256 VI. HEMOGLOBIN 



be immediately reversed. If oxyhemoglobin is added to 30% potassium or 

 sodium hydroxide at — 5° C, the spectrum of hemoglobin changes to that 

 of the hemochrome and the pigment precipitates. If the solution is then 

 neutralized by ammonium sulfate and diluted, hemoglobin is re-formed and 

 may be oxygenated to give oxyhemoglobin. If the temperature is allowed 

 to rise above 0° C, irreversible changes set in, and the native protein cannot 

 be recovered. This phenomenon has received little attention. Holden 

 {1320) has recently observed that a similar compound is formed by the 

 action of strong ammonia on hemoglobin in nitrogen, while Legge {1667) 

 found that if the reaction is carried out in potassium chloride solution (60% 

 saturated) native protein can be recovered after treatment with 1% alkali. 

 This finding may, perhaps, be associated with the increased resistance to 

 denaturation by alkali which other workers have observed in strong salt 

 .solutions. 



This behavior can be explained on the imidazole hypothesis by assuming 

 that the distance between the imidazoles on either side of the heme may be 

 lessened by a shrinkage of the protein in solutions of high ionic strength. 

 The closer approach of the imidazoles may allow a firm hemochrome linkage 

 to be established which may be strong enough to hold the structure of the 

 protein during the denaturation, so that, on renaturation, the original link- 

 ages are formed. Zeynek (quoted by Haurowitz, 1165) observed a similar 

 phenomenon when hemoglobin was dehydrated in a high vacuum, the 

 hemochrome spectrum disappearing on admission of moisture. 



In cytochrome c (Chapter \TII) the heme is not only firmly held between 

 two imidazoles but in addition is bound to the protein through a thioether 

 linkage in a side chain. The denaturation of cytochrome c is fully reversible, 

 probably because of the stability of the heme protein linkage which prevents 

 the unfolding of the molecule. 



These experiments indicate that heme and its linkages to the 

 protein stabilizes the structure of hemoglobin; this is also borne out 

 by the fact that native globin is far less stable and more readily 

 denatured than hemoglobin. 



4.3.3.2. Action of Acid. At the same time as Hill provided spectro- 

 scopic evidence that hemoglobin might be recovered from denatured 

 globin hemochrome, Anson and Mirsky {68) obtained crystalline 

 oxyhemoglobin by renaturation of the protein after it had been 

 denatured by a variety of procedures. Their renatured hemoglobin 

 was able to combine with oxygen, although Hill (quoted in 11^25) 

 found the dissociation curve to be hyperbolic {cf. Section 5.). The 

 results of Anson and Mirsky were confirmed in 1927 by Wu and Lin 

 {3130) and by Holden and Freeman {1321). The reversible denatura- 

 tion of hemoglobin derivatives in acid solutions, now more thoroughly 



