The isolation and characterization of bilirubin diglucuronide, the major bilirubin conjugate in dog and human bile.

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by t.l.c. into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields two types of product, those soluble in water and those soluble in organic solvents. The organic-soluble fraction was shown by t.l.c. and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analysed by enzymic procedures, t.l.c., n.m.r. spectrometry and combined g.l.c. and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and n.m.r. spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid had been released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.     

Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).     

Occurrence of ditaurobilirubin, bilirubin conjugated with two moles of taurine, in the gallbladder bile of yellowtail, Seriola quinqueradiata

An unknown bilirubin conjugate was detected by HPLC analysis in the bile of yellowtail, Seriola quinqueradiata. The retention time of this unknown conjugate was identical with that of authentic ditaurobilirubin, a bilirubin conjugated with 2 mol of taurine. The retention times of the ethyl anthranilate diazo derivatives of both bilirubin conjugates were the same. The azo derivatives of this unknown conjugate were purified by Amberlite XAD-2 treatment and preparative TLC and hydrolyzed with 6 N HCl at 105 degrees C for 12 h. The hydrolyzates obtained were analyzed with an amino acid analyzer. The ninhydrin-positive substance in the hydrolyzates was purified by column chromatography and analyzed by TLC and mass spectrometry. The results of these analyses indicated that this ninhydrin-positive substance was taurine. Therefore, this unknown bilirubin conjugate was identified as ditaurobilirubin.     

Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease.

We have developed an extremely rapid and efficient reverse-phase h.p.l.c. method for the measurement of bilirubin and its conjugates in human bile and in model bile systems. Our method involves the use of a Perkin-Elmer 3 mu C18 column and a methanol/sodium acetate/aq. ammonium acetate buffer system. Three isomers of bilirubin diglucuronide (BDG), two isomers of bilirubin monoglucuronide (BMG), three isomers of unconjugated bilirubin (UCB) and minor conjugates containing glucose and xylose were separated in 12 min. Initial quantification of BDG and BMG was based on the use of the ethyl anthranilate azo derivative of bilirubin (AZO UCB); however, the standard curves for BDG, BMG and UCB were similar enough to permit quantification to be later based on the UCB standard curve only, thereby simplifying the quantification process. Routine direct injection of 6 or 10 microliter of crude undiluted or diluted (1:1) bile sample was sufficient for analysis. The method was helpful in diagnosing biliary-tract obstruction in a newborn and a partial deficiency state of bilirubin conjugation (Crigler-Najjar syndrome) in a 10-year-old male. When the method was applied to biles of patients both with and without gallstones, levels of UCB were less than 2% of total pigment, consistent with previous reports. Because of its speed and efficiency, this method has the potential for a broad range of applications including enzymic, kinetic and bile sample analyses.     

Mitochondrial RNA Polymerase from <Emphasis Type="Italic">Neurospora crassa</Emphasis>

The enzyme consists of a single polypeptide chain of molecular weight of 64,000 and tends to form heavy aggregates. It is sensitive to rifampicin and resistant to α-amanitin. It prefers native mitochondrial DNA and d(AT) copolymer as template.     

Reverse-phase h.p.l.c. separation, quantification and preparation of bilirubin and its conjugates from native bile. Quantitative analysis of the intact tetrapyrroles based on h.p.l.c. of their ethyl anthranilate azo derivatives.

