60 HEMOGLOBIN 



simplest terms by using haemoglobin made directly from blood without 

 any crystallisation previous to his determinations. In this way he 

 avoided the contamination of the pigment with a substance, as yet 

 little studied, namely, "inactive haemoglobin." This substance has 

 been handled by most workers interested in blood pigments, the 

 clearest statement of its properties coming from the Rockefeller 

 Institute. My own acquaintance with it recalls an interesting 

 reminiscence. When A. V. Hill sat for his final examination in 

 Cambridge, I had the experience of examining him, and I set this 

 question in the practical examination : " Prepare a 5 per cent, solution 

 of haemoglobin." My idea was that, starting with blood, the candidates 

 should measure the oxygen capacity, assume that each cubic centi- 

 metre of oxygen which they obtained represented 1/1-34 grams of 

 haemoglobin and, having f oiuid the percentage of haemoglobin present 

 (presumably about 15 per cent.), dilute the blood with water tiU it 

 became 5 per cent. Incidentally the blood would be laked and 

 the haemoglobin thrown into solution. Hill, however, proceeded 

 otherwise. He made the crystals, dried and weighed them and then 

 dissolved up five grams of the weighed crystals in water. The colour 

 of the final solution did not commend itself to me, and wishing to 

 ascertain the amount of methaemoglobin which it contained I en- 

 deavoured to test its oxygen capacity with ferricyanide. To my great 

 surprise it yielded no oxygen: at the same time its spectrum, on a 

 casual examination, was that of oxyhaemoglobin. Bayliss(2i), I know, 

 also observed a similar phenomenon. 



The following is the statement made on the subject by Van Slyke, 

 Hastings, Heidelberger and Neill(22): 



If dried, even at high vacuum and in the cold, haemoglobin is almost completely 

 inactivated, although in solubility and colour it still resembles oxyhaemoglobin. Slow 

 loss of activity occurs even while standing in solution. The maximum loss in our 

 preparations was one-fifth of the theoretical oxygen capacity. 



The presence of this tautomeric form may have had something 

 to do with the anomalous results obtained by Bohr, his instinct 

 would have been to purify his haemoglobin to the maximum extent 

 possible by crystallisation. It is one of the ironies of haemoglobin 

 that the greater the pains at which you are to make it pure the 

 greater is the extent to which you are likely to contaminate it by 

 the formation of the "inactive" variety. 



Whether inactive haemoglobin occurs in nature we do not know, 

 obviously it was not present in more than the most trifling quantities 



