Cross-linking protein-formaldehyde reactions

This process of cross-linking does not appear have a major effect on protein secondary structure at room temperature. However, cross-links formed by reactions of formaldehyde with proteins retard, but do not eliminate, protein denaturation that occurs when proteins are heated to a temperature of approximately 70°C or above.7... 

Metabolically, formaldehyde is rapidly oxidized to formic acid (see Section 14.7), which is responsible in large part for its toxicity. Formaldehyde reacts by addition and condensation reactions with a variety of biocompounds, including DNA and proteins, and in so doing forms adducts and DNA-protein cross-links.9 Formaldehyde is incorporated into proteins and nucleic acids as the -CH3 group. Reactive formaldehyde has a short systemic lifetime of only about 1 min its formic acid metabolic product has a longer metabolic lifetime. 

Fraenkel-Conrat H, Olcott HS. Reaction of formaldehyde with proteins. VI. Cross-linking of amino groups with phenol, imidazole, or indole groups. J. Biol. Chem. 1948 174 827-843. 

Fraenkel-Conrat H, Olcott HS. The reaction of formaldehyde with proteins. V. Cross-linking between amino and primary amide or guanidyl groups. J. Am. Chem. Soc. 1948 70 2673-2684. 

Fraenkel-Conrat H, Mecham DK. The reaction of formaldehyde with proteins demonstration of intermolecular cross-linking by means of osmotic pressure measurements. J. Biol. Chem. 1949 177 477-486. 

These initial findings do not exclude other possible formaldehyde-induced reactions with tissue proteins. Notably, this first model system was not designed to detect the role of lysine residues. Lysine has a propensity to react with and form a variety of different types of cross-links with other amino acids in the presence of formaldehyde.1,3 417 Therefore, it is likely to also be important in reactions with formaldehyde. In fact, peptides with internal lysine residues were purposefully excluded from this initial study for technical reasons. To explore the importance of lysine residues in antigen retrieval, an alternative method was employed. 

These data suggest that the loss of immunoreactivity after formalin fixation involves a protein cross-linking reaction. The first amino acid is at or near the antibody epitope. The second can be on the same protein or on another nearby protein. A variety of different chemical reactions with formaldehyde can be occurring. The common theme is that regardless of the details of the formaldehyde-induced cross-linking reaction, steric interference prevents antibodies from gaining access to the epitope. 

Formaldehyde reacts with proteins to form adducts and cross-links.31516 Metz et al.3 have identified three types of chemical modifications after treatment of proteins with formaldehyde (a) methylol (hydroxymethyl) adducts, (b) Schiff bases, and (c) methylene bridges. The reaction of formaldehyde with proteins is summarized in Figure 19.1, but briefly, formaldehyde reacts primarily with lysine and cysteine to form methylol adducts. The methylol adduct can subsequently undergo a dehydration reaction to form a Schiff base. Adducted primary amine and thiol groups can undergo a second reaction with arginine,... 

The cross-linking scheme using this method can make use of the native e- and N-terminal amines on carrier proteins as the source of primary amine for the condensation reaction. Added to the conjugation reaction is formaldehyde and the desired hapten to be coupled containing an appropriately active hydrogen. 

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