HALDANE EFFECT 271 



almost independent of pH within the region in which the redox potential of 

 the hemoglobin-hem/globin system shows marked changes (c/. Sections 5.2.5. 

 and 6.2.8.). 



5.1.9. Attempt to Discover Intermediates Directly. When Adair's hypoth- 

 esis was first announced, a careful reinvestigation was made of the more 

 obvious properties of hemoglobin in an endeavor to find discontinuities which 

 would be accounted for by the existence of these intermediates. Spectro- 

 scopic observations failed to provide such evidence, since, as Hart ridge and 

 Roughton {llJi-O) pointed out, the light absorption of partially oxygenated 

 hemoglobin could be accounted for by the presence of only two species, fully 

 oxygenated and fully reduced hemoglobin. 



More recently the change in the magnetic properties of the pigment 

 during reactions in which it changed from one bond type to another has been 

 investigated {500). Certain anomalies in the values found for the magnetic 

 susceptibility of a number of hemoglobin derivatives were provisionally 

 attributed to the possibility that magnetic moments of the hemes were not 

 independent but were coupled to give a resultant moment. Careful investi- 

 gation of the susceptibility during such reactions failed to provide any 

 evidence for this, and since similar anomalies were found in the magnetic 

 moments of myohemoglobin derivatives they are now attributed to orbital 

 contribution {27J^7). 



The most ambitious attempt to gain direct experimental evidence for the 

 existence of the intermediates was that of Conant and McGrew {J^76), on the 

 basis of measurement of the solubility of fully oxygenated hemoglobin when 

 the mother liquor was partially reduced. Their work was criticized by 

 Roughton on experimental grounds {918, p. 257), while, later, Conant pointed 

 out that subsequent work had probably rendered their initial assumptions 

 incorrect. 



In view of the rapidity with which equilibrium between the different 

 intermediates would be expected to be established in comparison with the 

 relatively slow manipulations involved in such experiments, it seems unlikely 

 that such attempts will succeed. 



5.2. Hemoglobin Systems Containing More Than 

 Six Species of Intermediate 



5.2.1. Introduction. If two compounds such as oxygen and carbon 

 monoxide react with hemoglobin, we have 21 classes of intermediates, 

 comprising all possible combinations in which the four, hemes are 

 free, or are combined with oxygen, carbon monoxide, or with both. 

 The heme-heme interaction in such systems need not necessarily be 

 the same as that found in the simple systems when the hemes are 

 combined with only one type of molecule. 



5.2.2. Haldane Eflfect. Long before Adair's intermediate com- 

 pound hypothesis was presented, J. S. Haldane {1103) discovered a 



