Bilirubin oxidase

Manufactured by Merck Group

Sourced in United States

Bilirubin oxidase is an enzyme that catalyzes the oxidation of bilirubin, a breakdown product of heme, to biliverdin. It is used in laboratory settings to measure bilirubin levels, which can be an indicator of liver or bile duct function.

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Bilirubin

7 protocols using bilirubin oxidase

Measuring VWF, ADAMTS13, and Platelet Activation

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Plasma levels of VWF were determined with an in-house enzyme-linked immunosorbent assay (ELISA) using commercially available polyclonal antibodies (A0082 for coating and P0226 for detection, both are rabbit anti-human antibodies, P0226 is a horseradish-peroxidase conjugated version of A0082 (RRID:AB_579516), DAKO, Glostrup, Denmark). A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity was measured in plasma which was pretreated for 30 minutes at 37°C with bilirubin oxidase (10U/mL; Sigma-Aldrich, Zwijndrecht, The Netherlands) to avoid interference of bilirubin with the assay. ADAMTS13 activity was assessed using the FRETS-VWF73 assay (Peptanova, Sandhausen, Germany) based on method described by Kokame
et al.^{16 (link)}. The antigen levels of VWF and the activity of ADAMTS13 in pooled normal plasma were set at 100%, and values obtained in test plasmas were expressed as a percentage of pooled normal plasma.
Platelet activation was assessed by measuring plasma levels of soluble P-selectin and platelet factor 4 (PF4) with a commercially available ELISAs (R&D Systems, Abingdon, United Kingdom).

Arshad F., Adelmeijer J., Blokzijl H., van den Berg A., Porte R, & Lisman T. (2014). Abnormal hemostatic function one year after orthotopic liver transplantation can be fully attributed to endothelial cell activation. F1000Research, 3, 103.

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Enzymatic Biofuel Cell Components

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Glucose dehydrogenase (GDH; flavin adenine dinucleotide (FAD)-dependent) from Aspergillus oryzae was purchased from Toyobo Enzyme, Inc. (Japan). The glucose oxidase (GOD; FAD-dependent) from Aspergillus niger was purchased from Amano Enzyme, Inc. (Japan). The activity of the anode enzymes was certificated by the company (GDH = 584 U/mg, GOD = 243 U/mg). Bilirubin oxidase (BOD; 25 U/mg) from Myrothecium verrucaria, poly(ethylene glycol) diglycidyl ether (PEGDGE), sodium hydrosulfite, 1-vinylimidazole, acrylamide, N,N,N′,N′-tetramethyl ethylenediamine, and ammonium persulfate were purchased from Sigma-Aldrich Co. (Milwaukee, WI, USA). All other solutions including phosphate-buffered saline (PBS) were prepared using deionized Milli-Q water (DW; Millipore, Japan). PAA-PVI-[Os(dmo-bpy)₂Cl]^+/2+ (−0.012 V vs. Ag/AgCl) and PAA-PVI-[Os(dCl-bpy)₂Cl]^+/2+ (0.355 V vs. Ag/AgCl) as anode and cathode mediators were synthesized by modifying previously described methods (Fig. S2A)^{31 (link)}.

Jeon W.Y., Lee J.H., Dashnyam K., Choi Y.B., Kim T.H., Lee H.H., Kim H.W, & Kim H.H. (2019). Performance of a glucose-reactive enzyme-based biofuel cell system for biomedical applications. Scientific Reports, 9, 10872.

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Graphene Oxide-Enzyme Hybrid Biosensor

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Graphene oxide (GO > 99 wt% purity and total thickness < 3 nm. Average dimensions of individual flakes ranged from 300–800 nm, Glucose oxidase (EC 1.1.3.4 from Aspergillus niger), Bilirubin Oxidase (EC 1.3.3.5 from Myrothecium verrucaria), Iron (III) chloride hexahydrate (FeCl₃.6H₂O), 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC),N-hydroxysuccinimide (NHS), Ammonium hydroxide (NH₄OH), 1,6-Hexanediamine, Glucose, Sodium acetate (NaAc) and Ethylene glycol (EG) were purchased from Sigma Aldrich and used without further purification. A 0.1 M phosphate buffer saline PBS, pH 7.0 was prepared by mixing solutions of Na₂HPO₄ and NaH₂PO₄. The Glucose solution was prepared overnight before use to allow mutarotation for 24 hours. All solutions were prepared with deionized water (DI).

