Bilirubin Binding Research Articles

Combined fluorometric analysis of biliverdin and bilirubin by the recombinant protein HUG

Biliverdin is a secondary metabolite of heme catabolism. It is formed by the reaction catalyzed by heme oxygenase, which converts the heme group contained in proteins such as hemoglobin, myoglobin, cytochromes, and catalase into biliverdin, iron (II) and CO in equimolar amounts, consuming NADPH. Biliverdin is then reduced to bilirubin by biliverdin reductase. Biliverdin and bilirubin form a redox couple and are important for the redox homeostasis of cells. Heme oxygenase-1 is an inducible enzyme that is induced by hypoxic conditions, increased availability of heme or proinflammatory mechanisms such as LPS, UV radiation, etc. In addition, both heme oxygenase-1 and biliverdin reductase play roles other than catalysis by modulating specific metabolic pathways at the transcriptional level. There is a need for affordable assays to analyze these bile pigments in biological and clinical samples. Here we present a method for the combined determination of biliverdin and bilirubin that utilizes the specific binding of bilirubin to the fluorescent recombinant fusion protein HUG and the enzymatic conversion of biliverdin to bilirubin.•This method enables the combined measurement of bilirubin and biliverdin in the nM range.•The method does not require solvent extraction or protein precipitation of the samples.

Theory-Driven Tailoring of the Microenvironment of Quaternary Ammonium Binding Sites on Electrospun Nanofibers for Efficient Bilirubin Removal in Hemoperfusion.

Efficient adsorbents for excess bilirubin removal are extremely important for the treatment of hyperbilirubinemia. However, traditional adsorbents, such as activated carbons and ion-exchange resins, still suffer from dissatisfactory adsorption performance and poor blood compatibility. Herein, we adopted a rational design strategy guided by density functional theory (DFT) calculations to prepare blood-compatible quaternary ammonium group grafted electrospun polyacrylonitrile nanofiber adsorbents. The calculation analysis and adsorption experiments were used to investigate the structure-function relationship between group types and bilirubin adsorption, both indicating that quaternary ammonium groups with suitable configurations played a crucial role in bilirubin binding. The obtained nanofiber adsorbents showed the bilirubin removal efficiency above 90% even at a coexisting BSA concentration of 50 g L-1. The maximum adsorption capacities were 818.9 mg g-1 in free bilirubin solution and 163.7 mg g-1 in albumin bound bilirubin solution. The nanofiber adsorbents also showed considerable bilirubin removal in dynamic adsorption to reduce the bilirubin concentration to a normal level, which was better than commercial activated carbons. Our study demonstrates the high feasibility of a theory-driven design method for the development of grafted electrospun nanofibers, which have good potential as bilirubin adsorbents in hemoperfusion applications.

The fluorescence signal of albumin-bound bilirubin to monitor drug-induced bilirubin displacement from human serum albumin

Bilirubin (BR) binding to human serum albumin (HSA) may be affected by drugs, which may cause hyperbilirubinemia. A new method is proposed to investigate BR displacement from HSA using the characteristic fluorescence signal of albumin-bound BR at 533 nm upon 460 nm excitation. Any decrease in the fluorescence intensity at 533 nm upon drug addiction is direct evidence of BR displacement. Drug-induced BR displacement was tested using the well-known drug markers, warfarin (WFN) for Site I, ketoprofen (KTN) for Site II and hemin (HMN) for Site III of HSA. The maximum quenching (93%) and the highest Ka value (1.13 × 105 M1), obtained with HMN, suggested a common binding site of BR and HMN. Relatively lower BR displacement by WFN and KTN and lowered Ka values can be attributed to allosteric phenomena. These results showed the importance of the albumin-bound BR fluorescence signal in testing BR displacement from albumin.