We describe a facile and sensitive reverse-phase h.p.l.c. method for analytical separation of biliary bile pigments and direct quantification of unconjugated bilirubin (UCB) and its monoglucuronide (BMG) and diglucuronide (BDG) conjugates in bile. The method can be 'scaled up' for preparative isolation of pure BDG and BMG from pigment-enriched biles. We employed an Altex ultrasphere ODS column in the preparative steps and a Waters mu-Bondapak C18 column in the separatory and analytical procedures. Bile pigments were eluted with ammonium acetate buffer, pH 4.5, and a 20 min linear gradient of 60-100% (v/v) methanol at a flow rate of 2.0 ml/min for the preparative separations and 1.0 ml/min for the analytical separations. Bile pigments were eluted in order of decreasing polarity (glucuronide greater than glucose greater than xylose conjugates greater than UCB) and were chemically identified by t.l.c. of their respective ethyl anthranilate azo derivatives. Quantification of UCB was carried out by using a standard curve relating a range of h.p.l.c. integrated peak areas to concentrations of pure crystalline UCB. A pure crystalline ethyl anthranilate azo derivative of UCB (AZO . UCB) was employed as a single h.p.l.c. reference standard for quantification of BMG and BDG. We demonstrate that: separation and quantification of biliary bile pigments are rapid (approximately 25 min); bile pigment concentrations ranging from 1-500 microM can be determined 'on line' by using 5 microliters of bile without sample pretreatment; bilirubin conjugates can be obtained preparatively in milligram quantities without degradation or contamination by other components of bile. H.p.l.c. analyses of a series of mammalian biles show that biliary UCB concentrations generally range from 1 to 17 microM. These values are considerably lower than those estimated previously by t.l.c. BMG is the predominant, if not exclusive, bilirubin conjugate in the biles of a number of rodents (guinea pig, hamster, mouse, prairie dog) that are experimental models of both pigment and cholesterol gallstone formation. Conjugated bilirubins in the biles of other animals (human, monkey, pony, cat, rat and dog) are chemically more diverse and include mono-, di- and mixed disconjugates of glucuronic acid, xylose and glucose in proportions that give distinct patterns for each species.     

Bilirubin excretion into bile after partial hepatectomy in rats

Biliary excretion of bilirubin was studied in Wistar rats at different intervals following two-thirds hepatectomy. Endogenous bilirubin excretion was not significantly modified during liver regeneration. The maximal biliary excretion rate (Tm) of bilirubin, expressed per 100 g of body weight, significantly decreased immediately after hepatectomy with a recovery to presurgery values by 16 days. Values expressed per g of liver were significantly lowered only by 12- and 24h posthepatectomy. In these early periods there was a significant increase in the liver concentration of unconjugated bilirubin and a decrease in bile flow following bile pigment infusion. Maximal excretion of bilirubin in the rat seems to change parallel to the regeneration of liver mass except during the first 24h in which the inhibitory effects exerted by the high load of exogenous bilirubin would cause a decline in its own biliary output with an added cholestatic effect.     

Structural studies of a branchiopod crustacean (Lepidurus bilobatus) extracellular hemoglobin. Evidence for oxygen-binding domains.

The extracellular hemoglobin of the notostracan branchiopod Lepidurus bilobatus has an apparent molecular weight of 680,000 and may exist in a dissociation-association equilibrium dependent on pH and ligand state. The pigment contains one heme per 18,000 g protein. However, attempts to dissociate the hemoglobin by harsh denaturing conditions results in a 33-34,000 molecular weight polypeptide chain as well as traces of some 62-64,000 molecular weight material. Limited proteolysis of this hemoglobin with subtilisin produces 14,800 and 16,500 dalton heme-containing polypeptides (domains) which bind oxygen reversibly. These domains, isolated by column chromatography, have a heme content similar to the intact pigment. It is proposed that the intact 34,000 dalton subunit of Lepidurus hemoglobin consists of two linearly linked oxygen binding domains. Oxygen binding properties of the intact hemoglobin show a low oxygen affinity with a slight Bohr effect. In contrast, the isolated domains display a relatively high oxygen affinity and lack a Bohr effect between pH 7.0 and 8.0. It is apparent that the intact 34,000 dalton polypeptide is necessary for the expression of the heterotropic interactions of the native pigment.     

