280 VI. HEMOGLOBIN 



and following the rate of appearance of oxyhemoglobin (11^9). 

 Hemoglobin was prepared both by evacuation and by reduction with 

 ammonium sulfide. Hartridge and Roughton found that the rate at 

 which oxyhemoglobin was reduced by ammonium sulfide was so 

 slow that the back reduction with this reagent could be neglected 

 during the course of the reaction with oxygen. A twelvefold altera- 

 tion in the concentration of ammonium sulfide used had no effect on 

 the velocity of the association reaction, which was found to be much 

 faster than the reverse reaction first investigated. The velocity was 

 about 50% faster at pH 10 than at pH 5.6 and was unaffected by 

 the presence or absence of 0.067 M sodium chloride. The rate is 

 described by the equation : 



d[nhO,]/dt = ka [Hb] [O2] - ki [HbOa] 



When the ratio of the hemoglobin to oxygen was increased fourfold, 

 the rate increased likewise during the initial period of the reaction. 

 This finding excludes Hill's hypothesis on which the first term on 

 the right-hand side of the above equation would be ko [Hb] [O2]". 

 If this described the reaction, a fourfold variation in the ratio of 

 oxygen to hemoglobin should alter the rate by a factor 4", where n 

 is between 2 and 3. 



Since from their previous work they had obtained a value for the 

 rate of dissociation of oxyhemoglobin, they were able for the first 

 time to put the kinetic basis of Hiifner's theory to experimental test. 

 They measured the dissociation curve and the rate constants on the 

 same samples of hemoglobin. They found the dissociation curve to 

 be hyperbolic and fitted it with the Hiifner equation. The value of 

 the equilibrium constant determined from the rate constants was 

 then compared with that found experimentally. 



Sample 1 Sample 2 



pH K (exper.) K = k2 ki K (exp?r.) K = kz, ki 



7.7 218 164 112 148 



10.0 730 700 336 438 



In view of the experimental difficulties the agreement must be con- 

 sidered good, and under the conditions in which they worked the 

 kinetic evidence, therefore, supported Hiifner's theory, which, as we 

 have seen, had been discarded because of the inability to predict the 

 sigmoid dissociation curve obtained under most conditions. We shall 

 examine this paradox in Section 7.1.1. 