Synergistic effect of chlorogenic acid and vitamin D3 (cholecalciferol) on in-vitro glycation may assist in prevention of Polycystic Ovarian Syndrome (PCOS) progression - A biophysical, biochemical and in-silico study

Advanced glycation end products (AGEs) are formed through non-enzymatic glycation, that have been linked to various diseases, including polycystic ovarian syndrome (PCOS) playing a critical role leading to secondary comorbidities such as diabetes-related problems, cardiovascular complications, infertility, etc. As a result, there has been a lot of research into AGE-inhibiting phytochemicals for the remediation and obstruct progression of glycation-related illnesses. The current study is based on in-vitro protein model, in which human serum albumin have been used to investigate the cumulative effect of chlorogenic acid (CGA) and cholecalciferol (vitamin D) on glycation and evaluate their inhibitory impact on AGEs production in the presence of methylglyoxal. Through the application of several biochemical and biophysical techniques, we were able to examine the synergistic impact of both the compounds on albumin structure and its biochemical properties during different stages of glycation. According to Nitro-blue tetrazolium assay results indicate that CGA and vitamin D inhibited fructosamine (early glycation product) production. Moreover, free thiol and lysine residues were significantly increased whereas protein carbonyl levels were significantly decreased. Additive effect of CGA and vitamin D were associated with reduced AGEs fluorescence and increased tryptophan and tyrosine fluorescence. Amadori-albumin after treatment showed some evidence of regaining its alpha-helicity as measured by far-UV CD spectrum. Furthermore, secondary structural alterations were confirmed by Fourier transform infrared spectroscopy (FTIR). ANS (1-anilinonaphthalene-8-sulfonic acid) fluorescence spectra also displayed less revelation of hydrophobic patches. Bilirubin binding capacity was also restored which showed functional recovery of HSA. The electrophoretic mobility was also restored which is portrayed by SDS-PAGE. Additionally, to predict the anti-aggregation potential of CGA and vitamin D, congo red assay and ThT fluorescence was performed which reveal low aggregate formation after treatment. These results corroborated with scanning electron microscopy and confocal microscopy. Docking and simulation results also reveal spontaneous binding of CGA and vitamin D on subdomain IIA of HSA favoring their binding thermodynamically. All the findings suggest that chlorogenic acid and cholecalciferol given in combination might help in prevention of PCOS progression and its related complications.

The Impact of Bilirubin on 7α- and 7β-Hydroxysteroid Dehydrogenases: Spectra and Docking Analysis

7α- and 7β-hydroxysteroid dehydrogenases (HSDHs) are enzymes that can catalyze the isomerization of hydroxyl groups at site seven of bile acids. In a previous study, we found that the activities of 7α- and 7β-HSDHs can be inhibited by bilirubin. In order to clarify the impact, the effects of bilirubin on enzymes were studied by kinetics, spectrum, and docking analysis. The relative activity of 7α-HSDH remained less than 40% under 1 mM bilirubin, and only 18% activity of 7β-HSDH kept in the same condition. Using taurochenodeoxycholic acid (TCDCA) as substrate, the Km of 7α-HSDH was up to 0.63 mM from 0.24 mM after binding with bilirubin and the Km of 7β-HSDH rose from 1.14 mM to 1.87 mM for the catalysis of tauroursodeoxycholic acid (TUDCA). The affinity of 7α- and 7β-HSDHs to substrates decreased with the effect of bilirubin. The binding of bilirubin with 7α- or 7β-HSDHs was analyzed by UV–vis, fluorescence, and circular dichroism (CD) spectroscopy. The results reflected that bilirubin caused a slight change in the secondary structure of 7α- or 7β-HSDHs, and the changes were correlated with the ratio of bilirubin to enzymes. Ten candidate molecular docking results were presented to reflect the binding of bilirubin with 7α- or 7β-HSDHs and to explore the inhibition mechanism. This research provides a more in-depth understanding of the effect of bilirubin on 7α- and 7β-HSDHs.

Superior Dialytic Removal of Bilirubin and Bile Acids by Free Fatty Acid Displacement and Its Synergy With Albumin-Based Dialysis.

