries to the medium was observed at 37°C but not at 4°C. The uptake of 2-deoxyglucose by the isolated brain capillaries was inhibited by bilirubin in a noncompetitive manner, giving an apparent K_i for the pigment of 137 μM.These results suggest that bilirubin may be responsible for the decreased 2-deoxyglucose uptake in the brain capillaries by disturbing the membrane structure of the capillary endothelial cells.     

Photooxidation of human serum albumin and its complex with bilirubin.

Irradiation with visible light of human serum albumin in aqueous solution at pH 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole O2, and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen. Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non-oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole O2. Parallel photooxidation of the protein carrier could not be detected. It is considered possible that the fast, anaerobic process is operative in phototherapy of hyperbilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment.     

Porphyrin-induced photooxidation of conjugated bilirubin

Visible light irradiation of 18 microM bilirubin ditaurate (BR-DT) at pH 7.0 for 30 min showed a 10% decrease in absorbance at 445 nm. When the reaction was carried out in the presence of a trace amount of uroporphyrin (UP), the spectrum of BR-DT disappeared without a concomitant formation of biliverdin. Photooxidation products were confirmed to be dipyrrole-containing compounds. Photo-bleaching of BR-DT was accelerated by the increasing concentration of UP and was inhibited, when UP was replaced by Cu2+UP. Formation of 2,2,6,6-tetramethylpiperidine N-oxyl through the irradiation of UP was diminished by sodium azide, a potent scavenger of singlet oxygen. The efficiency of singlet oxygen formation through visible light irradiation was in the order UP, coproporphyrin > Cu2+UP. Both bilirubin and BR-DT bound to human serum albumin (HSA) were photooxidized effectively in the presence of UP. The results indicate that irradiation of UP produces singlet oxygen with high efficiency which then rapidly oxidizes free and conjugated bilirubin.     

Bilirubin. Solubility and interaction with albumin and phospholipid.

Bilirubin is generally considered a lipophilic substance, and its neurotoxicity is ascribed to an affinity for lipids in the central nervous system. In the present paper, it is shown that the solubility of bilirubin in apolar solvents and in triglycerides is low and increases with solvent polarity. Consequently, bilirubin should not be characterized as lipophilic. The solubility in aqueous buffers was studied under exclusion of light and was found lower than previously reported, about 7 nM at pH 7.4, temperature 37 degrees C, increasing with higher pH, approximately in inverse proportion with the squared hydrogen ion concentration. Binding of bilirubin to human serum albumin was studied by the rate of oxidation with peroxidase of the free ligand at equilibrium. The stoichiometry of proton involvement in the binding process was investigated by acidimetric titration. It is concluded that precipitation of bilirubin in vivo is thermodynamically possible. It was further demonstrated by light absorption spectroscopy that bilirubin forms a complex with phosphatidylcholine in diethyl ether and that an aqueous suspension of phosphatidylcholine enhanced aggregation of bilirubin. Transfer of bilirubin dianion from its complex with plasma albumin and precipitation of bilirubin acid with phosphatidylcholine in membranes of nerve cells is therefore possible and may account for the neurotoxicity.     

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.     

Kinetics and mechanism of bilirubin binding to human serum albumin

The kinetics of bilirubin binding to human serum albumin at pH 7.40, 4 degrees C, was studied by monitoring changes in bilirubin absorbance. The time course of the absorbance change at 380 nm was complex: at least three kinetic events were detected including the bimolecular association (k1 = 3.8 +/- 2.0 X 10(7) M-1 S-1) and two relaxation steps (52 = 40.2 +/- 9.4 s-1 and k3 = 3.8 +/- 0.5 s-1). The presence of the two slow relaxations was confirmed under pseudo-first order conditions with excess albumin. Curve-fitting procedures allowed the assignment of absorption coefficients to the intermediate species. When the bilirubin-albumin binding kinetics was observed at 420 nm, only the two relaxations were seen; apparently the second order association step was isosbestic at this wavelength. The rate of albumin-bound bilirubin dissociation was measured by mixing the pre-equilibrated human albumin-bilirubin complex with bovine albumin. The rate constant for bilirubin dissociation measured at 485 nm was k-3 = 0.01 s-1 at 4 degrees C. A minimum value of the equilibrium constant for bilirubin binding to human albumin determined from the ratio k1/k-3 is therefore approximately 4 X 10(9) M-1.     

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.     

Stopped-flow studies of spectral changes in bilirubin-human serum albumin following an alkaline pH jump and following binding of bilirubin

A stopped-flow technique was used to study the spectral changes occurring in bilirubin-albumin following a pH jump as well as following binding of bilirubin at 25 degrees C. The changes were studied in two wavelength ranges, 280-310 nm (tyrosine residues) and 400-510 nm (bound bilirubin). The changes were analyzed according to a scheme of consecutive unimolecular reactions. Spectral monitoring of a pH jump from 11.3 to 11.8 reveals that the bilirubin-albumin complex changes its structure in several steps. The UV absorption spectra show that 3.8 tyrosine residues ionize in the first step, 2.5 in the second, none in the third, and 0.8 in the fourth and following steps. The visible absorption spectrum of bound bilirubin changes in the second, third, and fourth steps. The bilirubin spectra of the different bilirubin-albumin complexes occurring in the transition show a common isosbestic point at 445 nm, indicating a change of the dihedral angle between the two bilirubin chromophores in a three-step reaction. It is suggested that 1 tyrosine residue is located close to the bilirubin site and is externalized in the second step. Bilirubin binding to albumin was monitored at two pH values, 11.3 and 11.8. At pH 11.3 the complex changes its structure in a three-step relaxation sequence. A change of the dihedral angle between the bilirubin chromophores can explain the spectral changes observed in the second and third relaxations. Protonation of 0.7 tyrosine residues occurs in the third relaxation, suggesting internalization of a tyrosine residue as a late consequence of bilirubin binding. At pH 11.8 a two-step relaxation sequence follows bilirubin binding. No tyrosine protonation occurs. Bilirubin is probably bound more superficially at pH 11.8 than at pH 11.3.     

