Modification of avidin

Modification of avidin or streptavidin with Texas Red sulfonyl chloride may be done similarly, except the fluorophore is first dissolved in acetonitrile prior to addition to the aqueous reaction mixture. 

Conjugates of avidin or streptavidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with SPDP to create a sulfhydryl-reactive derivative, followed by modification of avidin or streptavidin 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 avidin or streptavidin can be found in Section... 

The activation of enzymes using adipic acid dihydrazide and EDC is identical to the procedure outlined for the modification of avidin or streptavidin (Chapter 13, Section 5). 

G. Paganelli. 1998. Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immuno-genicity. Br. J. Cancer 78 189-197. 

Chinol M, Casalini P, Maggiolo M, Canevari S, Omodeo ES, Caliceti P, Veronese FM, Cremonesi M, Chiolerio F, Nardone E, Siccardi AG, Paganelli G. Biochemical modifications of avidin improve pharmacokinetics and biodistribution, and reduce immunogenicity. Br J Cancer 1998 78 189-197. 

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.

As schematically depicted in Figure 5, two different routes are available for immobilizing biotin-labeled enzymes on the support through avidin-biotin complexation. The first procedure employs the biotin-modified surface on which biotin-labeled enzymes are immobilized through avidin as binder protein. For this procedure, the covalent linkage of biotin onto the surface of a carbon electrode and the preparation of biotin-labeled lipid bilayer on electrode have been studied. An alternative way involves the direct modification of an electrode surface with avidin. If avidin could be immobilized directly without loss of the binding activity to biotin, biotin-labeled enzymes could be loaded more easily on the electrode surface. 

Biotin s interaction with the proteins avidin and streptavidin is among the strongest noncovalent affinities known (K l = 1015 M l). The binding occurs between the bicyclic ring of biotin and a pocket within each of the four subunits of the proteins. The valeric acid portion is not involved or required for the interaction (Green, 1975 Wilchek and Bayer, 1988). This characteristic allows modification of the valeric acid side chain without affecting the binding potential toward avidin or streptavidin. 

Streptavidin is another biotin binding protein isolated from Streptomyces avidinii that can overcome some of the nonspecificities of avidin (Chaiet and Wolf, 1964). Similar to avidin, streptavidin contains four subunits, each with a single biotin binding site. After some postsecretory modifications, the intact tetrameric protein has a molecular mass of about 60,000 D, slightly less than that of avidin (Bayer et al., 1986, 1989). 

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