1 98 IMMUNO-CATALYSIS 



nitrogen of the toxin is slowly and irreversibly bound. In view of the 

 irreversibility of the union of the toxin with formaldehyde, Pappen- 

 heimer considered this reaction not to be due to the mere formation of 

 methylene linkages to the nitrogen. Hydroxymethyl compounds re- 

 sulting from this reaction with free amino groups are unstable combina- 

 tions (French and Edsall, 1945) and should therefore be reversible. 



While with low concentrations of formaldehyde the toxin did not 

 lose the ability to flocculate with antitoxin, the higher concentrations 

 caused the destruction of antigenic properties. Horsfall (1934), and 

 Jacobs and Sommers (1939) also reported serologically demonstrable 

 changes with formolized proteins involving combinations other than 

 with free amino groups. 



The reviewers of the reactions with formaldehyde point out that the 

 types of reactions it enters into is governed by the H+ and 0H~ con- 

 centration, period of treatment and the type of protein treated. In 

 neutral solution, the immediate reaction with proteins is a reversible 

 combination with the free amino groups. With longer time of reaction, 

 as in the case of the preparation of toxoids and vaccines, the formalde- 

 hyde slowly becomes more firmly bound with decrease of amino nitro- 

 gen; under these conditions, not only the amino, but also the indole, 

 amide, and guanidyl groups react with formaldehyde, forming cross- 

 linkages and more stable combinations and, therefore, antigenically 

 modified proteins. There has been indication that formaldehyde reacts 

 also with sulfhydryl groups (Anson, 1945). 



In view of the many complications resulting from the action of 

 formaldehyde on the various groups in the protein molecule, it is 

 impossible to correlate any specific group in the protein molecule with 

 its loss of viral, enzyme, toxic, serological and antigenic properties. 



c. Acylation of Proteins. The principal reagents used for the acyla- 

 tion of proteins are ketene [(HoC-C-A)] and acetic anhydride 

 (CH3CO)20. Ketene has been extensively used with aqueous protein 

 solutions. At a pH above 5.0, ketene reacts with NH2, SH and 

 tyrosine-OH groups in proteins. With NH2 groups it yields pro- 

 tein-NH-COCH3. 



Acylation at pH 5.5 of the amino groups of pepsin causes no inactiva- 

 tion (Herriott, 1935). The number of amino plus tyrosine groups 

 covered was less than the number of acetyl groups, indicating that some 

 other protein groups had reacted. It has been found that ketene reacts 