Metabolism of bilirubin by a clonal strain of rat hepatoma cells

These studies demonstrate that the MH₁C₁ strain of rat hepatoma cells has the ability to take up and conjugate bilirubin and then excrete the conjugated pigment into the culture medium. On incubation with unconjugated bilirubin, the average rate of appearance of conjugated bilirubin in the medium was 4.4 ± 0.20 µg per mg of cell protein per hour (mean ± SE). The products formed from bilirubin by MH₁C₁ cells were chromatographically identical to those found in normal rat bile. Assay of bilirubin UDP glucuronyl transferase activity in homogenates of MH₁C₁ cells gave a value of 3.3 ± 0.50 µg of conjugated pigment formed per mg protein per hour, only moderately less than the enzyme activity of liver from normal rats. Rat fibroblasts in culture did not conjugate bilirubin, nor did they contain bilirubin UDP-glucuronyl transferase activity. As in living animals, flavaspidic acid inhibited bilirubin metabolism by MH₁C₁ cells, suggesting that the mechanism for bilirubin uptake is similar to that of normal liver. In contrast to the findings in animals, however, preincubation of MH₁C₁ cells with phenobarbital led to only minimal enhancement of pigment conjugation. MH₁C₁ cells represent the first example of a clonal strain of cells in culture in which many of the pathways of hepatic bilirubin metabolism remain intact. They should, therefore, serve as a useful model for studies of bile pigment metabolism which are not easily performed in the living animal.     

Photodynamic action of bilirubin on human erythrocyte membranes. Modification of polypeptide constituents.

The photodynamic action of bilirubin on isolated human erythrocyte membranes (ghosts) has been studied. When incorporated into ghosts (pH 8.0,10 degrees) the bile pigment photosensitizes in blue light the peroxidation of unsaturated lipids, as evidenced by a positive color reaction with 2-thiobarbituric acid. Accompanying lipid peroxidation was the disappearance of most of the major membrane proteins (Coomassie Blue staining in sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and appearance of polypeptide photoproducts of greater size (mol wt greater than 250,000). The association of membrane proteins (presumably by cross-linking) was insignificant when bilirubin-ghost suspensions were kept in the dark, or when ghosts were irradiated in the absence of bilirubin. Electrophoretic bands 1 and 2 (Fairbanks, G., Steck, T.L., and Wallach, D. F.H (1971), Biochemistry 10, 2606) diminished rapidly during the photoreaction, whereas band 3 and the three sialoglycoproteins disappeared at a much slower rate. Dispersal of membrane consituents by treatment with sodium dodecyl sulfate prior to irradiation resulted in relatively little peroxidation and no noticeable formation of high molecular weight polypeptide complexes. The possibility that malonaldehyde, a product of lipid peroxidation, is involved in cross-linking during irradiation was studied by incubating ghosts with exogenous malonaldehyde. Although the reagent did cross-link membrane proteins (electrophoretic bands 1, 2, 2.1 2.2, and 4.1 diminished most rapidly and high molecular weight bands appeared), the reaction could only be demonstrated with malonaldehyde concentrations several orders of magnitude greater than those detected in irradiation experiments. If malonaldehyde cross-linking occurs, it does not appeare to be the predominant mechanism of polypeptide association during irradiation of bilirubin-containing ghosts.     

Glyceraldehyde-3-phosphate dehydrogenase in the isolated human erthrocyte membrane: selective displacement by bilirubin.

When unsealed erythrocyte ghosts in 6 mm phosphate buffer (pH 8.0, 4 °C) were incubated with bilirubin in excess of 0.1 mm and washed with buffer, a single polypeptide component (band 6 in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis) diminished and was recovered in the supernatant fraction. Release of this component was virtually complete at 1 mm initial bile pigment. Since band 6 was believed to be the protomer of membrane-bound glyceraldehyde-3-phosphate dehydrogenase (G3PD), assays for this enzyme in bilirubin-treated ghosts were carried out. These revealed that enzymatic activity decreased concurrently with the disappearance of band 6. The molecular weight of the eluted polypeptide was found to be 36,000, in agreement with the known value for the G3PD protomer. When Mg²⁺-resealed ghosts were washed after exposure to 1 mm bilirubin, less than 20% of the G3PD was eluted, which is consistent with the fact that the enzyme is attached to the cytoplasmic face of the membrane. NAD⁺ in concentrations up to 2 mm displaced no more than 15% of the G3PD from unsealed ghosts. However, after preincubation with NAD⁺ (1 mm) followed by bilirubin (1 Mm) and washing, loss of G3PD was similar to that observed in the absence of cofactor. Since NAD⁺ did not attenuate release of the enzyme, it appears unlikely that such release is attributable to binding of bilirubin at the active site. Protoporphyrin acted similarly to bilirubin on unsealed ghosts, whereas rose bengal had a more pronounced effect, removing all enzymatic activity when the dye concentration reached 0.2 mm. Electrophoretic analysis of ghosts after rose bengal treatment, however, revealed a diminution not only of band 6 but bands 1, 2, and 5 as well.     