One of the cardinal features of any liver replacement therapy is the ability to remove accumulated metabolites. However, an unsolved problem is the low dialyzability of lipophilic toxins. This study aimed to explore whether bilirubin and bile acids removal can be increased by free fatty acid (FFA) displacement and its synergy with albumin dialysis. First, we found that the protein binding of both bilirubin and bile acids decreased significantly with increasing FFA concentrations when co-incubated directly. Then, in vitro dialysis showed that fatty acid mixtures infusion prefilter effectively increased the fractional removals of bilirubin and bile acids, showing higher efficiency compared with albumin-based hemodialysis (HD); in vivo dialysis in liver failure rats showed that lipid emulsion administration resulted in higher reduction ratios and more total solute removals for bilirubin and bile acids after 4 h HD compared with control, which were also superior to albumin-based HD. Finally, the highest dialysis efficacy was always observed by their synergy whether in vitro or in vivo . These findings highlight that FFA displacement-based HD could efficiently improve the dialytic removal of bilirubin and bile acids, which might even be more efficient than albumin-based HD. Their synergy may represent a promising strategy to maximize the removal of circulating bilirubin and bile acids accumulated in liver failure.

Narrative review of bilirubin measurement and binding

: It is well accepted that infants with an imbalance in bilirubin production and excretion and/or poor plasma bilirubin binding have an increased risk of bilirubin-induced neurological dysfunction (BIND). The objective of this narrative review is to provide current literature on bilirubin measurement and bilirubin binding in premature and term infants with unconjugated hyperbilirubinemia. We provide evidence to support the notion that both total serum bilirubin (TSB) and bilirubin binding measurement contribute critical information about the risk of BIND in premature and term infants. We have reviewed and provided the evidence supporting the usefulness of free or unbound bilirubin as a better predictor of BIND compared to TSB in premature and term infants. We have also discussed promising newer developing technologies as point-of-care (POC) methods for bilirubin and bilirubin binding measurement. Overall, the improved knowledge of bilirubin binding over the last 20 years and recent development of promising newer point of care technologies for bilirubin and binding measurement augur well for development of pragmatic intervention trials to reduce mortality and morbidity associated with unconjugated hyperbilirubinemia in premature and term infants.

Gilbert's syndrome: hyperbilirubinemia enemy or friend

Gilbert's syndrome is a kind of benign inherited disease of bilirubin binding disorder, mainly due to the homozygous polymorphism A(TA)7TAA in the promoter of the gene for uridine diphosphate -glucuronosyltransferase 1A1 (UGT1A1), which is a TA insertion into the promoter, designated as UGT1A1*28, with UGT activity reduction to 30% of the normal value. Therefore, circulating fat-soluble unconjugated bilirubin cannot be converted into water-soluble conjugated bilirubin, leading to unconjugated hyperbilirubinemia. Bilirubin has a strong affinity for erythrocyte phospholipids, which interferes with membrane composition and dynamics, resulting in increased erythrocytes fragility, easy rupture, and gradual shortening of survival time. However, there are no obvious sign of hemolysis or abnormal iron metabolism, erythrocytes and bone marrow morphology. A small amount of chronic hemolysis stimulates extramedullary (normal bone marrow morphology) hematopoiesis, ensuing compensatory increase in circulating erythrocytes and hemoglobin. Hyperbilirubinemia may also weaken gastrointestinal motility, increase passive diffusion and absorption across the intestinal mucosal epithelium by 1.5 to 2 times, thereby aggravating or worsening hyperbilirubinemia mainly with unconjugated bilirubin circulation, which indicates that there is a causal relationship between the circulating bilirubin concentration and rapid erythrocytes turnover and hemolysis rate in patients with Gilbert's syndrome. Interestingly, bilirubin also has significant antioxidant and anti-mutagenic activities, and the potential health benefits of mild hyperbilirubinemia in Gilbert's syndrome include reduced prevalence of cardiovascular disease, type 2 diabetes mellitus (and related risk factors), certain cancers, and cardiovascular-related and all-cause mortality. Exogenous bilirubin and biliverdin supplements in intestinal epithelial cells can be absorbed and may increase circulating concentration of these antioxidant compounds. With this information, we hope to raise awareness of the potentially harmful and beneficial effects of benign hyperbilirubinemia, and explore and develop beneficial medical interventions.

