Bilirubin dehydrogenase,an enzyme in Aspergillus ochraceus IB-3 useful for diagnostic measurement of bilirubin

Bilirubin dehydrogenase, a membrane-bound enzyme that catalyzes the one-step oxidation of ditaurobilirubin and bilirubin to ditaurobiliverdin and biliverdin, respectively, in the presence of an electron acceptor, was found in Aspergillus ochraceus IB-3, and purified from the membrane fraction through solubilization by Triton X-100. Phenazine and quinone derivatives acted as electron acceptors. Accumulation of ditaurobiliverdin and biliverdin by enzyme catalysis increased the absorbance at 660 nm, which is far from the range of wavelengths affected by serum ingredients. The enzyme selectively oxidized ditaurobilirubin at low pH, so changes in the reaction pH enable the enzyme to discriminate between the bilirubin fractions ditaurobilirubin (an example of conjugated bilirubin) and bilirubin (an example of unconjugated bilirubin). Using the enzyme, 2 to 80 microM of ditaurobilirubin were measured accurately by monitoring the changes in absorbance at 660 nm.     

Candida Rugosa lipase reactions in nonionic w/o-microemulsion with a technical surfactant

In enzyme catalysis there is a great interest in finding suitable organic media for less water soluble substrates in order to increase the substrate concentration and therefore the reaction rates. These requirements are fulfilled by using w/o-microemulsions as reaction media. The influences of pH, temperature, water concentration and the kinetic parameters of Candida Rugosa Lipase in a nonionic w/o-microemulsion with a surfactant of technical grade, Marlipal O13-60, are presented. In an example the enantiospecific esterification of racemic menthol with propionic anhydride using this nonionic microemulsion likely to be affordable in large scale applications is shown. For a continuous process an ultrafiltration unit is attached to a reactor within a loop. In this way, the reverse micelles containing the enzymes can be separated from the oil, containing the product, and reused afterwards.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

Peroxidative oxidation of bilirubin during prostaglandin biosynthesis

The peroxidative oxidation of bilirubin has been characterized in the ram seminal vesicle microsomal system. The oxidation was monitored by following the loss in absorbance of bilirubin at 440 nm. Bilirubin behaves as a peroxidase substrate for prostaglandin H synthase. The oxidation may be initiated by the addition of arachidonic acid or peroxides to incubations containing ram seminal vesicle microsomes and bilirubin, and is sensitive to inhibition by reduced glutathione. The arachidonate-dependent oxidation, but not the peroxide-initiated case, is inhibited by indomethacin. Similar results were obtained using microsomal preparations from mouse, rat, and pig lungs. Spectral and chromatographic examination of the products of bilirubin oxidation in the ram seminal vesicle system demonstrate that biliverdin is produced in this system by the dehydrogenation of bilirubin, but that this product accounts for only about 15% of the bilirubin consumed. Biliverdin itself is not oxidized in this system. At least three highly polar, fluorescent products also are formed from bilirubin. Though not identified, these polar products differ markedly in chromatographic behavior from the major fluorescent products obtained following the singlet oxygen oxidation or the autoxidation of bilirubin.     

