DENATURATION 257 



investigated, showed the following features: Below pH 5.7 hemoglobin 

 dissociates into half molecules which in the pH range of 4.6 to 4.1 

 reciuire a far lower energy for denaturation than above pH 5.7 (517). 

 Ox hemiglobin, which is stable at pll 4, is completely converted to 

 "acid hematin" at pH 3. If the pH is shifted back to 4 without 

 precipitation of the protein, the renaturation is immediate. If, after 

 denaturation at pH 3, the solution is neutralized, the denatured 

 protein precipitates. Renaturation from the precipitated protein is 

 slow, taking up to six hours if it is dissolved at pH 4. If dissolved in 

 a slightly alkaline solution, the renaturation is complete in three 

 hours. The rate at which renaturation proceeds is of importance in 

 the preparation of native globin (Section 4.3.5.). 



Oxyhemoglobin is more resistant to denaturation by acid than is 

 hemoglobin; renaturation of only 60-70% of the pigment is possible. 

 This is due to the irreversible oxidation of the protein (1702,2279). 

 If the presence of oxygen is avoided, either by using carbon monoxide 

 hemoglobin or hemoglobin, recoveries of 90-95% of native protein 

 may be obtained. In the presence of ascorbic acid, the oxidation of 

 globin by the oxygen liberated when oxyhemoglobin is acidified is 

 prevented, the ascorbic acid being oxidized itself and protecting other 

 oxidizable groups (Lemberg and Legge, 1702; cf. also Chapter VIII). 

 Holden (private communication) has observed that, in the presence 

 of ascorbic acid, up to 90% of denatured oxyhemoglobin may be 

 renatured. 



One aspect of the renaturation needs further investigation. Wu and Lin 

 as well as Anson and Mirsky (71) have claimed that addition of dithionite 

 and buffered cyanide increases the yield of native protein. The effect of 

 cyanide appears to be on the protein. The yields which these workers have 

 obtained, however, are rather low and without dithionite or cyanide they 

 were able to obtain very little renatured protein at all. Since Holden is able 

 to obtain virtually complete renaturation without the use of these reagents 

 (1309, p. 48), their mode of action requires reinvestigation. 



4. .3.3. 3. Other Reagents. Oxyhemoglobin may be denatured in neutral 

 solution by organic solvents such as alcohol or acetone, anionic and cationic 

 detergents, salicylate {1964,2280,2305,2310,2312), or amides such as urea, ace- 

 tamide, and particularly guanidines (2621). Denaturation by urea is accom- 

 panied by -splitting into half molecules with the hemoglobin of some, but not 

 of all, species {93,1352,2621). Renaturation of a variable fraction can be 

 obtained on dialysis; the reversal of denaturation by salicylate appears to 

 yield hemoglobin indistinguishable from native hemoglobin (Roche, 2305, 

 2310,2312). 