Investigation of mutations (L41F, F17M, N57E, Y99F_Y134W) effects on the TolAIII-UnaG fluorescence protein's unconjugated bilirubin (UC-BR) binding ability and thermal stability properties

The UnaG protein is a ligand (unconjugated bilirubin) dependent fluorescence protein isolated from Unagi freshwater eel larvae and expressed as fusion in heterologous expression systems. Bilirubin is a tetrapyrrole molecule mainly produced from heme catabolism by the destruction of erythrocytes in the body. Bilirubin can cause kernicterus, a serious condition associated with permanent neurological damage in neonates with the passage of brain tissue. Different methods have been developed for plasma bilirubin analysis and quantification. The use of UnaG fluorescence protein triggered by bilirubin has become a new approach in bilirubin studies. In this study, we aimed to investigate the biophysical characterization of ligand interactions with the proteins obtained as a result of mutations (UnaGY99F_Y134W, UnaGN57E, UnaGL41F, and UnaGF17M) on the amino acid sequence of TolAIII-UnaG protein. After the purity levels of the expressed proteins have been analyzed by SDS-PAGE, secondary structures and thermal melting temperatures of the proteins have been examined by circular dichroism spectroscopy. Then determination of excitation and emission points by fluorescence spectroscopy, titration studies have been performed with bilirubin, and dissociation constant was calculated. According to the biophysical characterization studies, UnaGL41F has the highest affinity and stability among the mutants.

Withdrawal

One of the cardinal features of any liver replacement therapy is the ability to remove accumulated metabolites. The removal of water-soluble substances by dialysis procedures is well established. However, an unsolved problem is the low dialyzability of lipophilic toxins. This study aimed to explore whether bilirubin and bile acids removal can be increased by free fatty acid (FFA) displacement and its combination with albumin dialysis. First, we found that the protein binding of both bilirubin and bile acids decreased significantly with increasing FFA concentrations when co-incubated directly. Then, in vitro dialysis showed that fatty acid mixtures ([linoleic acid] / [oleic acid] = 2) infusion pre-filter effectively increased the fractional removal of bilirubin and bile acids from 1.43 ± 0.07% and 2.50 ± 0.12%, to 10.04 ± 0.29% and 6.12 ± 0.07%, respectively, which were significantly higher than albumin-based hemodialysis (HD); in vivo dialysis showed that lipid emulsion administration resulted in higher reduction ratios and more total solute removals for bilirubin and bile acids after 4 h HD compared with control, which was also superior to albumin-based HD. Finally, the highest dialysis efficacy was always observed by their combination whether in vitro or in vivo. These findings highlight that FFA displacement-based HD could efficiently improve the dialytic removal of bilirubin and bile acids, whilst its dialysis efficacy might even be superior to albumin-based HD. The concept of their combination may open up new avenues and possibilities in the field of dialysis to further enhance lipophilic toxins removal in liver failure.

Identification of Binding Regions of Bilirubin in the Ligand-Binding Pocket of the Peroxisome Proliferator-Activated Receptor-A (PPARalpha).

Recent work has shown that bilirubin has a hormonal function by binding to the peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor that drives the transcription of genes to control adiposity. Our previous in silico work predicted three potential amino acids that bilirubin may interact with by hydrogen bonding in the PPARα ligand-binding domain (LBD), which could be responsible for the ligand-induced function. To further reveal the amino acids that bilirubin interacts with in the PPARα LBD, we harnessed bilirubin’s known fluorescent properties when bound to proteins such as albumin. Our work here revealed that bilirubin interacts with threonine 283 (T283) and alanine 333 (A333) for ligand binding. Mutational analysis of T283 and A333 showed significantly reduced bilirubin binding, reductions of 11.4% and 17.0%, respectively. Fenofibrate competitive binding studies for the PPARα LBD showed that bilirubin and fenofibrate possibly interact with different amino acid residues. Furthermore, bilirubin showed no interaction with PPARγ. This is the first study to reveal the amino acids responsible for bilirubin binding in the ligand-binding pocket of PPARα. Our work offers new insight into the mechanistic actions of a well-known molecule, bilirubin, and new fronts into its mechanisms.

