Bilirubin is a class of linear tetrapyrrole derivatives, and is one of the metabolites of iron porphyrin compounds in the human body. When the bilirubin production is excessive, or the bilirubin uptake, combination and excretion of liver cells is disorder, the blood bilirubin concentration is increased and resulting in hyperbilirubinemia. Therefore, the clinical determination of bilirubin is important for detection of hepatobiliary disease, hemolysis and other related diseases. Bilirubin oxidase is a commonly used tool enzyme that catalyzes the oxidation of bilirubin to produce biliverdin, which is used in clinical diagnostics to determine serum bilirubin and total bilirubin levels. It is more sensitive to monitor liver diseases than non-specific chemical methods, and serum bilirubin studies provide more detailed information on the pathology of jaundice.
Structure
Holoenzyme of bilirubin oxidase consists of proteins, divalent copper ions and carbohydrates. The protein consists of 17 amino acids with no cysteine, low levels of lysine and methionine, and high levels of aspartic acid, glutamic acid and alanine. Precursor of bilirubin oxidase is composed of 572 amino acid residues, including 534 amino acids and 38 amino acid leader. The 19 hydrophobic amino acids at the C-terminal in the leader are similar in character to known signal peptides and may also play a role as signal peptides. Spectral analysis demonstrated that divalent copper ions can bind to Cys, Met and His residues. The gene sequence of bilirubin oxidase is divided into 6 exons by 5 introns. All intron borders are marked with double-stranded nucleotide with GT and AG. Four transcriptional start sites were found by primer extension analysis and the major transcriptional start site was 93 bases upstream of the ATG start codon.
Properties
The isoelectric point of bilirubin oxidase is around 4.1. The enzyme's Michaelis constant is slightly different due to different strains. Usually, the nature of bilirubin oxidase is stable at 37°C and the pH between 5.0 to 9.7, and most stable at 5°C and pH 9.2 to 9.7. The optimum reaction temperature of bilirubin oxidase is about 40°C, the optimum reaction pH is about 8, and the enzyme is not easily to dissolve in the acidic range. Reported molecular weight of bilirubin oxidase is 52kDa and 68kDa, the difference between the two molecular weight may be due to the existence of natural bilirubin oxidase oligosaccharide chain. The bilirubin oxidase molecular weight in Penicillium janthinellum is 2000kDa, which is due to the metal ions that crosslink the enzyme protein. Bilirubin oxidase will lost its activity when utilize ascorbate and potassium cyanide to remove divalent copper ions. Apo-derived bilirubin oxidase regains activity to varying degrees if added with cobalt, cadmium and iron ions, respectively. It indicates that catalytic performance of bilirubin oxidase are closely related with metal ions. Mercury ions can significantly promote the increase of enzyme activity, because mercury ion and substrate combine to form more suitable enzyme-catalyzed intermediates. Potassium cyanide, sodium azide and thiourea can inhibit the reaction. Bilirubin oxidase specifically reacts with tetrapyrrole-containing compounds, and it can also oxidize some laccase substrates.
Catalytic Mechanism
Bilirubin oxidase is a copper-containing polyphenol oxidase that contains three types of copper ions. A type I copper ion mediates the transfer of electrons from the substrate to other copper sites. Oxygen molecules are bound to a trinuclear center with one type II copper ion and two type III copper ions, which are reduced to two water molecules. So its catalytic process is an oxidation reaction that utilizes molecular oxygen as an electron acceptor. Detected by electron absorption spectroscopy and thin-layer chromatography, the specific reaction is: bilirubin + 1/2 O2 → biliverdin + H2O. The process is that bilirubin oxidase catalyzes bilirubin to generate bilirubin, the bilirubin is further oxidized to form a kind of colorless material. Studies have shown that the catalytic mechanism of bilirubin oxidase is a secondary reaction to the copper ion bound to the protein. The donor structure is of the CuSS*N2 (S = Cys, S* = Met, N = His) type, which is the same as the ceruloplasmin family. In bilirubin oxidase reaction against jaundice blood, the bilirubin concentration in the blood was measured by HPLC, and bilirubin oxidase was found to oxidize bound bilirubin as well as can oxidize non-conjugated bilirubin. Bilirubin oxidases can selectively oxidize bound bilirubin in the presence of reagents such as sodium chloride.
Clinical Application
A variety of different liver diseases can cause metabolic disorder of bilirubin. Increasing bilirubin will lead to the appearance of jaundice. Therefore, the determination of bilirubin is an important indicator of liver function. Bilirubin oxidase can catalyze the bilirubin to produce bilirubin. The change of absorbance value at 440 nm and 450 nm was detected to determine the content of bilirubin in serum. Therefore, bilirubin oxidase can be used for clinical diagnosis of jaundice disease, and is expected to be used for neonatal jaundice and hyperbilirubinemia treatment.