Pakapongpan S., Tuantranont A, & Poo-arporn R.P. (2017). Magnetic Nanoparticle-Reduced Graphene Oxide Nanocomposite as a Novel Bioelectrode for Mediatorless-Membraneless Glucose Enzymatic Biofuel Cells. Scientific Reports, 7, 12882.

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Unconjugated Bilirubin Binding Assay

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Unconjugated bilirubin and bilirubin oxidase were purchased from Sigma-Aldrich (St. Louis, MO). Fluorecene-5’-maleimide and DyLight633-maleimide were from Thermo Scientific (Waltham, MA). Metal ion chelating resin was purchased from GE Healthcare Life sciences (Mickleton, NJ). Pooled normal human plasma (NHP) was obtained from George King Biotechnology (Overland Park, KS). SeaKem HGT (P) agarose (Lonza, Rockland, ME), nitrocellulose membrane (Bio-Rad, Hercules, CA), Pefablock (Sigma-Aldrich), rabbit anti-human VWF antibody (Dako, Carpinteria, CA), and IRDye-800CW-labeled goat anti-rabbit IgG (LI-COR Biotech, Lincoln, NE) were all commercially available. Plasma VWF [15 (link)] and recombinant human ADAMTS13 (rA13) [16 (link)] were purified to homogeneity using the methods previously described. GST-VWF73-H [17 (link)] and GST-rVWF71-11K [8 (link)] were prepared according to methods described previously. Leftover de-identified plasma samples anti-coagulated with sodium heparin after chemistry tests were collected from hospitalized patients with significantly elevated serum levels of Unconjugated bilirubin.

Lu R.N., Yang S., Wu H.M, & Zheng X.L. (2015). Unconjugated Bilirubin Inhibits Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Protease. Journal of thrombosis and haemostasis : JTH, 13(6), 1064-1072.

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Enzymatic Glucose Sensing Nanomaterials

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N-methyl-2-pyrrolidone (NMP), Nafion™, perfluorinated resin solution (5% wt), mesoporous carbon nanoparticles (MPCNPs, d < 500 nm, average pore size 6.4 nm), glucose oxidase (GOx, Type-VII, from Aspergillus niger, 100 000 units per g), horseradish peroxidase (HRP, Type VI-A), albumin (from human serum), bilirubin oxidase (BOD, from Myrothecium verrucaria), potassium hexachloroplatinate, glucose, fructose, polyvinylidene fluoride (PVDF), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS²⁻) were purchased from Sigma.

Trifonov A., Stemmer A, & Tel-Vered R. (2018). Enzymatic self-wiring in nanopores and its application in direct electron transfer biofuel cells. Nanoscale Advances, 1(1), 347-356.

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Enzymatic Bilirubin Oxidation Assay

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NaH₂PO₄, Na₂HPO₄, H₂SO₄, and KMnO₄ were obtained from Synth. Bilirubin oxidase from Myrothecium verrucaria (the cofactor structure was probed by electron paramagnetic resonance spectroscopy presented in Supplementary Fig. 5), isopropanol, and carbon nanoparticles were obtained from Sigma-Aldrich and used without further purification. All solutions were prepared with deionized water (18 MΩ cm at 25 °C).

Macedo L.J., Hassan A., Sedenho G.C, & Crespilho F.N. (2020). Assessing electron transfer reactions and catalysis in multicopper oxidases with operando X-ray absorption spectroscopy. Nature Communications, 11, 316.

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Quantification of Soluble P-selectin, VWF, and ADAMTS13

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Soluble P-selectin (sP-selectin) levels were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Abingdon, United Kingdom). VWF levels were assessed with in-house ELISA using commercially available polyclonal antibodies against VWF (DAKO, Glostrup, Denmark). Plasma activity of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) was measured using the FRETS-VWF73 assay (Peptanova, Sandhausen, Germany) which has been described in detail previously. 23 Plasma samples were pre-treated with bilirubin oxidase (2.5 U/mL, Sigma-Aldrich, Zwijndrecht, The Netherlands) for 30 minutes at 37°C to avoid interference of bilirubin with the FRETS-VWF73 assay. 24 Levels of VWF and ADAMTS13 in pooled normal plasma were set at 100%, and values obtained in test plasmas were expressed as a percentage of pooled normal plasma.

Bos S., van den Boom B., Kamphuisen P.W., Adelmeijer J., Blokzijl H., Schreuder T, & Lisman T. (2019). Haemostatic Profiles are Similar across All Aetiologies of Cirrhosis. Thrombosis and haemostasis, 119(2).

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