Homobifunctional reagents

a dialdehyde with the basic structure of pentanedial, exists mostly in a hydrated form at or below room temperature (Korn et al., 1972). Monsan et al. (1975) analyzed commercial preparations of aqueous 25% GA by NMR spectroscopy and found 79% water, 18% polymers and only 3% free GA. The polymers, which regenerate easily at higher temperatures to the free monomeric form (Korn et al., 1972), are formed particularly at higher pH, and will precipitate in alkaline conditions as poly(GA). GA should, therefore, be stored as a slightly acidic solution (pH 3-4). Purification of GA by vacuum distillation does not increase its cross-linking ability (Siess et al., 1971). The monomer absorbs at 280 nm (4.2 M cm ), while its polymers absorb strongly at 235 nm (1.53 x 10 M cm ). 

GA reacts irreversibly with the e-amino group of lysine (Quiocho and Richards, 1966). Since this bond is stable to hydrolysis, formation 

The reaction of a protein with GA is favored by a rise of (i) the pH above 7.0 (stable rate below pH 7.0 Molin et al., 1978) (ii) of the temperature (Bowes and Cater, 1966) and, (iii) of the concentrations of GA (law of mass action). However, the enzymatic and antibody activity of the conjugates may decrease significantly when the formation of polymers is beyond control (Boorsma and Streefkerk, 1976 Molin et al., 1978). 

The GA method is most suitable for APase (one-step procedure), and for the antibody-chimera method (Section 11.5), whereas for EIH the POase-Fab or POase-IgG conjugates are frequently prepared by the two-step GA procedure. 

The technical details of the one-step procedures (Avrameas, 1969) for various enzymes are given in Tables 11.7 and 11.8. This method cannot be recommended for POase only 1% of the POase and 5% of the immunoreactivity were recovered in the conjugates (Clyne 

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The use of homobifunctional reagents in two-step protocols still creates many of the problems associated with single-step procedures, because the first protein can crosslink and... 

Figure 1.23 Protein crosslinking reactions done using homobifunctional reagents can result in large polymeric complexes of multiple sizes and indefinite structure.

In two-step protocols, one of the proteins to be conjugated is reacted with the homobifunctional reagent and excess crosslinker and by-products are removed. In the second stage, the activated protein is mixed with the other protein or molecule to be conjugated, and the final conjugation process occurs (Figure 4.2). 

The particular crosslinkers discussed in this section are the types most often referred to in the literature or are commercially available. Many other forms of homobifunctional reagents containing almost every conceivable chain length and reactivity can be found mentioned in the scientific literature. 

Figure 4.2 Homobifunctional crosslinkers may be used in a two-step process to conjugate two proteins or other molecules. In the first step, one of the two proteins is reacted with the crosslinker in excess to create an active intermediate. After removal of remaining crosslinker, a second protein is added to effect the final conjugate. Two-step reaction schemes somewhat limit the degree of polymerization obtained when using homobifunctional reagents, but can t entirely prevent it.

B/s-maleimidohexane (BMH) is a homobifunctional reagent containing a non-cleavable, 6-atom spacer between terminal maleimides (Thermo Fisher). The maleimide groups can react... 

Avoid covalently linking the A and B chains together during the crosslinking process. This can be done by using heterobifunctional crosslinkers that are more controllable in their reactivity than homobifunctional reagents. 

Bz s-imidoesters like DMS may be used to couple proteins to PE-containing liposomes by crosslinking with the amines on both molecules (Figure 22.24). However, single-step crosslinking procedures using homobifunctional reagents are particularly subject to uncontrollable polymerization of protein in solution. Polymerization is possible because the procedure is done with the liposomes, protein, and crosslinker all in solution at the same time. 

Figure 25.10 PEG-amine compounds may be reacted with this heterobifunctional crosslinker to form amide bond derivatives terminating in maleimide groups. This results in a homobifunctional reagent capable of crosslinking thiol molecules. Subsequent reaction with sulfhydryl-containing molecules yields thioether linkages.

The problems of indeterminate conjugation products are amplified in single-step reaction procedures using homobifunctional reagents (Chapter 4). Single-step procedures involve the addition of all reagents at the same time to the reaction mixture. This technique provides the least control over the cross-linking process and invariably leads... 

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