Biotin Biocytin

Glncose-6-phosphatase Biotin (biocytin) CO, Propionyl-CoA carboxylase... 

Prepare the labeling reagent stock solution. Biotin, biocytin, and desthiobiotin hydrazide derivatives are typically used at a labeling reagent antibody MR of 20-25. Biotin and desthiobiotin hydrazide stock solutions should be prepared at 10-20 mg/mL in DMSO or DMF while biocytin hydrazide can be dissolved in the reaction... 

Most biotin in foods is present as biocytin, incorporated into enzymes, which is released on proteolysis, then hydrolyzed by biotinidase in the pancreatic juice and intestinal mucosal secretions to yield free biotin. Biocytin is not absorbed to any significant extent. 

The studies of Reed and co-workers on the nature of protein-bound lipoic acid and its enzymatic release and reincorporation may be applicable to biotin-containing enzymes. It is pertinent to note that a conjugated form of biotin, biocytin, has been isolated from yeast autolyzate and identified as A -biotinyl-L-lysine (Wright et ah, 19r)2 Peck et al., 1952). Biotin is now known to be the prosthetic group of several carboxylases (see Ochoa and Kaziro, 1961, for a review of these enzymes). Although the nature of the moiety to which biotin is bound has not been established, it seems highly probable that it is the -amino group of a lysine residue. 

Many foodstuffs contain a metabolic intermediate of biotin, biocytin s-N-biotinyUysine), which is cleaved in the intestinal tract by the enzyme biotinidase. Only free biotin can be resorbed in the proximal small intestine, a process, which can be blocked by avidin, a glycoprotein with a molar mass of ca. 70,000. Avidin occurs in greater amounts in egg-white, and forms with biotin an extraordinarily stable molecular complex (dissociation constant at 25 °C K = 10 M), which can be cleaved neither by acids nor by peptidases. Only irradiation or longer exposure to heat leads to denaturation of avidin and thereby the release of biotin. This is another reason why a breakfast egg ought to be cooked for at least AVi minutes. In this way avidin is denatured and loses its harmful effect. Similarly stable complexes are formed by biotin with neutravidin (de-glycosyl-ated avidin), streptavidin and stravidin from certain Streptomyces and Saccharo-myces species respectively. 

The vitamin, biotin, has been shown to infiuence a variety of enzymatic reactions. Although a bound form of biotin, biocytin, has been obtained in crystalline form, no conclusive proof of the existence of a coenzyme form... 

Biotin was originally discovered as part of the complex called bios, which promoted the growth of yeast and, separately, as vitamin H, the protective or curative factor in egg white injury — the disease caused in man and experimental animals by feeding diets containing large amounts of uncooked egg white. The structures of biotin, biocytin and carboxybiocytin (the active metabolic intermediate) are shown in Figure 11.25. 

Amide bonds resulting from the condensation of amino groups of a-amino acids with carboxyhc groups of organic acids are found in many other compounds but these are not classified as peptides. Examples are some vitamins such as pantothenic acid, tetrahydrofolic acid (folic acid with two or more molecules of glutamic acid are already peptides) and the active form of biotin (biocytin). 

Biotin Biocytin Acetyl-CoA carboxylase pyruvate carboxylase Fatty-acid synthesis from glucose... 

Since the neurons targeted by whole-cell patch clamp recording were also filled with biocytin (0.2 %) we were also able to identify the shape and position of these P -GFP neurons within the cortical network. We used streptavidin conjugated to a red fluorescent probe (Alexa 568). Streptavidin reacts directly with biotin (biocytin) within the filled neuron, labeling only the filled neuron in the red fluorescence channel of the wide-field microscope. Typically we obtained recovery percentages of 80-100 % of neurons using the protocol below. 

Biotin Methylcobalamin Biotin-lysine complexes (biocytin)... 

Biotin can be synthesized by the human colon flora. The question to which extent this production contributes to covering the host-organism s requirements is, however, subject to discussion. In most foods of animal origin as well as in cereals, biotin prevails in the protein (= enzyme)-bound form as e-N-biotinyl-L-lysine (= biocytin). Brewer s yeast, liver, soya beans, and peanuts number among the biotin rich foods [1]. 

Biotin functions to transfer carbon dioxide in a small number of carboxylation reactions. A holocarboxylase synthetase acts on a lysine residue of the apoenzymes of acetyl-CoA carboxylase, pymvate carboxylase, propi-onyl-CoA carboxylase, or methylcrotonyl-CoA carboxylase to react with free biotin to form the biocytin residue of the holoenzyme. The reactive intermediate is 1-7V-carboxybiocytin, formed from bicarbonate in an ATP-dependent reaction. The carboxyl group is then transferred to the substrate for carboxylation (Figure 21—1). 

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). 

Biocytin should not be used in a carbodiimide reaction to modify proteins or other molecules, since it contains both a carboxylate and an amine group. A carbodiimide-mediated reaction, as suggested for D-biotin previously, would cause self-conjugation and polymerization of this reagent. 

The reagent is similar to another maleimide-containing biotinylation reagent, 3-(N-maleimi-dopropionyl) biocytin, a compound used to detect sulfhydryl-containing molecules on nitrocellulose blots after SDS-electrophoresis separation (Bayer et al., 1987). Biotin-BMCC should be useful in similar detection procedures. 

The reactivity and use of biocytin-hydrazide is similar to that described for biotin-hydrazide in Section 3, this chapter. The following protocol for labeling glycoproteins at oxidized carbohydrate (galactose) sites is from Bayer and Wilchek (1992). 

Bayer, E.A., Ben-Hur, H., and Wilchek, M. (1988) Biocytin hydrazide—a selective label for sialic acids, galactose, and other sugars in glycoconjugates using avidin-biotin technology. Anal. Biochem. 170, 271-281. 

Dethiobiotin, the sulfur-free analog of biotin, competitively inhibits the growth of O. danica the inhibition index is 10. Biocytin (e-N-biotinyl-L-lysine) stoichiometrically replaced biotin for O. danica. Because O. danica is phagotrophic (A2), it can probably ingest low-molecular forms of biotin, e.g., biocytin. Other forms of biotin were not studied. 

The way biotin participates in carbon dioxide fixation was established in the early 1960s. In 1961 Kaziro and Ochoa using propionyl CoA carboxylase provided evidence for 14C02 binding in an enzyme-biotin complex. With excess propionyl CoA the 14C label moved into a stable position in methyl malonyl CoA. In the same year Lynen found biotin itself could act as a C02 acceptor in a fixation reaction catalyzed by B-methylcrotonyl CoA carboxylase. The labile C02 adduct was stabilized by esterification with diazomethane and the dimethyl ester shown to be identical with the chemically synthesized molecule. X-ray analysis of the bis-p-bromanilide confirmed the carbon dioxide had been incorporated into the N opposite to the point of attachment of the side chain. Proteolytic digestion and the isolation of biocytin established the biotin was bound to the e-NH2 of lysine. 

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