REACTIONS WITH PROTEINS 755 



less affinity for the mercurial continue to react, a sloping or curved line will 

 be obtained (curve B). It is important to construct such curves whenever 

 possible in order to understand the binding characteristics. 



(B) Hemoglobin. Crystalline human oxyhemoglobin reacts with 2 moles 

 of MM per mole of protein but the rate is rather slow at pH 7.5 (W. L. 

 Hughes, 1950). The hemoglobins of other species may contain either two 

 or four SH groups that react readily. Green et al. (1954) crystallized a de- 

 rivative of horse hemoglobin in which two SH groups had been reacted 

 with p-MB and showed that, although the crystals are isomorphous with 

 normal hemoglobin, the X-ray diffraction pattern is somewhat different. 

 Ingram (1955) established that Hg+^, MM, and p-MB all combine readily 

 with four SH groups of horse hemoglobin in the native state, and with six 

 in the denatured form; ox and human hemoglobins are similar but the 

 latter presents eight SH groups when denatured. In native hemoglobin, 

 Hg++ probably reacts with two SH groups simultaneously; at least 2 equiv- 

 alents of Hg+^ reduce the free SH groups to zero. However, p-MB like- 

 wise blocks two SH groups and since reaction of a molecule of p-MB with 

 two SH groups is impossible, it is likely that a pair of SH groups is so close 



Fig. 7-4. The reactions of the SH group 

 pairs on hemoglobin with Hg++ and p-MB, 

 according to the concept of Ingram (19.55). 



that each group cannot react with the large mercurial. It is difficult to say 

 in the case of Hg++ if the effect is steric or due to the formation of a bridge 

 between the two SH groups in a pair, but the latter mechanism is favored. 

 The situation as represented by Ingram is shown in Fig. 7-4. It is interesting 

 that Hg++ and p-MB compete for the SH groups, and that the former is 

 bound more tightly, probably due to the reaction with two of the SH groups. 

 Since the earliest studies of hemoglobin SH groups, there has been diffi- 

 culty in establishing the exact number of reactive and unreactive groups, 

 due to the fact that the nature of the reactions and the stoichiometry were 

 uncertain. The details are not pertinent to our purpose and have been well 

 reviewed by Huisman (1959). Further complications have arisen in the re- 

 cognition that different types of hemoglobin react differently with the mer- 

 curials and that the pH is an important factor. Murayama (1958) claimed 



