Glutaraldehyde protein crosslinking

Schiff base interactions between aldehydes and amines typically are not stable enough to form irreversible linkages. These bonds may be reduced with sodium cyanoborohydride or a number of other suitable reductants (Chapter 2, Section 5) to form permanent secondary amine bonds. However, proteins crosslinked by glutaraldehyde without reduction nevertheless show stabilities unexplainable by simple Schiff base formation. The stability of such unreduced glutaraldehyde conjugates has been postulated to be due to the vinyl addition mechanism, which doesn t depend on the creation of Schiff bases. 

Branner-Jorgensen, S. (1978) On the mechanism of protein crosslinking with glutaraldehyde. 4 Enzyme Engineering Conference, M4. 

Formaldehyde, a monoaldehyde, is preferred over glutaraldehyde (a dialdehyde) because the latter introduces mostly irreversible protein crosslinks, masking the epitopes. Formaldehyde penetrates rapidly into the tissue but crosslinks proteins slowly. Most of these crosslinks are reversible, and therefore masked epitopes can be easily unmasked by treatments such as heating. However, for better preservation of cell morphology, a mixture of formaldehyde (4%) and glutaraldehyde (0.01-0.1%) can be tried. Bouin s fixative with its acidic pH should not be used. 

As explained in this chapter, glutaraldehyde introduces mostly irreversible protein crosslinks that may alter the conformation of the antigen (epitope) molecule. Such extensive crosslinkages become a barrier to the antibody penetration, and thus its accessibility to the epitope is hindered. This impediment becomes a serious problem when monoclonal antibodies specific for only one epitope type are used and/or when epitopes are not located at the surface of the antigen molecule. 

It is assumed that the partially denaturated protein binds the inhibitor whereby a new active center is formed which is similar to that of the model enzyme. In one of several examples transformation of a trypsin to a chymo-trypsin-active protein is described with indole as inhibitor and glutaraldehyde as crosslinking agent. The new product showed an increase of chymotrypsin activity of A00% and a decrease of trypsin activity of 1AZ. 

Glutaraldehyde is the most popular b/s-aldchydc homobifunctional crosslinker in use today. Flowever, a glance at glutaraldehyde s structure is not indicative of the complexity of its possible reaction mechanisms. Reactions with proteins and other amine-containing molecules would be expected to proceed through the formation of Schiff bases. Subsequent reduction with sodium cyanoborohydride or another suitable reductant would yield stable secondary amine... 

The following protocol describes the 2-step method wherein the liposome is glutaraldehyde-activated, purified away from excess crosslinker, and then coupled to a protein by reductive amination (Figure 22.23). 

Both cells and enzymes can be crosslinked with glutaraldehyde. Inert protein or polyamines may be added to the reaction mixture. 

---