Alkali-induced conformational transition in different domains of bovine serum albumin

Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to N<-->B transition (pH 7.5 to 9.0) and the other to B --> U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.     

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.     

On the modulation of photoinduced fluorescence enhancement and conformational stability of albumin-bound bilirubin: effect of epsilon-NH(2) groups blocking and chloroform binding

Photoinduced fluorescence enhancement of bilirubin bound to primary binding site on human serum albumin (HSA) was completely ceased when epsilon-NH(2) groups of its internal lysine residues were covalently blocked by acetylation or succinylation though the pigment bound to these derivatives in a folded conformation akin to that bound to HSA. These photoinduced fluorescence modulations cannot be ascribed to the binding of bilirubin to secondary low affinity sites as the CD spectrum of bilirubin bound to these derivatives showed complete inversion upon addition of chloroform which binds to subdomain IIA in HSA where high affinity bilirubin binding site is located. Presence of chloroform reconciled the photoinduced alterations in the CD spectrum observed in its absence, suggesting that chloroform stabilized the bound ligand against light but the fluorescence properties of bilirubin complexed with acetylated or succinylated derivatives remained unchanged. Guanidination of internal epsilon-NH(2) groups in HSA by O-methylisourea did not alter the spectral properties of the bound ligand. These results suggest that salt linkage(s) existing between epsilon-NH(2) groups of lysine residues in HSA and carboxyl groups of bilirubin, act(s) as a potential barrier during conformational rotation of the bound ligand assisted by photoactivation and their abolishment can alter its dynamics and stereoselectivity, a hitherto unnoticed implication of salt linkage(s) in BR-HSA complex.     

Binding of bilirubin by bovine and human alpha-fetoprotein.

alpha-Fetoprotein, a fetal protein associated with certain tumors, was found to bind bilirubin. Addition of human or bovine alpha-fetoprotein to bilirubin solutions enhanced the light absorbance of bilirubin and shifted its maximum. Bovine alpha-fetoprotein caused a marked shift towards shorter wavelengths, while human alpha-fetoprotein gave a slight red shift. The spectral changes were used to study the characteristics of the binding of bilirubin by bovine alpha-fetoprotein. These studies indicated the presence of one binding site/molecule of alpha-fetoprotein with an association constant of about 1.1 . 10(6) M-1. A difference between the spectral changes brought about by alpha-fetoprotein and albumin allowed comparison of their relative affinities for bilirubin. The spectrum approximated the average between the spectra induced by the two proteins when the ratio of bovine alpha-fetoprotein to bovine albumin was 6.3 : 1, and of the human proteins 21 : 1, respectively. These results show that alpha-fetoprotein from two species binds bilirubin with an affinity somewhat lower than that of albumin. Binding of bilirubin by alpha-fetoprotein is in agreement with the recent demonstration of structural homology between alpha-fetoprotein and albumin. Whether alpha-fetoprotein plays a role in the metabolism of bilirubin or other degradation products of heme remains to be investigated.     

Albumin prevents nonspecific transferrin binding and iron uptake by isolated hepatocytes

Bovine serum albumin inhibits binding of transferrin by hepatocytes in suspension by 60-70%. Iron uptake is inhibited by less than 20%. A Scatchard analysis of the transferrin-binding data reveals a biphasic plot in the absence of bovine serum albumin, but a monophasic plot in the presence of bovine serum albumin. Bovine serum albumin inhibits low-affinity binding of transferrin (125000 molecules/cell), but has no effect on high-affinity binding (38000 molecules/cell). In pronase-treated cells, transferrin binding is reduced by 40%, and when bovine serum albumin is added, the binding is reduced by a further 40%. Corresponding figures for iron uptake are 70 and 10%, respectively. The results are strong evidence that the major part of iron uptake by hepatocytes occurs from transferrin bound to the plasma membrane transferrin receptor.     

Cobinding of bilirubin and sulfonamide and of two bilirubin molecules to human serum albumin: a site model

Differential light absorption spectra of the bilirubin-albumin 1:1 complex, obtained on addition of 20 different sulfonamides, differ with respect to shape and amplitude. This finding seems to indicate that the sulfonamide molecule is bound in direct touch with the bilirubin. The light absorption spectrum of bilirubin-albumin 1:1 undergoes changes on cobinding of a fatty acid anion, laurate, and on variation of pH, previously explained by a change of dihedral angle between the two chromophores of the bilirubin molecule. In bilirubin-albumin 2:1, binding of laurate and variation of pH cause little change of the spectrum. This is best explained by binding of the two bilirubin molecules in close proximity, preventing conformational changes in the complex. From measurements of fluorescence of the lone tryptophan group in albumin and quenching on binding of bilirubin, we calculated the distance of 22 A from tryptophan to the first bound bilirubin molecule, and of 18 A to the second. Mutual quenching of the bilirubin fluorescence from two bound bilirubin molecules seemed to indicate that the two are bound closely together. A model of bilirubin-albumin with a binding site capable of accommodating one bilirubin and one sulfonamide molecule, or two molecules of bilirubin, is compatible with our findings.     

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.