Microsomal conjugation and oxidation of bilirubin

Bilirubin diglucuronide and bilirubin monoglucuronide are formed on incubation of microsomal preparations from rat liver with bilirubin and UDPglucuronate. Microsomal diglucuronide formation is a two-step reaction: first monoglucuronide is formed and this is subsequently converted to diglucuronide. Both steps require UDPglucuronate and have a similar pH optimum at pH 7.8. Albumin inhibits the conversion of monoto diglucuronide. Factors favouring diglucuronide formation are: (a) low bilirubin concentration; (b) relatively high UDPglucuronate concentration; (c) complete removal of UDPglucuronyltransferase latency. For the latter, trypsin-treatment appeared superior over digitonin or UDP-N-acetylglucosamine. Trypsin-treatment had to be done under strictly anaerobic conditions. If trypsin treatment was done under aerobic conditions, reactive molecules were formed which initiated the rapid oxidation of bilirubin and its glucuronides. Microsomal oxidation of bilirubin and glucuronides also occurred in untreated and digitonin-treated microsomes and was stimulated by NADPH and by the cytochrome P-450 inhibitor, metyrapone. This suggests that lipid peroxides act as initiators of bilirubin oxidation. Indirect evidence was found that trypsin inactivates nucleotide pyrophosphatase. This is an active UDPglucuronate-consuming enzyme in microsomal preparations which must be inactivated before meaningful kinetic studies can be done. With trypsin-treated microsomal preparations the Vmax for bilirubin monoglucuronide formation was 1.7 X 10(-9) mol . mg protein-1 . min-1 and KUDPglucuronatem 43 X 10(-6) M. For bilirubin diglucoronide formation the apparent Vmax was 0.7 X 10(-9) mol . mg protein-1 . min-1 and the apparent KUDPglucuronate m 1.0 X 10(-3) M.     

Stability of bilirubin oxidase in organic solvent media: a comparative study on two low-water systems

The storage stability of bilirubin oxidase was studied in water-in-oil CTAB microemulsions with a chloroformrich continuous organic phase. The kinetics of the inactivation process were best described by a double exponential equation. Approximately half of enzymatic activity was lost during a "fast" phase with a half life of ca. 50 min, whereas the remaining activity was lost much more slowly (half life ca. 1000 min). Rates of inactivation were not affected significantly by variation of either solvent composition or concentration of water droplets, but inactivation was more rapid when droplet size was very small. Steady-state enzyme kinetics were studied at various stages in the inactivation process, and it was shown that inactivation occurred without change in the K(m) of the enzyme for bilirubin. Stability was also studied in a liquid/solid two-phase system; it was found that the inactivation process in this system; it was found that the inactivation process in this system was best described by a single exponential term. The rate was similar to the "fast" phase rate observed in the water-in-oil microemulsion system. Inactivation of the enzyme slow. Addition of the surfactant CTAB to the aqueous environment increased the rate of inactivation to levels comparable to those of the "slow" phase observed in water-in-oil microemulsions. (c) 1993 Wiley & Sons, Inc.     

An immobilized enzyme reactor for the detoxification of bilirubin

An immobilized enzyme reactor has been developed for the degradation of bilirubin as a potential treatment for neonatal jaundice. It utilizes the enzyme bilirubin oxidase from Myrothecium verrucaria, which in the presence of molecular oxygen converts bilirubin to biliverdin and other products that are much less toxic than bilirubin. Bilirubin oxidase was covalently attached to agarose beads using cyano transfer activation. Forty percent of the specific activity of bilirubin oxidase was retained after immmobilization, and preparations with 20 units of enzymatic activity per gram of drained wet weight of gel were obtained. The stability of bilirubin oxidase at pH 7.4 and 37 degrees C was improved fivefold by immobilization. A 15-mL column containing immobilized bilirubin oxidase, through which a 37 degrees C solution of 332muM bilirubin and 450muM human serum albumin in 0.05M phosphate buffer (pH 7.4) was passed at 1 mL/min, converted more than 60 percent of the bilirubin per pass. The substrate specificity of the enzyme and the small volume of the reactor are important characteristics for this clinical application where it is desirable to remove only one compound from the blood and to minimize the volume of blood in the extracorporeal circuit. This reactor, by detoxifying the jaundiced infant's blood of bilirubin, would eliminate the risks associated with the use of donor blood as is done currently in treating severe neonatal jaundice.     

