MACROMOLECULES 101 



sulphur (around 2%); when the different amino acids have been deter- 

 mined these figures allow one to check that the sum of these amino acids 

 accounts completely for the composition of the protein. 



(b) Titration 



The reactive groups of the protein may be determined by titration, with 

 acids and dilute alkalies when dealing with acid or basic groups, and by 

 means of silver nitrate, iodine, etc., when dealing with thiol groups. 



The potentiometric titration of a protein in aqueous solution with an acid 

 or dilute alkali is carried out by measuring the change in pH which results 

 from the addition of a known amount of the acid or base to the isoionic 

 protein. The isoionic state exists when the number of protons attached to 

 the basic groups (e.g. -NHg + H+ -> -NH+g) is equal to the number of 

 protons removed from the acidic groups (e.g. -COOH -^ -COO" + H+). 

 If there are no other ions apart from protons fixed on to the protein, 

 then the isoionic state coincides with the isoelectric state, defined by charge 

 O. The results of the titration are expressed in terms of change in pH/unit 

 of acid added and change in pH/unit of base added. Figure 12 shows the 

 dissociation curve of the protein. 



There are quite a number of ionizing groups present in proteins : 

 a-COOH the terminal group of polypeptide chains; 

 ^-COOH or y-COOH of aspartic or glutamic acid; 



n NH of histidine; 



N 



H 



a-NH+3 the terminal group of polypeptide chains; 

 €-NH+3 of lysine; 



-<^ ^-OH of tyrosine; 



SH of cysteine ; 



---NH 



\ + . • • 

 C=NHo of argmme. 



/ 



H2N 



When the pH is very acid, around 1-0, for example, all these groups are in 

 the undissociated state. At pH 14-0, they are all completely dissociated. 

 As one passes from pH 1-0 to pH 14-0 each ionizing group will dissociate 



