Albumin bilirubin binding

J. Jacobsen and R, Broderson, Albumin-bilirubin binding mechanism Kinetic and spectroscopic studies of binding of bilirubin and xanthobilirubic acid to human serum albumin, J. Biol. Chem., 258 6319 (1983). 

Mir MM, Fazili KM, Abul Qasim M. Chemical modification of buried lysine residues of bovine serum albumin and its influence on protein conformation and bilirubin binding. Biochim. Biophys. Acta 1992 1119 261-267. 

FlC. 9. Nonparallel regeneration of the functions for bilirubin binding and fatty acid binding in bovine plasma albumin. The oxidative regeneration of reduced albumin was carried out at protein concentrations of 1 ft,M at 25°C in 0.10 M Tris-chloride buffer, pH 8.0, containing 1 mM EDTA and 1 mM reduced and 0.10 mM oxidized glutathione (Johanson et al, 1977, 1981). 

The most frequent protein in the plasma, at around 45 g is albumin. Due to its high concentration, it plays a crucial role in maintaining the blood s colloid osmotic pressure and represents an important amino acid reserve for the body. Albumin has binding sites for apolar substances and therefore functions as a transport protein for long-chain fatty acids, bilirubin, drugs, and some steroid hormones and vitamins. In addition, serum albumin binds Ca "" and Mg "" ions. It is the only important plasma protein that is not glycosylated. 

Albumin is a major transport facilitator of hydrophobic compounds which would otherwise disrupt cellular membranes. These compounds include free fatty acids and bilirubin as well as hormones such as cortisol, aldosterone, and thyroxine when these materials have exceeded the capacity of proteins normally associated with them. Albumin also binds ions, including toxic heavy metals and metals such as copper and zinc which are essential for normal physiological functioning but may be toxic in quantities in excess of their binding capacity for their carrier proteins. Binding of protons is the basis for the buffering capacity of albumin. 

D. compete for bilirubin binding sites on plasma albumin. 

Since binding of bilirubin to albumin is usually reversible, a small amount of free bilirubin is present in plasma in equilibrium with albumin-bound bilirubin. It is probably this free bilirubin that is taken up at a rate determined by its plasma concentration. As this free bilirubin concentration decreases, more bilirubin is released from albumin and becomes available for uptake. Alternatively, the albumin-bilirubin complex may bind to specific hepa-tocyte plasma membrane receptors, and thereby bilirubin is released to enter the cell. Both models are consistent... 

Plasma albumin has binding sites for various natural metabolic substances (e.g., glucose, fatty acids, bilirubin) as well as xenobiotics and drugs (e.g., digitoxin, Warfarin). While chromatographic and solubility differences from pure human serum albumin occur in some glycosylated albumins, there does... 

H45. Hirano, K., Watanabe, Y., Adachi, T., and Sugiura, M., Carrier proteins in human fetal serum Bilirubin-binding abilities of albumin, alpha-fetoprotein and ligandin. Chem. Pharm. Bull. 32, 708-715 (1984). 

Serum albumin is the most abundant plasma protein (3.5-5.5%) and is responsible for the binding and transport of various metabolically and pharmacologically active molecules, e.g., bilirubin, uric acid, vitamin C, acetylcholine, cholinesterase, adenosine, aureomycin, barbiturate, Chloromycetin, digitonin, fatty acids, atabrine, neosilversalvarsan, penicillin, salicylate,/ -aminosalicylate, sulfonamide, streptomycin, acid dyes, histamine, triiodothyronine, and thyroxine (Bennhold, 1962 Putnam, 1975). Moreover, albumin tightly binds various metal ions, e.g., Zn (Giroux,... 

Jacobsen, C. Chemical Modification of the High-Affinity Bilirubin Binding Site of Human Serum Albumin. Eur. J. Biochem. 27, 513-519 (1972). 

Some Conjugated Bilirubin Can Bind Covalently to Albumin... 

