Biotin reactive derivatives

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

Other reactive derivatives can be used to attach biotin to phenolic, thiol, or carbonyl groups. 

The pH-dependent binding of 2-imino-biotin (Fig. 3g) has been used in affinity chromatography to purify strept(avidin) because binding is easily reversible [85]. Specifically,bin ding occurs under alkaline conditions (pH 10) and release occurs under acidic conditions (pH 4). An amine-reactive derivative of desthiobiotin (Fig. 3h), a non-sulfur containing metabolic precursor to biotin that is bound to strept(avidin) with lower affinity than biotin, has been used to form reversible complexes with strept(avidin) [74]. Release is achieved by incubation in the presence of free desthiobiotin or biotin. Recently developed lower affinity derivatives include 9-methyl biotins [86]. Another approach covalently attaches a photocleavable biotin derivative (Fig. 3h) to molecules that are released following exposure to 200 nm light [75]. 

There are many ways of attaching biotin to such molecules via a variety of chemically reactive biotincommercially available from Sigma Chemical Co. (St. Louis, MO), Pierce Chemical Co. (Rockford, IL), Calbiochem AG (Lucerne, Switzerland), Molecular Probes, Inc. (Eugene, OR), and others. 

The following sections discuss some of the more common biotinylation reagents available for modification of proteins and other biomolecules. Each biotin derivative contains a reactive portion (or can be made to contain a reactive group) that is specific for coupling to a particular functional group on another molecule. Careful choice of the correct biotinylation reagent can result in directed modification away from active centers or binding sites, and thus preserve the activity of the modified molecule. 

Figure 11.16 The aminophenyl group of this biotin derivative can be transformed into a diazonium reactive group by treatment with sodium nitrite in dilute HC1.

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. 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

NHS-LC-biotin is an extended spacer arm derivative of biotin containing an amine-reactive NHS ester (Chapter 11, Section 1). The compound is a popular choice for biotinylating a wide... 

AT-hiotinylaminoethylmethanethiosulfonate (MTSEA-biotin) is a relatively membrane-impermeant MTS derivative to which a hiotin moiety is conjugated (Seal et al., 1998 Boileau et al., 2002). It is composed of two structural domains a flexible tail 12-14A in length, 2.5A in diameter, and 4 x 5A planar head group (Teissere and Czajkowski, 2001 Wagner and Czajkowski, 2001). The reactive disulfide is near the end of the tail 12A from the head group. 

---