CHEMICAL PROPERTIES 17 



are protected as a result of interactions between the a- and /5-chains. Since 

 carboxymethylation of the free SH groups alters heme-heme interactions 

 and the binding of O2, it is not surprising that oxygenation of hemoglobin 

 affects the reactivity with SH reagents (R. E. Benesch and R. Benesch, 

 1962). lodoacetamide does not attack hemoglobin unless it is oxygenated 

 (or complexed with other molecules) under the conditions used, and this 

 was interpreted as due to configurational changes brought about by the 

 oxygenation. Whatever the mechanism, it is interesting that oxygenation 

 does not promote reaction with iodoacetate or p-mercuribenzoate. 



One of the important aspects of the reaction of iodoacetate and related 

 compounds with enzymes is that certain groups are attached to the enzyme, 

 these groups altering the configuration and properties of the enzyme sur- 

 face. The introduction of negatively charged carboxy methyl groups with 

 iodoacetate modifies the electric field and this must often have some effect 

 on reactions occurring at the active center. Thus it is of interest to inquire 

 how the properties of proteins are changed by alkylation. The studies on 

 keratin by Goddard and Michaelis (1935) are pertinent in this connection, 

 since they determined changes in the isoelectric point following reaction 

 with a variety of agents introducing different groups (see accompanying 

 tabulation). It may also be noted that no detectable reaction with amino 



Agent Group introduced Isoelectric point 



groups occurs even under these rather vigorous conditions (40 hr at pH 

 9-9.5). Proteins with fewer reactive SH groups than keratin would not be 

 altered so much, but changes in these directions must be expected in all 

 cases. Pillemer et al. (1939) confirmed these results and found no amino 

 groups reacted after 12 hr at pH 8. Not only the isoelectric point but also 

 the solubility and immunological specificity of proteins are changed by 

 reaction with iodoacetate. The coagulation of serum proteins upon heating 

 is abolished by iodoacetate (15 min at 12 mM and pH 7.4), whereas lodo- 

 acetamide enhances clotting (Huggins and Jensen, 1949). After incubation 

 with lodoacetamide, iodoacetate does not produce its usual effect, indicating 

 that both react with the same groups. The difference presumably lies in the 

 negative charges introduced by iodoacetate. lodoacetamide also alters the 

 nature of the clots, making them more firm and elastic, and increasing the 

 binding of water (Jensen et al, 1950). The inhibiting effect of iodoacetate 