When levels of conjugated bilirubin remain high in plasma, a fraction can bind covalently to albumin (delta bilirubin). Because it is bound covalently to albumin, this fraction has a longer half-life in plasma than does conventional conjugated bilirubin. Thus, it remains elevated during the recovery phase of obstructive jaundice after the remainder of the conjugated bilirubin has declined to normal levels this explains why some patients continue to appear jaundiced after conjugated bilirubin levels have returned to normal. 

Another important function of albumin is its ability to bind various ligands. These include free fatty acids (FFA), calcium, certain steroid hormones, bilirubin, and some of the plasma tryptophan. In addition, albumin appears to play an important role in transport of copper in the human body (see below). A vatiety of drugs, including sulfonamides, penicilhn G, dicumarol, and aspirin, are bound to albumin this finding has important pharmacologic implications. 

Albumin, which is not glycosylated, is the major protein and is the principal determinant of intravascular osmotic pressure it also binds many Hgands, such as drugs and bilirubin. 

Fig. 7.10 Matching of RR from 7 a (thick lines) with the extended form of bilirubin IX (dashed lines). The extended conformation of bilirubin is one of several possibilities and it is uncertain which of these it adopts when binding to albumin. The iron atom of 7a is placed at the central methylene of bilirubin. A number of the peripheral groups have been omitted from the structures for clarity. Reproduced with permission from R. B. Lauffer, A. C. Vincent, S. Padmanabhan and T. I. Meade, J. Amer. Chem. Soc. 109, 2216 (1987). (1987) American Chemical Society.

For further degradation, bilirubin is transported to the liver via the blood. As bilirubin is poorly soluble, it is bound to albumin for transport. Some drugs that also bind to albumin can lead to an increase in free bilirubin. 

Bilirubin effects depend on the method used for analysis. Interferences in direct serum protein methods are observed at bilirubin levels greater than 5 mg/100 ml (K7). A sample containing 20 mg of bilirubin per 100 ml increased the apparent total protein by 0.2 g/100 ml. Concentrations of bilirubin as high as 20 mg/ml do not effect albumin assays using bromocresol green binding (D12), but have a marked effect on these assays when [2-(p-hydroxyphenylazo)-benzoic acid] (HABA) dye is used (A7b). 

Bilirubin derives largely from senescent erythrocyte hemoglobin [for reviews, see references (F9, G7, L6, S3, Wll)]. When transported in plasma the pigment is bound to albumin (B9, 04). Dissociation from albumin precedes rapid uptake by liver tissues (B4, Bll, B29). This uptake and the transfer of bilirubin to its sites of metabolism may be mediated by cytoplasmic binding proteins (G8, Gll, L8, M9). 

Most frequently, binding protein is added to the incubation mixtures as either serum or purified serum albumin. With human serum albumin, at equilibrium, the acceptor substrate will largely be protein-bound, when the bilirubin albumin molecular ratio is smaller than one (the dissociation constant of the first binding site of purified human serum albumin is approximately 7 X 10 M with 2 X 10 M for two additional binding sites) (J2). The first binding site of albumin, measured with rat serum, has a dissociation constant of about 5 X 10" M (M8). The unbound fraction will normally not exceed the very low solubility of the pigment. Addition of albumin to an alkaline solution of bilirubin, or its addition immediately after neutralization, prevents colloid formation, if the bilirubin albumin molecular ratio is smaller than one (B25). However, colloidal bilirubin, once formed, cannot be redissolved by the addition of albumin (B26). 

At a bilirubin albumin molecular ratio below one the added binding protein will thus act as a kind of buffer, keeping the concentration of unbound substrate sufficiently low to inhibit colloid formation (B25) or precipitation onto bound bilirubin (B26), and will prevent aspecific binding to cell particulates. The binding protein can also be thought of as a reservoir providing a continuous stream of molecularly dispersed sub-... 

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