Pyruvate carboxylase from a thermophilic Bacillus: some molecular characteristics.

Analysis of the native enzyme and of the subunits produced upon its denaturation shows that pyruvate carboxylase from a thermophilic Bacillus is a tetramer with a molecular weight (mean value) of 558,000 and that the four polypeptide subunits are probably identical. The three functions (carboxyl carrier, carboxylation, and carboxyl transfer) in the pyruvate carboxylation reaction must therefore reside in this quarter-molecular polypeptide. The enzyme molecule contains four atoms of zinc and four molecules of D-biotin, and in the electron microscope the disposition of its four subunits presents a rhombic appearance. Reaction of the denatured enzyme with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reveals 10 sulfhydryl groups/subunit. In the native enzyme less than one of these groups reacts with DTNB. By contrast, all of these groups (11/subunit) of the native chicken liver pyruvate carboxylase are accessible to DTNB. The thermophile enzyme is also more resistant to other sulfhydryl reagents and to denaturation under certain conditions than the avian enzyme.     

Nuclear-magnetic-resonance investigations of the biliverdin-apomyoglobin complex

The recently described biliverdin-apomyoglobin complex has been investigated by two-dimensional NMR methods and molecular modeling with respect to the geometry of the chromophore and its position within the myoglobin pocket. Nuclear Overhauser effect correlations between the ligand and apoprotein amino acid residues prove that the bile pigment assumes a cyclic helical conformation and a position similar to heme in native myoglobin.     

Inhibition of cholesterol crystallization under bilirubin deconjugation: partial characterization of mechanisms whereby infected bile accelerates pigment stone formation

Pigment gallstones have been reported to be closely associated with biliary tract infection. We previously reported that addition of unconjugated bilirubin (UCB), which is deconjugated by beta-glucuronidase in infected bile, could enhance cholesterol crystal formation in supersaturated model bile (MB). The present study evaluated the effect of beta-glucuronidase on the processes of pigment gallstone formation and cholesterol crystallization. Supersaturated MB (taurocholate/lecithin/cholesterol at 71:18:11, a total lipid concentration of 10.0 g/dl and a cholesterol saturation index (CSI) of 2.0) and native rat bile were mixed at a ratio of 3:1. Then, mixed bile was incubated with or without beta-glucuronidase and changes of the following parameters were investigated over time: (1) the UCB/total bilirubin ratio; (2) cholesterol crystal formation; (3) the precipitate weight and the cholesterol concentration in the precipitate and supernatant; and (4) the lipid distribution of vesicles in the supernatant. Compared with beta-glucuronidase-free bile, (1) beta-glucuronidase-containing bile showed a significant increase of the UCB/total bilirubin ratio, (2) as well as a significantly longer nucleation time (96+/-17.0 vs. 114+/-20.0) and fewer cholesterol crystals. (3) The precipitate weight and the cholesterol concentration in the precipitate were significantly increased, while the cholesterol concentration in supernatant was decreased. (4) When mixed bile was incubated with beta-glucuronidase, the cholesterol concentration in the vesicles was lower than in bile without beta-glucuronidase. The precipitate weight and the cholesterol concentration in the precipitate was increased by incubation with beta-glucuronidase, while cholesterol concentration was decreased in the supernatant (especially in the vesicles). This means that bile vesicles were more stable and it was more difficult for cholesterol crystals to form. Thus, the presence of beta-glucuronidase may inhibit the formation of pure cholesterol stones even in the presence of cholesterol supersaturation.     

Non-enzymic hydrolysis of bilirubin mono- and diglucuronide to unconjugated bilirubin in model and native bile systems. Potential role in the formation of gallstones.

