280 CELL HEREDITY 



that the enzymes themseKes have been altered, but they tell us very little 

 about the precise chemical nature of the gene-induced alteration. 

 Changes in thermostability, serological specificity, and catalytic activity 

 all depend upon the folded three-dimensional configuration of the 

 enzyme. Do these altered properties result from substitutions of one or 

 more amino acids for others in the primary sequence of the protein? 

 Many current investigations are designed to answer this question. As 

 yet however, the relation between amino acid sequence and folded con- 

 figuration is not well established, nor have analyses been reported for 

 any of the enzymes listed in Table 10.1. Thus, it has not yet been shown 

 in any instance that the observed differences in behavior of wild type 

 and mutant enzymes result from altered amino acid sequences. 



HEMOGLOBIN 



One protein has been described, in which a single amino acid substi- 

 tution is characteristic of a single mutant allele; that protein is hemo- 

 globin. There is an altered form of hemoglobin, HbS, found in sickle 

 cell anemia, a human ailment inherited as a single gene difference from 

 normal. Sickle cell trait is a mild form of the disease, found in heterozy- 

 gotes; individuals with sickle cell anemia are homozygous for the sickling 

 gene. The anemia results from the presence of hemoglobin S (HbS) 

 which tends to crystallize out in red blood cells at low oxygen tension, 

 causing rupture of the cells. Heterozygotes contain, in each red blood 

 cell, both HbS and normal hemoglobin, HbA, resulting in mild symp- 

 toms, if any. 



The chemical distinction between HbA and HbS was first detected as 

 a slight difference in electrophoretic mobility, that is, in their rate of 

 movement in an electric field resulting from a net charge difference on 

 the molecules. It was postulated that this charge difference resulted 

 from a change in amino acid composition, but attempts to demonstrate 

 such a change by analysis of total amino acid composition revealed no 

 differences. A more sensitive method was needed. 



The method that has worked so beautifully in the analysis of hemo- 

 globin is called "fingerprinting, ' developed by Sanger for the sequence 

 analysis of insulin. The protein is digested by trypsin, which breaks only 

 those peptide bonds involving either lysine or arginine. The resulting 

 peptides, most of which differ from one another in net charge, can be 

 separated by paper electrophoresis, the peptides migrating in one dimen- 

 sion on wet filter paper. Subsequently, the paper is rotated through 90°, 



