254 VI. HEMOGLOBIN 



therefore not safe to rely on one criterion alone in establishing dena- 

 turation experimentally. The reader is referred to the review on 

 denaturation by Neurath and co-workers {i204-6). 



The two most readily observable alterations which hemoglobin 

 suffers in denaturation are changes in solubility and loss of its ability 

 to combine reversibly with oxygen. The latter change may be 

 demonstrated directly by gas analysis or indirectly by spectroscopic 

 identification as denatured globin hemochrome, which is unable to 

 combine reversibly with oxygen. The other changes enumerated 

 above have also been observed to occur on the denaturation of hemo- 

 globin. There is no longer any doubt that denaturation as defined 

 above can be reversed, though it has not been proved that a completely 

 unfolded peptide chain can be rearranged to form the native protein. 



Neurath and co-workers {201^6) point out that it can never be proved that 

 a renatured protein is absolutely identical with the native protein, but does 

 this objection mean anything? If it is possible by the appropriate treatment 

 to recover from a denatured protein a certain proportion of a protein which 

 in most of its properties agrees with the native protein, the denaturation 

 will have been, to that extent, reversible; the remainder of the denatured 

 protein will have been irreversibly denatured. 



While reversible denaturation of proteins had been described pre- 

 viously', the investigations of Anson and Mirsky on reversible dena- 

 turation of hemoglobin and other proteins have been of particular 

 importance. In 1925 these workers succeeded in preparing crystalline 

 oxyhemoglobin from heat-coagulated hemoglobin. Later they and 

 other workers have shown that hemoglobin, myohemoglobin, and 

 other proteins can be denatured by a variety of agents in such a way 

 that the products can be reconverted to native proteins, denaturation 

 and reversal being established by several criteria. 



4.3.2. Bond Changes on Denaturation. Two types of linkage are present in 

 a native protein in addition to the peptide linkage: those which may be 

 ruptured by pH change alone and those linkages which require reduction 

 for their rupture. The rupture of the latter linkage may be facilitated by 

 changes in pH which in themselves are unable to bring it about {378). In 

 the first group are placed salt linkages {728) or hydrogen bonds {1964.) and, 

 in hemoglobin, the linkages between the heme iron and the histidine imi- 

 dazoles; in the second are the disulfide groups. Denaturation appears to 

 involve the first type of linkages, although it may facilitate the attack on 

 the sulfhydryl and disulfide groups by means of oxidizing or reducing sub- 

 stances. There is no evidence that peptide linkages are hydrolyzed. 



The character of the bond changes on denaturation is at present the 

 subject of controversy. This centers on the high energy of activation found 