Pigment gallstones contain considerable amounts of unconjugated bilirubin (UCB) in the form of calcium bilirubinate and/or bilirubin polymers. Since more than 98% of bile pigments are excreted as conjugates of bilirubin, the source of this UCB needs to be identified. By using a rapid h.p.l.c. method, we compared the non-enzymic hydrolysis of bilirubin monoglucuronide (BMG) and bilirubin diglucuronide (BDG) to UCB in model bile and in native guinea-pig bile. Model biles containing 50 microM solutions of pure BMG and BDG were individually incubated in 25 mM-sodium taurocholate (NaTC) and 0.4 M-imidazole/5 mM-ascorbate buffer (TC-BUF) at 37 degrees C. Over an 8 h period, BMG hydrolysis produced 4-6 times more UCB than BDG hydrolysis. At pH 7.4, 25% of the BMG was converted into UCB, whereas only 4.5% of BDG was converted into UCB. Hydrolysis rates for both BMG and BDG followed the pH order 7.8 greater than 7.6 approximately equal to 7.4 greater than 7.1 Incubation with Ca2+ (6.2 mM) at pH 7.4 in TC-BUF resulted in precipitated bile pigment which, at 100 X magnification, appeared similar to precipitates seen in the bile of patients with pigment gallstones. At pH 7.4, lecithin (crude phosphatidylcholine) (4.2 mM) was a potent inhibitor of hydrolysis of BMG and BDG. The addition of a concentration of cholesterol equimolar with that of lecithin eliminated this inhibitory effect. Guinea-pig gallbladder bile incubated with glucaro-1,4-lactone (an inhibitor of beta-glucuronidase) underwent hydrolysis similar to the model bile systems. The non-enzymic hydrolysis of bile pigments, especially BMG, may be an important mechanism of bile-pigment precipitation and, ultimately, of gallstone formation.     

pKa and aggregation of bilirubin: titrimetric and ultracentrifugation studies on water-soluble pegylated conjugates of bilirubin and fatty acids

A water-soluble conjugate (1) with intact carboxyl groups was prepared by addition of poly(ethylene glycol) thiol (MPEG-SH) regiospecifically to the exo vinyl group of bilirubin. (1)H and (13)C NMR and absorbance spectroscopy in CDCl(3) and DMSO-d(6) confirmed the assigned structure and showed that pegylation did not disrupt the hydrogen-bonded ridge-tile conformation of the pigment moiety. Aqueous solutions of 1 were optically clear, but NMR signals were seen only from the MPEG portion and none from the tetrapyrrole, consistent with dissolved assemblies containing aggregated bilirubin cores within mobile polyether chains. On alkalinization (pH >12), signals from the pigment moiety reappeared. Titrimetric measurements on 1 in water showed the pK(a)'s of the two carboxyl groups to be similar (average 6.42). Control studies with pegylated half-esters of succinic, suberic, brassylic, thapsic, and 1,20-eicosanedioic acid showed that pegylation per se has little, if any, effect on carboxyl ionization. However, aggregation increases the apparent pK(a) by approximately 1-2 units. The molecularity of bilirubin in solution was further characterized by ultracentrifugation. Over the pH range 8.5-10 in buffer, bilirubin formed multimers with aggregation numbers ranging from approximately 2-7. Bilirubin is monomeric in DMSO or CHCl(3) at approximately 2 x 10(-)(5) M, but aggregation occurred when the CHCl(3) was contaminated with trace adventitious (perhaps lipoidal) impurities. These observations show that aggregation increases the pK(a)'s of aliphatic carboxylic acids relative to their monomer values in water. They are consistent with earlier (13)C NMR-based estimates of approximately 4.2 and approximately 4.9 for the aqueous pK(a)'s of bilirubin and similar studies of bilirubin in micellar bile-salt solutions. Together with earlier work, they confirm that the pK(a)'s of bilirubin are about normal for aliphatic carboxyls and suggest that the high (>7.5) values occasionally reported, including those based on CHCl(3) partitioning, are artifacts of aggregation or technique.     