Influence of mutations caused by radiation exposure on the bilirubin binding sites of human serum albumin

In this article we analyze the bilirubin binding sites of human serum albumin from the point of view of the secondary structure instability, as well as the effect of amino acid substitutions caused by radiation exposure on the ability of albumin to bind bilirubin-IX-alpha. Based on calculations of binding energy and inhibition constants of bilirubin-albumin complexes before and after the amino acid substitutions, it was found that amino acid substitutions have different effects on the ability of human serum albumin to bind bilirubin. Amino acid substitutions Asp269-Gly269 (Nagasaki-1), Glu354-Lys354 (Hiroshima-1), Asp375-Asn375 (Nagasaki-2) reduce the binding free energy of bilirubin with human serum albumin, and the amino acid substitutions His3-Gln3 (Nagasaki-3) and Glu382-Lys382 (Hiroshima-2) increase it during molecular docking with the corresponding areas of the protein surface. The inhibition constants are significantly higher than with known binding sites. In general, mutations caused by radiation exposure cannot effect on bilirubin binding sites of human serum albumin, since the amino acid residues that are replaced do not interact with the amino acid residues from the binding sites (Leu115, Arg117, Phe134, Tyr138, Ile142, Phe149, Phe157, Tyr161, Arg186, Lys190, Lys240, Arg222). All amino acid residues from known binding sites are located in stable elements of the secondary structure of human serum albumin.The data obtained are important for understanding the impact of radiation exposure on the development of bilirubin encephalopathy in the population of the Chernobyl region and Japan.

Bilirubin, Interleukin 6 (IL 6) and Lipopolysaccharide �Binding Protein (LBP) � Biomarkers of Sepsis in Appendicular Peritonitis

Peritonitis remains, despite the diagnostic and therapeutic developments, a serious condition, the main cause of septic shock with lethal potential in abdominal surgery Identifying and characterizing the dynamics of early biomarkers of the inflammatory response in abdominal sepsis is important to better follow-up evolution and postoperative infectious complications. The paper aims to study the dynamics of serum concentration of biomarkers LBP, IL6 and bilirubin in patients with acute peritonitis of appendicular cause, in the pre and postoperative stage and the correlation with the clinical evolution of inflammatory syndrome. A prospective 6-month study was performed, including patients for appendicular peritonitis. LBP, IL6, bilirubin, leukocytes were sampled preoperatively, then postoperatively at 72 h, and repeated at 72 h until values returned to normal. Clinical evolution and leukocytes were correlated with biochemical parameters. The mean preoperative values for the studied biochemical parameters were for Il6 of 14.81 +/- 4,047 pg / mL, for LBP of 33,826 +/- 5.5.02 microgram / mL and for bilirubin of 1.77 +/- 0.55mg / mL. The postoperative values decreased in all cases, but remained above normal limits in cases with septic complications. LBP, Il 6 and bilirubin are biomarkers useful in the pre- and postoperative evaluation of sepsis in appendicular peritonitis, correlating more accurately with the evolution of the inflammatory response than leukocytosis. Bilirubin is useful in monitoring intraperitoneal infection, but is not influenced by the existence of extraperitoneal infection. The dynamics of LBP is the most accurate description of bacterial infectious factor exposure.

Bilirubin binding affects the structure and function of alpha-2-macroglobulin

ABSTRACT Bilirubin is an endogenous antioxidant that is a metabolite of the heme in red blood cells (RBC). In blood, bilirubin is associated with albumin to form a water-soluble complex, known as unconjugated bilirubin. Alpha-2-macroglobulin (α2M) is a proteinase inhibitor found in the plasma of vertebrates. In the present study, we have investigated the interaction of photo-illuminated bilirubin with serum α2M using various biophysical and thermodynamic techniques. The binding of bilirubin to α2M leads to various functional and structural changes in α2M protein. The result of ultraviolet (UV) and fluorescence spectroscopy suggests that binding of bilirubin to α2M induces a conformational change in the secondary structure of protein which was corroborated by circular dichroism (CD) and Fourier-transform infrared spectroscopy (FT-IR). This binding leads to the conversion of β-sheet into α-helical conformation and subsequently loss in protein activity. The thermodynamic parameters of bilirubin-α2M binding indicate that the binding is exothermic, and the reaction spontaneous. Our studies show that binding of bilirubin with α2M in the presence of light induces structural and functional modifications in the protein. Bilirubin possesses multiple biological activities, including immunomodulatory property which has not been extensively explored and which may be of interest for further study.

