258 VI. HEMOGLOBIN 



4.3.4. Chemical Changes on Denaturation. Anson and Mirsky 

 (75), in contradiction to an earlier report {1901), found sulfhydryl 

 groups in denatured globin. Their procedure was criticized by 

 Meldrum (1899), but reinvestigation by different methods (1054^,1963) 

 have confirmed their original finding. The sulfhydryl groups in the 

 native protein do not reduce cystine or ferricyanide at pH 6.5, but 

 react at pH 9.5, the protein structure altering sufficiently to allow 

 the reaction to take place. At this pH, they will also react with 

 iodoacetic acid. These reactions do not, in themselves, bring about 

 denaturation of the protein (cf. Chapter VIII). On denaturation, 

 however, sulfhydryl groups become reactive, irrespective of pH. In 

 solutions of guanidine or methylguanidine hydrochlorides, 75% of 

 the total alkali-labile sulfur in horse globin is estimated as cysteine 

 by porphyrindine (1054). If> before globin is prepared by the acetone 

 procedure, the sulfhydryl groups are oxidized, they can no longer be 

 detected in the denatured protein. 



Haurowitz and collaborators (1175) have shown that globin may 

 react with iodine or small amounts of formalin and that groups may 

 be inserted by diazotization without denaturation of the protein or 

 loss of oxygen-combining power. 



4.3.5. Reaction between Cephalin and Oxyhemoglobin. An interesting 

 reaction between cephalin and oxyhemoglobin was discovered by ChargafF 

 and co-workers (4^9). Addition of a dilute solution of cephalin to oxyhemo- 

 globin causes the slow disappearance of the oxyhemoglobin bands and their 

 replacement by a spectrum resembling that of heme. Carboxyhemoglobin 

 reacts similarly, while the band of hemoglobin does not change. Additions 

 of dithionite do not cause the appearance of the spectrum of denatured globin 

 hemochrome, but, if a base is provided with which the heme can combine, 

 by the use, for example, of ammonium hydrosulfide as reducer, the hemo- 

 chrome spectrum appears. 



On the evidence of spectroscopic change and altered solubility the reaction 

 would be classed as one of denaturation. Yet the fact that no denatured 

 globin hemochrome is formed speaks against this. Chargaff has not reported 

 attempts to recover native hemoglobin from the compounds. A reinvesti- 

 gation of this reaction would be of interest. 



4.4. Preparation of Native Globin 



In the light of present knowledge of the renaturation of denatured 

 globin it is probable that both Bertin-Sans and de Moitessier (351) 

 in 1892, and Schultz (2472a) in 1898, did in fact succeed in obtaining 