An in vitro model of aneurysmal subarachnoid hemorrhage: oxidation of unconjugated bilirubin by cytochrome oxidase

Aneurysmal subarachnoid hemorrhage is a stroke subtype with high rates of mortality and morbidity. Cerebral vasospasm can lead to ischemic injury or death and is a common complication of aneurysmal subarachnoid hemorrhage, usually occurring 3-9 days afterwards. The cause of vasospasm is not known. Recently, there has been strong evidence that vasoactive oxidation products of bilirubin may be involved. Currently, the factors that lead to bilirubin oxidation are poorly characterized. In this study, we have designed an in vitro model of hemorrhagic stroke in order to investigate conditions that promote the oxidation of bilirubin to form vasoactive compounds. Using our model, we created a basic hematoma system of blood, CSF, and hemeoxygenase-1. We manipulated this system in various ways, incubated it and determined the concentration of vasoactive bilirubin oxidation products that resulted. Conditions where cytochrome oxidase was stimulated caused an increase bilirubin oxidation products (292.6 +/- 39.9 micromol/L respectively, vs. 79.3 +/- 1.3 micromol/L for the basic reaction, p < 0.05), which was attenuated by cyanide. Our data suggest that bilirubin oxidation products may be produced by oxidation(s) requiring an oxygen-utilizing enzyme like cytochrome oxidase.     

The water-dependence of the catalytic activity of bilirubin oxidase suspensions in low-water systems.

We investigated the enzymic activity of bilirubin oxidase when it is suspended as a lyophilized powder in a low-water system. The enzyme required buffer salts and a source of water to show activity. This study investigated the complete range of water thermodynamic activity (a(w)) by combining the use of salt hydrates and two-phase systems with concentrated solutes in the aqueous phase. When free water was added, activity reached a maximum at a defined water content, but this maximum increased with buffer content, suggesting that there was competition for water with the buffer salts from which the enzyme was lyophilized. Alternatively, a range of salt hydrates was used, each able to fix the water activity (a(w)) at a different value. By providing water to the organic solvent phase in this way, the dependency of enzyme activity upon a(w) was investigated and shown to be independent of buffer concentration. However, the optimum a(w) was uncertain because the available a(w) range for salt hydrates is < or = 0.90. Investigation of the remaining water activity range was made possible by using an a(w) depressor (sorbitol) to lower the a(w) of a two-phase system. The optimum a(w) for the bilirubin oxidase activity in this two-phase system was a(w) = 0.936, independent of buffer concentration. The study therefore confirmed the need to control the water 'available' to low-water systems and the dependence of enzyme activity on water thermodynamic activity (a(w)) not water content.     

Characterization of alkaliphilic laccase activity in the culture supernatant of Myrothecium verrucaria 24G-4 in comparison with bilirubin oxidase

An enzyme showing alkaliphilic laccase activity was purified from the culture supernatant of Myrothecium verrucaria 24G-4. The enzyme was highly stable under alkaline conditions, showed an optimum reaction pH of 9.0 for 4-aminoantipyrine/phenol coupling, and decolorized synthetic dyes under alkaline conditions. It showed structural and catalytic similarities with bilirubin oxidase, but preferably oxidized phenolic compounds. The enzyme catalyzed veratryl alcohol oxidation at pH 9.0 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a mediator, suggesting that the laccase mediator system functioned well under alkaline conditions.     

The formation of bilirubin and p-nitrophenyl glucuronides by rabbit liver

1. Glucuronide formation of bilirubin and p-nitrophenol in vitro with excess of UDP-glucuronic acid by UDP-glucuronyltransferase from livers of young and adult rabbits was studied. 2. The development of UDP-glucuronyltransferase for the two substrates followed a markedly different pattern during maturation of young rabbits, p-nitrophenol-conjugation ability being much higher at birth than that for bilirubin. 3. Mg(2+) increased bilirubin conjugation, but inhibited p-nitrophenyl glucuronide formation. 4. p-Nitrophenol acted as a potent non-competitive inhibitor for bilirubin conjugation but bilirubin did not affect p-nitrophenyl glucuronidation. 5. The enzyme for bilirubin conjugation was inactivated at pH9 during treatment with snake venom, whereas in the same preparation the activity of the corresponding enzyme for p-nitrophenol was enhanced. In addition, some solubilization of the latter enzyme could be achieved by this method. 6. The possibility of the existence of more than one enzyme system for the formation of O-glucuronides is discussed.     