Competitive Binding of Bilirubin and Fatty Acid on Serum Albumin Affects Wear of UHMWPE.

Total Joint Replacement (TJR) devices undergo standardized wear testing in mechanical simulators while submerged in a proteinaceous testing solution to mimic the environmental conditions of artificial joints in the human body. Typically, bovine calf serum is used to provide the required protein content. However, due to lot-to-lot variability, an undesirable variance in testing outcome is observed. Based on an earlier finding that yellowish-orange serum color saturation is associated with wear rate, we examined potential sources of this variability, by running a comparative wear test with bilirubin; hemin; and a fatty acid, oleic acid, in the lubricant. All these compounds readily bind to albumin, the most abundant protein in bovine serum. Ultrahigh molecular weight polyethylene (UHMWPE) pins were articulated against CoCrMo discs in a pin-on-disc tribometer, and the UHMWPE wear rates were compared between lubricants. We found that the addition of bilirubin increased wear by 121%, while hemin had a much weaker, insignificant effect. When added at the same molar ratio as bilirubin, the fatty acid tended to reduce wear. Additionally, there was a significant interaction with respect to bilirubin and hemin in that UHMWPE wear rate decreased with increasing fatty acid concentration. We believe the conformational change in albumin by binding bilirubin makes it more likely to form molecular bridges between UHMWPE and the metal counterface, thus increasing adhesive wear. However, fatty acids compete for binding sites on albumin, and can prevent this conformational change. Hence, the protein is stabilized, and the chance for albumin to form bridges is lowered. Ultimately, UHMWPE wear rate is driven by the competitive binding of bilirubin and fatty acid to albumin.

Bilirubin: A Fat Busting Metabolic Hormone?

Obesity is an epidemic in the United States, which has led to an increased prevalence of insulin‐resistant type II diabetes and heart disease, heightening patient morbidity and mortality. Current treatments of obesity are not sufficient, often requiring combinations for beneficial results, and most have the negative side effect of increasing blood pressure. It has been recently shown that bilirubin, a red blood cell metabolite that at very high levels is commonly associated with liver pathology, prevents cardiovascular disease, diabetes, and obesity at modest levels. We have found that bilirubin induces transcriptional activity via the fat‐burning nuclear receptor, peroxisome proliferator‐activated receptor α (PPARα). RNA‐sequencing in HepG2 hepatocytes showed that bilirubin‐induced transcriptome responses that are predominantly PPARα‐dependent (~95%). From our investigations, we were able to demonstrate a preference for binding of bilirubin to PPARα compared to the other PPAR isoforms (gamma and delta). Utilizing a completely new fluorescent technique established by our lab, we found that bilirubin directly binds to the ligand‐binding domain (LBD) of PPARα. As measured by Pamgene Pamstation Nuclear Hormone Receptor (NHR) chip analysis, we also found that bilirubin functions as a ligand for PPARα and induces the recruitment and dissociation of coregulators that regulate gene function. Subsequent chromatin immunoprecipitation assays showed that bilirubin drives PPARα to the promoters of genes involved in mitochondrial respiration, including the uncoupling protein 1 (Ucp1) and carnitine palmitoyltransferase I A (Cpt1a). We affirmed that the effect of bilirubin on mitochondrial function, analyzed by oxygen consumption rate (OCR) using Seahorse analysis, was higher in the cells that expressed PPARα, but not PPARg2. These findings aid in defining the role of bilirubin in altering the metabolic function and, more specifically, through the regulation of mitochondrial‐mediated mechanisms. The investigation of the bilirubin‐PPARα axis reveals a newfound position of bilirubin as a metabolic hormone mediating protection from lipid accumulation and subsequent metabolic dysfunction.

Crigler-Najjar Syndrome Type 1: Pathophysiology, Natural History, and Therapeutic Frontier.

