28 II. METHODS OF INVESTIGATION 



7.1.4. Differential Titration of Proteins. When a protein enters 

 into combination in such a manner that one or more of its dis- 

 sociable acidic or basic groups are involved, there will in general 

 be foun<l differences between the titration curve of the original 

 protein and that of the compound. Differences in the curves in a 

 particular pH region indicate that groups dissociating in this region 

 are in some way involved in the combination. It has been found 

 possible by this means to suggest the particular amino acid residues 

 responsible for combination with the prosthetic group in a number of 

 hemoproteins, and also those involved in combination with oxygen 

 or in changes of linkage associated with change of valency of the iron 

 atom. In the hemoproteins, the picture is complicated by the pres- 

 ence of carboxyl groups in the hematin side chains, and of hydroxyl 

 groups attached to the iron atom in the ferric compounds. Examples 

 of the use of the method and the results obtained are given in 

 Chapters VI and IX. 



7.2. Oxidation-Reduction Potentials 



Measurement of oxidation-reduction potentials has contributed 

 important data toward the solution of numerous problems encoun- 

 tered in the chemistry of hematin compounds. Its use depends on 

 the capacity of the iron atom (or some other metal atom in synthetic 

 metalloporphyrins) to exist in either oxidized or reduced state. The 

 problem is by no means a simple one since a great number of other 

 variables have to be taken into account, including, for example, 

 change of pH, ionization of one or both of the components of the 

 reaction, coordination of components with other substances, and 

 change of aggregation accompanying reduction or coordination. 

 Consequently although the use of a general equation expressing the 

 effect of all possible variables is theoretically admissible, the mathe- 

 matical manipulations involved in this are prohibitively compli- 

 cated. The experimental treatment likewise recjuires some simpli- 

 fication of the problem, since it is seldom possible to secure adequate 

 data^ on all aspects of such a complex system. 



The practice is therefore adopted of "breaking down the con- 

 tinuum into a plexus of equations" (Clark) and of utilizing graphical 

 methods for the interpretation of results. 



It is obviously outside the scope of this elementary review to 

 attempt a complete or even entirely rigorous exposition of the 

 theory of oxidation-reduction potentials. The reader is referred in 



