300 BIOCHEMICAL GENETICS 



variants that can be easily isolated from other biological contaminants without de- 

 naturation in quantities sufficiently large for comparative physical and chemical analy- 

 ses. Among other molecules, mammalian hemoglobins seem to satisfy these criteria. 



Hemoglobin is a conjugated protein composed of heme prosthetic groups, which 

 are united to the polypeptide chains of a globin. The physical structure, four heme 

 units associated with four globin coils, appears to be similar for all mammalian hemoglo- 

 bins. 998 Heme is compsed of four pyrole groups with one molecule of iron and is similar 

 in all mammals. Each of the four polypeptide chains of the globin molecule is composed 

 of approximately 150 amino acids. In contrast to heme, the globin moiety differs 

 among species and frequently occurs in more than one form within a species. 



Establishment of homogeneity or heterogeneity of hemoglobin may require the 

 use of many physical and chemical methods, since small interspecies variations fre- 

 quently cannot be revealed by use of a single method. Intraspecies heterogeneity 

 is often more difficult to demonstrate; however, the genetic implications that might be 

 derived only through complete characterization of intraspecies differences become 

 the reward for the added effort required to elucidate such variations. Ultimately, 

 only a complete analysis of the amino-acid sequence will establish unequivocally the 

 homogeneity or heterogeneity of some intraspecies hemoglobin variants. Use of pooled 

 samples should be avoided if at all possible, since properties of individual samples may 

 be obscured. However, pooling of samples may be permitted if strains of laboratory 

 animals are used in which homogeneity of individuals has been established. 



The objectives of the investigator should be decided at the outset, since the informa- 

 tion being sought usually dictates the analytical procedures. Simplicity is desirable, 

 but simple methods frequently yield equivocal results. Every method has its limitations 

 and sources of error, and the investigator should be acquainted with them. Moreover, 

 hemoglobin is a relatively labile protein and may therefore be altered during some 

 preparative or analytical procedures. Hence, it may be necessary on occasion to 

 establish that minor hemoglobin variations observed with use of a particular technique 

 reflect actual hemoglobin differences per se and are not modified forms or complexes 

 of hemoglobin with other molecules, such as haptoglobins. The validity of the results 

 is strengthened, however, if similar results are obtained by more than one method. 



For the purposes of presentation, the methods are classified according to whether 

 they are used to study a physical or a chemical property of the molecule; some methods 

 could be placed under either category. An evaluation and discussion of each method is 

 presented, and several references are cited for the application of each method to the 

 analysis of mammalian hemoglobins. 



PHYSICAL METHODS 



Electrophoretic analysis 



Electrophoresis is commonly used to determine the isoelectric point and electro- 

 phoretic mobility of biological materials. Knowledge of the isoelectric point is of 