We describe the pathophysiology, treatment, and outcome of Crigler-Najjar type 1 syndrome (CN1) in 28 UGT1A1 c.222C>A homozygotes followed for 520 aggregate patient-years. Unbound ("free") bilirubin (Bf ) was measured in patient sera to characterize the binding of unconjugated bilirubin (BT ) to albumin (A) and validate their molar concentration ratio (BT /A) as an index of neurological risk. Two custom phototherapy systems were constructed from affordable materials to provide high irradiance in the outpatient setting; light dose was titrated to keep BT /A at least 30% below intravascular BT binding capacity (i.e., BT /A=1.0). Categorical clinical outcomes were ascertained by chart review, and a measure (Lf ) was used to quantify liver fibrosis. Unbound bilirubin had a nonlinear relationship to BT (R2 =0.71) and BT /A (R2 =0.76), and Bf as a percentage of BT correlated inversely to the bilirubin-albumin equilibrium association binding constant (R2 =0.69), which varied 10-fold among individuals. In newborns with CN1, unconjugated bilirubin increased 4.3±1.1mg/dL per day. Four (14%) neonates developed kernicterus between days 14 and 45 postnatal days of life; peak BT ≥30mg/dL and BT /A≥1.0 mol:mol were equally predictive of perinatal brain injury (sensitivity 100%, specificity 93.3%, positive predictive value 88.0%), and starting phototherapy after age 13days increased this risk 3.5-fold. Consistent phototherapy with 33-103µW/cm2 •nm for 9.2±1.1hours/day kept BT and BT /A within safe limits throughout childhood, but BT increased 0.46mg/dL per year to reach dangerous concentrations by 18years of age. Liver transplantation (n=17) normalized BT and eliminated phototherapy dependence. Liver explants showed fibrosis ranging from mild to severe. Seven decades after its discovery, CN1 remains a morbid and potentially fatal disorder.

A Dual-Ligand-Modulable Functional Protein Based on Ligand-inducible Green Fluorescent Protein and Calmodulin

UnaG, a superfamily of fatty-acid-binding protein (FABP), is a green-emitting fluorescent protein that utilizes unconjugated bilirubin (BR) as its fluorophore. Physiologically, BR is a notable antioxidant to scavenge reactive oxygen species in biological membranes, although excessive accumulation of BR causes several clinical symptoms such as jaundice. The optimal regulation of the BR concentration in biological tissues would result in clinical therapies for aging as well as reduce risks of clinical symptoms. However, UnaG hardly releases BR because of its high affinity for BR (Kd= 98 pM). In this study, we generated Ca2+-inducible BR releasing protein by genetically insertion of calmodulin (CaM) into UnaG. We speculated that the insertion of calmodulin (CaM) near the BR binding pocket of UnaG would result in the production of a novel fluorescent protein that releases the fluorophore in a Ca2+-dependent manner. In addition, as the site is close to Ser80/Asp81 residues, which anchor the propionate carboxylate from the exo-vinyl dipyrrinone of BR, the Ca2+-dependent conformational change on the inserted CaM was expected to affect the BR affinity. The resultant UnaG/CaM hybrid protein, named BReleaCa (BR+releaser+Ca2+), is a dual-ligand modulable fluorescent protein; binding of the fluorogenic ligand BR is negatively regulated by the other ligand, Ca2+ion. The affinity for BR differed by three orders of magnitude between the Ca2+-free state (Kd= 9.70 nM) and Ca2+-saturated state (Kd= 9.65 μM). The chimeric protein can release nano- to micro-molar levels of BR with Ca2+control. Such a protein hybridization technique will be applicable as a method of changing the ligand binding properties of different ligand-inducible protein.

Influence of pH and ionic strengths on bilirubin binding to sheep serum albumin

Despite great degree of similarity in the structure and conformation of different serum albumins, they often show marked difference in the ligand binding properties. These differences and similarities would be useful in understanding the molecular basis of ligand binding to serum albumins. In the present communication binding of bilirubin with sheep and bovine serum albumin have been studied by visible difference spectroscopy as a function of pH and ionic strength. The results suggest that the structure of bilirubin binding site in sheep serum albumin is similar to that in bovine serum albumin and that complex between sheep serum albumin and bilirubin is stabilized by electrostatic interactions. Keywords: Sheep serum albumin, Bovine serum albumin, Bilirubin binding, ionic strength, pH.

Trp\u2013His covalent adduct in bilirubin oxidase is crucial for effective bilirubin binding but has a minor role in electron transfer

Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396–His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp–His adduct mainly for binding and to a smaller extent for electron transfer.