Biotin lysine derivative

Said, H.M., Thuy, L.P., Sweetman, L., and Schatzman, B., 1993. Transport of the biotin dietary derivative biocytin (V-biotinyl-L-lysine) in rat small intestine. Gastroenterology. 104 75-80. 

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). 

The biochemical MS assay performance was studied for various biotin derivatives, such as biotin [m/z 245), N-biotinyl-6-aminocaproic acid hydrazide (m/z 372), biotin-hydrazide (m/z 259), N-biotinyl-L-lysine (m/z 373) and biotin-N-succinimi-dylester m/z 342). These five different bioactive compounds were consecutively injected into the biochemical MS assay. Figure 5.12 shows triplicate injections in the biochemical MS-based system of the different active compounds. Each compound binds to streptavidin, hence the MS responses of peaks of the reporter ligand (fluorescein-biotin, m/z 390) are similar. The use of SIM allows specific components to be selected and monitored, e.g. protonated molecule of the biotin derivatives. In this case, no peaks were observed for biotin-N-succinimidylester (m/z 342), because under the applied conditions fragmentation occurred to m/z 245. In combination with full-scan MS measurements, the molecular mass of active compounds can be determined simultaneously to the biochemical measurement. 

The enzymatic role of the thieno[3,4-d]imidazole derivative biotin as the coenzyme for the transfer of carbon dioxide in carboxylation reactions is well established. Strong acidic hydrolysis of biocytin, a naturally occurring complex of biotin, yields biotin and L-lysine. 

The -amino groups of lysyl residues serve as attachment sites of a number of coenzymes in proteins (e.g. biotin in pyruvate carboxylase, pyridoxal phosphate in phosphorylase, lipoic acid in lipoate acetyl-transferase) and form covalent intermediates in several enzymic reactions (e.g. transaldolase, aldolase, etc.). Discussion of all of these naturally-occuring derivatives of lysine will not be attempted in this treatise, but the investigator using chemical modification of proteins should be aware of their possible presence and effect on the results of his experiments. It should be noted that e-N-phospholysine has been reported in nucleoside diphosphate kinase (Walinder 1968). 

Many proteins have been labeled with organometallic complexes, mostly for analytical purposes. Some of those are mentioned in Section 1.31.5 of this chapter, and the topic has been comprehensively reviewed by Salmain. Ryabov published an earlier review on the topic.The labeling techniques are mostly the same as for organic derivatives, that is, cysteine-selective reactions (maleiimides, acetic acid halogenides), activated acids, aldehydes, or thiocyanates that react with lysines, biotin-(strept)avidine labeling, and others. 

Treatment of the biocytin derivative with biotini-dase, an enzyme that splits the bond between biotin and lysine, yields l -N-methoxy-Ci4-carbonylbiotin. This mode of attachment of CO2 to biotin (in the L-N position) and of biotin to the apoenzyme (6-N-(+) biotinyl lysyl) proved to be identical for j8-methyl-crotonyl CoA carboxylase, propionyl CoA carboxyyl-ase, and oxaloacetic transcarboxylase. The binding of CO2 with biotin is a high-energy binding ( — 4.7 kilocalories per mole) and the CO2 bond to the L-N-meth-oxy-Ci4-carbonylbiotin can be considered as a form of activated CO2. 

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