The enzyme-catalyzed formation of bilirubin diglucuronide by a solubilized preparation from cat liver microsomes

When bilirubin monoglucoronide is incubated with a preparation from the 105 000 × g-supernatant of deoxycholate-treated cat liver microsomes, bilirubin diglucuronide is formed. This is an UDPglucuronate-dependent reaction whereby bilirubin IXα monoglucuronide is stoichiometrically converted into bilirubin IXα diglucuronide.The pH optimum for the conversion of bilirubin into bilirubin monoglucuronide lies between pH 8.0 and pH 8.8. For the conversion of mono- into diglucuronide two optima were found, one at about pH 6.5 and another at pH 8.1.When incubation was performed at pH 6.5 and the enzyme protein concentration was lowered, bilirubin monoglucuronide started to isomerise. As a result of this isomerisation bilirubin diglucuronide is also formed. Diglucuronide formation according to this mechanism however, can be clearly differentiated from the enzyme-catalyzed diglucuronide formation.By the formation of bilirubin monoglucuronide, one monoglucuronide isomer is preferentially synthesized.The alkaline-labile bilirubin conjugates in the bile of cats and rats have mainly the IXα isomeric structure. This suggests that in these animals bilirubin diglucuronide is formed enzymically as the bilirubin moiety of diglucuronide, formed by means of the isomerisation reaction, has predominantly the XIIα structure.     

Bilirubin Dehydrogenase,an Enzyme in Aspergillus ochraceus IB-3 Useful for Diagnostic Measurement of Bilirubin

Bilirubin dehydrogenase, a membrane-bound enzyme that catalyzes the one-step oxidation of ditaurobilirubin and bilirubin to ditaurobiliverdin and biliverdin, respectively, in the presence of an electron acceptor, was found in Aspergillus ochraceus IB-3, and purified from the membrane fraction through solubilization by Triton X-100. Phenazine and quinone derivatives acted as electron acceptors. Accumulation of ditaurobiliverdin and biliverdin by enzyme catalysis increased the absorbance at 660 nm, which is far from the range of wavelengths affected by serum ingredients. The enzyme selectively oxidized ditaurobilirubin at low pH, so changes in the reaction pH enable the enzyme to discriminate between the bilirubin fractions ditaurobilirubin (an example of conjugated bilirubin) and bilirubin (an example of unconjugated bilirubin). Using the enzyme, 2 to 80 μM of ditaurobilirubin were measured accurately by monitoring the changes in absorbance at 660 nm.     

Characterization of lipase in reversed micelles formulated with cibacron blue F-3GA modified span 85

Sorbitan trioleate (Span 85) modified by Cibacron Blue F-3GA (CB) was prepared and used as an affinity surfactant to formulate a reversed micellar system for Candida rugosa lipase (CRL) solubilization. The system was characterized and evaluated by employing CRL-catalyzed hydrolysis of olive oil as a model reaction. The micellar hydrodynamic radius results reflected, to some extent, the redistribution of surfactant and water after enzyme addition, and the correlation between surfactant formulation, water content (W0), micellar size, and enzyme activity. An adequate modification density of CB was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited a different activity behavior versus W0. The optimal pH and temperature of the encapsulated lipase remained unchanged, but the apparent activity was significantly higher than that of the native enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant was found to decrease with increasing surfactant concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0.     

Determination of bilirubin glucuronide and assay of glucuronyltransferase with bilirubin as acceptor

1. Conjugated bilirubin is conveniently determined by coupling with the diazonium salt of ethyl anthranilate. 2. This method has been used in the development of assays for UDP-glucuronyltransferase (EC 2.4.1.17), with bilirubin as substrate, in rat liver homogenates, microsomal preparations and partly purified fractions. 3. Chromatographic analysis suggests that bilirubin monoglucuronide is the product of the enzyme systems studied.     

Enzyme function in organic solvents.

