IX. GENETICS AND HUMAN HEMOGLOBIN CHEMISTRY 409 



III. Normal Hemoglobins 



A. Hb-A 



Hb-A is the normal inajoi- c'oin]ioiU'nt of henioglohiii prepared from 

 the red cells of adult individuals. Hb-A is usually defined by its electro- 

 phoretic mobility, the isoelecti'ic i)oint being at pH 6.87, and by its rate 

 of alkali and acid denaturation. Most of the i)hysical and chemical 

 properties established by the protein chemists in the past for human 

 hemoglobin can be referred to Hb-A. Rhinesmith et al. (1957a) deter- 

 mined the N-terminal residues of Hb-A; four valine residues were ob- 

 tained indicating the presence of four jieptide chains. Rhinesmith et al. 

 (1957b) next found that two peptide chains on acid hydrolysis release 

 quickly the N-tcrminal dipeptide valijl-leucine and two chains release 

 valine only more slowly; these peptide chains have been designated a 

 chains and p chains, respectively. The N-terminal sequence of the latter 

 has been found to be Val.His.Leu. (Rhinesmith et al., 1958).- 



The presence of two types of peptide chains in Hb-A, demonstrated 

 by Rhinesmith et al. (1958), has been of particular importance in solving 

 some of the genetic and chemical problems presented by the abnormal 

 hemoglobins. The idea of a molecule composed of two pairs of identical 

 chains agreed particularly well with the X-ray crystallographic picture 

 of the horse hemoglobin molecule, obtained by Perutz and his collab- 

 orators (see Perutz et al., 1960). The horse hemoglobin molecule has the 

 shape of an ellipsoid composed of two identical parts. In each half- 

 molecule there are two different peptide chains. This type of structure is 

 probably common to all mammalian hemoglobins. A rather schematic 

 representation of the hemoglobin molecule is shown in Fig. 1. 



Wilson and Smith (1959) obtained pure preparations of the two 

 peptide chains of horse hemoglobin by ion exchange chromatography 

 in a urea gradient and by preparative boundary electrophoresis. Ingram 

 (1959a) applied the same methods to the preparation of the peptide 

 chains of human adult hemoglobin; two fractions were separated by free 

 boundary electrophoresis and l)y column chromatography. 



The fractions isolated by column chromatography were examined by 

 "fingerprinting." In the fingerprinting analysis a mixture of peptides, 

 obtained by tiyptic hydrolysis of denatured hemoglobin, is separated 

 by paper ionophoresis, followed by paper chromatography in a perpen- 

 dicular direction. The peptides are spread on paper sheets in character- 



■ Amino acids are indicated by the first tliree letters of their names. Asparagine 

 and gkitamine are indicated as Asp-NH2 and Gki-NH-;, respectively, in the text, 

 but as Asn and Gin in the illustrations. Isoleucine is indicated as Ilou. 