The ionization behavior of bile acids in different aqueous environments

The ionization behavior of cholic acid, deoxycholic acid, and chenodeoxycholic acid in a variety of physiologically important molecular environments was studied using 13C NMR spectroscopy. The apparent pKa of the carboxyl group was determined from titration curves obtained from the dependence of the carboxyl carbon chemical shift on pH. Using 90% 13C isotopic substitution of the carboxyl carbon, a complete titration curve was obtained for cholate at a concentration below its critical micelle concentration and solubility limit in water. Incorporation of 12 mole % bile acid into mixed micelles with its taurine conjugate prevented precipitation of the unconjugated bile acid, and titration curves for cholic, deoxycholic, and chenodeoxycholic acids in the mixed micelles were obtained. The apparent pKa was also determined for 13C-enriched bile acids complexed with bovine serum albumin and in egg phosphatidylcholine vesicles. For monomers, micelles, and BSA complexes of all three bile acids and for deoxycholic and chenodeoxycholic acid in vesicles, one magnetic environment was observed. In contrast, two environments, both titratable, were detected for cholic acid in phosphatidylcholine vesicles. The apparent pKa's of the bile acids in the different environments ranged from 4.2 to 7.3. At pH 7.4, as monomers or bound to albumin, the bile acids were fully ionized, but when associated with phosphatidylcholine vesicles they were only partially ionized. In addition, aspects of the molecular motion and relative hydrophobicity of the bile acid carboxyl group in the environments studied were discerned from chemical shift, line-width, and lineshape data.     

Urobilinogen-i is a major derivative of bilirubin in bile of homozygous Gunn rats.

Gunn rats lack bilirubin UDP-glycosyltransferases, but diazo-negative derivatives of bilirubin have been described in their bile. In order to investigate this alternative disposal of bilirubin, crude bile samples from Gunn and Wistar rats were directly analysed by h.p.l.c. Besides bilirubin (in Gunn rats) or its glycosides (in Wistar rats), two major compounds were detected. A yellow one corresponded to the previously documented vitamin B-2 and was equally prominent in Gunn rats or Wistar-rat bile. The other compound was colourless, but on standing in contact with air it was spontaneously oxidized to a pinkish-yellow pigment. It was far more prominent in Gunn-rat bile. Analysis of bile obtained after intravenous injection of [14C]bilirubin to Gunn rats demonstrated that this compound was highly labelled. Freezing and thawing of the bile resulted in the formation of a series of diazo-negative derivatives, demonstrating that the original compound was quite labile. Spectral (adsorption and fluorescent) and chromatographic (h.p.l.c., t.l.c. and paper chromatography) analysis of the oxidized form of the labelled compound allowed its identification as urobilin-i. The colourless compound secreted in bile was urobilinogen-i. Administration of neomycin and bacitracin to Gunn rats or gut resection suppressed the biliary excretion of urobilinogen and thus confirmed its intestinal origin. Urobilinogen seems thus to represent the major bilirubin derivative present in Gunn-rat bile. Its breakdown products might represent the so-far-unidentified diazo-negative polar bilirubin derivatives. Since only a small amount of bilirubin is present in Gunn-rat bile, the urobilinogen formed in the intestinal lumen seems to be derived from bilirubin reaching the gut via routes other than the biliary one.     

A physical-chemical and immunological comparison shows that native and renatured Escherichia coli tryptophan synthase beta 2 subunits are identical

An acid-denaturation of the beta 2 subunit of Escherichia coli tryptophan synthase has been recently described. In the present study, renaturation yield of acid-denaturated beta 2, and the influence of temperature, protein concentration and presence of ligands are investigated. It is also demonstrated that 3 forms of the protein are obtained at the end of the renaturation process: one is fully active, and is identical to native beta 2, as indicated by some of its chemical and physical properties, as well as by its immunological reactivity towards monoclonal antibodies specific for the native protein. A second form is composed of high molecular weight insoluble and inactive aggregates. A third form consists of low molecular weight soluble and inactive aggregates. The results obtained for the immunochemical reactivity of these small aggregates indicate that they are formed with partly correctly folded beta monomers assembled by specific but incorrect quaternary interactions. The capacity of monoclonal antibodies to detect such incorrect structures and to characterize renatured proteins is particularly emphasized.