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non-aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water less than 0.02% by vol.), perhaps reflects that the minimum necessity for water is for forming bonds with polar amino acids on the enzyme surface. The rigidity of enzyme structure at such low water content results in novel substrate specificities, pH memory and the possibility of techniques such as molecular imprinting. Limited data indicates that, while enhanced thermal stability invariably results, the optimum temperature for catalysis may not change. If true in general, this enhanced thermostability would have extremely limited benefits. Medium engineering and biocatalyst engineering are relevant techniques to improve the efficiency and stability of enzymes in such low water systems. Most promising, as part of the latter, is the technique of protein engineering. Finally, this review provides illustrations of applications of such systems in the diverse areas of organic synthesis, analysis and polymer chemistry.     

Factor affecting enzyme characteristics of bilirubin oxidase suspensions in organic solvents

The activity of bilirubin oxidase toward bilirubin was studied in a liquid/solid two-phase low-water organic system using a simple spectrophotometric assay to follow the reaction. The enzyme was lyophilized from aqueous solution before being suspended in the organic solvent reaction medium. The activity was significantly influenced by the properties of the aqueous medium from which the enzyme was lyophilized, specifically its pH, and the quantity and nature of the buffering species. Analyses of these effect showed that the role of buffering species in such systems went beyond their effect in fixing the protonation state of the enzyme. The activity was also influenced by the quantity of water added to the organic solvent reaction medium. The reaction was shown to follow Michaelis-Menten Kinetics, and K(m) and k(cat) were determined. The liquid/solid two-phase system studied was extensively compared to a previously studied water-in-oil microemulsion system (c) 1993 Wiley & Sons, Inc.     

Interaction of bilirubin with gangliosides

Gangliosides seem to play an important role in the interaction of the neurotoxic pigment bilirubin with the synaptosomal plasma membrane (Vázquez et al. [1988] J. Biol. Chem. 263, 1255-1265). In this report, a further characterization of the bilirubin-ganglioside interaction is presented. The interaction is fast, and it is observed at any pH in the range 7.0-9.0. The characteristics of the interaction are different from those observed with other membrane lipids, including sphingomyelin. A model of binding to a single population of sites is able to adequately fit the experimental data. This model predicts a decrease in the tendency of bilirubin to interact with gangliosides and an increase in the binding capacity as the pH is decreased from 8.0 to 7.0. Our data would suggest a role for gangliosides in explaining the preferential accumulation of bilirubin in some areas of the brain and the toxic effect of this pigment in neuronal membrane-related functions.     

Thermostability of α-chymotrypsin encapsulated in reversed micelles

Summary The influence of water content, additives, pH and substrate concentration on the thermostability of -chymotrypsin entrapped in a reversed micellar system of the cationic surfactant TTAB/heptane/ chloroform, was studied. Increasing the water level inside the reversed micelles diminishes the enzyme stability. Enzyme stability enhancement was achieved with the addition of glycerol, by increasing the nucleophile concentration or by decreasing the buffer pH.     

Evaluation of steady-state kinetic parameters for enzymes solubilized in water-in-oil microemulsion systems.

1. Equations are derived for the steady-state kinetics of substrate conversion by enzymes confined within the water-droplets of water-in-oil microemulsion systems. 2. Water-soluble substrates initially confined within droplets that do not contain enzyme are assumed to be converted into product only after they enter enzyme-containing droplets via the inter-droplet exchange process. 3. Hyperbolic (Michaelis-Menten) kinetics are predicted when the substrate concentration is varied in microemulsions of fixed composition. Both kcat. and Km are predicted to be dependent on the size and concentration of the water-droplets in the microemulsion. 4. The predicted behaviour is shown to be supported by published experimental data. A physical interpretation of the form of the rate equation is presented. 5. The rate equation for an oil-soluble substrate was derived assuming a pseudo-two-phase (oil & water) model for the microemulsion. Both kcat. and Km are shown to be independent of phi aq. Km is larger than the aqueous solution value by a factor approximately equal to the oil/water partition coefficient of the substrate. The validity of the rate equation is confirmed by published data.