244 VI. HEMOGLOBIN 



monoxide, has led, in carboxyhemalbumin, to the estabhshment of an 

 iron protein bond in addition to the other Hnkages. 



No observations have as yet been reported on the combination of 

 mesohematin with serum albumin but, in view of the ease with which 

 the linkage between hematin and albumin may be formed or broken, 

 it is unlikely that the vinyl groups play any role. In view of the 

 discussion in Section 3.3.4. the evidence points to linkage from the 

 carboxyl side chains of the hematin to the protein. Measurements 

 are not available from which the dissociation constant of the com- 

 pound may be calculated, nor has there been any quantitative investi- 

 gation of the acid-base-binding power of methemalbumin which 

 might throw light on the residues within the protein which are involved 

 in the linkage. 



It would be of interest to measure the relative affinities of caffeine 

 and albumin for hematin. In view of the occurrence of bilirubin in 

 the plasma as bilirubin albumin it is possible that both bilirubin and 

 hematin may be combined with the same groups in albumin. 



3.3.6. DiflFerential Titration of Globin and Hemiglobin. The imidazole 



groups in the globin which combine with tiie iron atom in the heme have been 

 called "heme-linked" groups because their ionization is affected by the 

 changes in the character of the iron bonds. The groups in the protein involved 

 in hematin carboxyl-protein linkage would not be "heme-linked" in this 

 sense. The carboxyl groups, insulated from the resonance system of the 

 porphyrin by being attached to propionic acid side chains, would be little 

 affected by changes in the character of the iron bonds, and hence would be 

 unable to affect the ionization within the protein. 



Changes in the position of the heme could, of course, alter the strength of 

 the carboxyl-protein bond. On entry of oxygen, the heme may be pushed 

 toward the proximal imidazole ring, but in view of the configurations which 

 the propionic acid side chain may take up it is most unlikely that appreciable 

 alteration of bond length occurs between the carboxyl and the protein. 



Differential electrometric titrations of hemoglobin and oxyhemoglobin 

 (Sections S.'i.'i.S. and 3.'i.2.5.) would therefore not be expected to throw 

 light on the groups within the protein which are linked to hematin carboxyls, 

 but investigation of the changes which take place in the globin on combination 

 with hematin might do so. 



Theoreil {277()) has investigated this important system. He titrated both 

 globin and hem/globin from pH o.ii to pH 11.3, working at 0° C. on account 

 of the instability of tlie globin. On completion of the titration of the globin 

 at /;H 11.3, the solutions were then immediately neutralized with hydro- 

 chloric acid and the globin was then coupled with heme. Theoreil does not 

 record the absence of denatured globin hemochrome after the procedure, but 

 states that no precipitate appears on neutralization, from which it may be 

 concluded that this difficult titration was carried out satisfactorily. 



