Biotin occurence

The chemical structure of biotin (hexahydro-2-oxo-IH-thieno [3,4-d] imidazol-4-valeric acid) is shown in Fig. 1. Of the eight stereoisomers, only d-(+)-biotin occurs naturally and is biologically active. 

Biotin occurs as a practically white, crystalline powder. It is stable to air and heat. One gram dissolves in about 5000 mL of water at 25° and in about 1300 mL of alcohol it is more soluble in hot water and in dilute alkali, and it is insoluble in other common organic solvents. 

The structural formula (Figure 9-23) contains three asymmetric carbon atoms, and eight different stereoisomers are possible. Only the dextrorotatory D-biotin occurs in nature and has biological activity. Biotin occurs in some products in free form (vegetables, milk, and fruits) and in other products is bound to protein (organ meats, seeds, and yeast). Good sources of the vitamin are meat, liver, kidney, milk, egg yolk, yeast, vegetables, and mushrooms (Table 9-27). 

Biotin is a water-soluble vitamin. It is a cofactor for four ATP-dependent carboxylases acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and p-methylcrotonyl-CoA carboxylase. Biotin occurs covalently bound to the enzymes via the terminal amino group of a lysine residue. With the normal and continual turnover of these enzymes in the body, the biotin is released, but then utilized again as a cofactor when the enzymes are re-synthesized. The structure of biotin is shown in Figure 9.32,... 

The enrichment of foods with biotin occurs only rarely. 

The proposed methods are based on spectrometric, chromatographic, and proteinbinding techniques. As biotin occurs in food both in free form and covalently bound to proteins, in many methods it is necessary to break these bonds by acid or enzymatic hydrolysis. The former is more frequently used than the latter, since acid treatment converts o-biocytin into o-biotin and allows a total determination of biotin. Enzymatic treatment with papain leaves o-biocytin unchanged and only D-biotin is determined. Despite this, some methods propose enzymatic hydrolysis rather than acid hydrolysis because it does not induce any degradation of biotin. 

Lipoic acid exists as a mixture of two structures a closed-ring disulfide form and an open-chain reduced form (Figure 18.33). Oxidation-reduction cycles interconvert these two species. As is the case for biotin, lipoic acid does not often occur free in nature, but rather is covalently attached in amide linkage with lysine residues on enzymes. The enzyme that catalyzes the formation of the lipoamide nk.2Lg c requires ATP and produces lipoamide-enzyme conjugates, AMP, and pyrophosphate as products of the reaction. 

In terms of amino acids bacterial protein is similar to fish protein. The yeast s protein is almost identical to soya protein fungal protein is lower than yeast protein. In addition, SCP is deficient in amino acids with a sulphur bridge, such as cystine, cysteine and methionine. SCP as a food may require supplements of cysteine and methionine whereas they have high levels of lysine vitamins and other amino acids. The vitamins of microorganisms are primarily of the B type. Vitamin B12 occurs mostly hi bacteria, whereas algae are usually rich in vitamin A. The most common vitamins in SCP are thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, choline, folic acid, inositol, biotin, B12 and P-aminobenzoic acid. Table 14.4 shows the essential amino acid analysis of SCP compared with several sources of protein. 

Alimentary biotin deficiency is rare. It may, however, occur in patients on long-term parenteral nutrition lacking biotin or in persons who frequently consume raw egg white. Raw egg white contains a biotin-binding glycoprotein, called avidin, which renders biotin biologically unavailable. Pharmacological doses of the vitamin (1-10 mg/d) are then used to treat deficiency symptoms. There are no reports of toxicity for daily oral doses up to 200 mg and daily intravenous doses of up to 20 mg [2]. 

The LANCE cAMP assay is a competitive assay in which cAMP produced by the cells competes with fluorescent-labeled acceptor cAMP for a cryptate tagged donor antibody. The principal of the assay is shown in Fig. 6. On the left strepta-vidin conjugated Europium binds to biotinylated cAMP. An antibody labeled with the fluorescent dye Alexa binds to the cAMP, bringing the donor and acceptor into close proximity, and energy transfer occurs. When the cell releases cAMP, it competes with the biotin-labeled cAMP for the antibody, and a signal decrease is observed. In the TR-FRET assay the antibody is directly labeled with either Eu or Tb. In this format an increase in cAMP also causes a decrease in signal. 

Biocytin is e-N-biotinyl-L-lysine, a derivative of D-biotin containing a lysine group coupled at its e-amino side chain to the valeric acid carboxylate. It is a naturally occurring complex of biotin that is typically found in serum and urine, and probably represents breakdown products of recycling biotinylated proteins. The enzyme biotinidase specifically cleaves the lysine residue and releases the biotin component from biocytin (Ebrahim and Dakshinamurti, 1986, 1987). 

A water-soluble analog of NHS-biotin containing a negatively charged sulfonate group on its NHS ring structure also is available. Sulfo-NHS-biotin may be added directly to aqueous reactions without the need for organic solvent dissolution. A concentrated stock solution may be prepared in water to facilitate the addition of a small quantity to a reaction, but hydrolysis of the NHS ester will occur at a rapid rate, so the solution must be used immediately. 

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

On rare occasions an organic aciduria occurs not because of an enzyme deficiency but from a failure to transport or activate a water-soluble vitamin that serves as a cofactor for the reaction in question. Thus, congenital deficiencies in the metabolism of vitamin B12 commonly give rise to methylmalonic aciduria (Fig. 40-1, Table 40-2). Similarly, deficiencies of biotin metabolism can cause a severe organic aciduria (Table 40-2). It is very important to be aware of the defects of vitamin metabolism because the administration of large doses of these cofactors may completely prevent brain damage. 

Spontaneous biotin deficiency is unlikely to occur as a result of simple dietary restriction, since biotin has high potency, is widely distributed in foods, and is also synthesized in the intestine. The human requirement for biotin is not known, but subjects on a low-biotin diet recovered in 3-5 days, when 75-300 jxg biotin was administered daily (S23, S24). 

Avidin-biotin complex (ABC) is based on the high affinity that streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) have for biotin. Biotin is a naturally occurring vitamin. One mole avidin will bind four moles biotin. ABC method affords a several-fold higher antigen detectability than those achieved in the standard indirect method. 

Some enzymes are nonfunctional until posttranslationally modified. Examples of these enzymes include the acyl- and carboxyltransferases. While lipoate and phosphopantetheine are necessary for acyl transfer chemistry, tethered biotin is used in carboxyl transfer chemistry. Biotin and lipoate tethering occur under a similar mechanism the natural small molecule is activated with ATP to form biotinyl-AMP or lipoyl-AMP (Scheme 20). A lysine from the target protein then attacks the activated acid and transfers the group to the protein. The phosphopantetheine moiety is transferred using its own enzyme, the phosphopantetheinyltrans-ferase (PPTase). The PPTase uses a nucleophilic hydroxy-containing amino acid, serine, to attach the phosphopantetheinyl (Ppant) arm found in coenzyme A to convert the apo (inactive) carrier protein to its holo (active) form. The reaction is Mg -dependent. 

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