102 



UNITY AND DIVERSITY IN BIOCHEMISTRY 



over a particular pH range corresponding to its pK value. Figure 13 

 illustrates this. In Fig. 12, three regions, labelled a, b and c, may be dis- 

 tinguished, in which a slight change in pH has had a marked effect on the 

 number of protons combined with the protein. Referring to Fig. 13, we can 

 see that region a corresponds to the dissociation of terminal free-carboxyl 

 groups, and region h to that of the dissociable groups of histidine and 

 terminal a-amino groups. Region c corresponds to the dissociation of the 

 groups of lysine, tyrosine, cysteine and arginine. To investigate the ques- 

 tion further and resolve the complexities of regions b and c it is necessary 



pH-* 



Fig. 12 (Springall) — Titration curve of a protein. Abscissae : pH values. Ordinates : 

 the number h of protons added or subtracted, starting from the isoionic state, to give these 

 pH values in aqueous solution. These values of h are obtained from the number of equiva- 

 lents necessary to bring the solvent to the same pH. 



to mask one or other of the groups. Thus, free amino groups may be re- 

 acted with formol or removed by enzymatic deamination, etc. The com- 

 plete and often arduous analysis of the titration curve of a protein gives us 

 information about the amount of arginine, histidine and lysine in that 

 protein, and also the total number of free carboxyl and a-amino groups. 

 Knowing the number of primary amide groups (from the amount of 

 ammonia in the hydrolysed protein — see later) and the amount of aspartic 

 and glutamic acids, it is possible to calculate the number of carboxyl 

 groups not involved in peptide bonds. If the number of free carboxyls and 

 the number of free amino groups are known some idea may be obtained of 

 the number of polypeptide chains present in the protein molecule. 



{c) Composition of the Hydrolysate 



When the titration of the native protein has been carried out the amino 

 acid composition is determined. Hydrolysis gives a mixture of amino 



