Biochemical Genetics (II) 



287 



chains are coiled about each other in a regular 

 way. Each chain has an iron-containing 

 heme group fitting into a pocket on the outer 

 surface of the coil it makes. In the whole 

 hemoglobin molecule, therefore, there are 

 four heme groups, one for each of the two 

 a and two (3 chains, and a total of about 600 

 amino acids. Henceforth, we shall be con- 

 cerned with the protein, or globin, part of 

 this molecule, since the heme groups are not 

 involved in the variations to be considered. 



It has been possible to partially digest the 

 globin portion of the molecule with trypsin, 

 an enzyme which splits a polypeptide at every 

 place where either of the amino acids lysine 

 or arginine is present. This produces some 

 28 smaller polypeptides, or peptides, in dupli- 

 cate (since there are two chains of each type), 

 plus an undigested core that makes up about 

 25% of the globin. The 28 peptides can be 

 separated from each other by their differential 

 migration on filter paper when the digest 

 containing them is subjected to an electrical 

 field and various solvents. The result is that 

 there are separate spots or "fingerprints" for 

 each of the peptides on the filter paper 

 (Figure 32-3). Each peptide (fingerprint) is 

 given a different number, and can be further 

 analyzed as to its amino acid content. Pep- 

 tide 4, for example, normally contains eight 

 amino acids in the following sequence: 



Val-His-Leu-Thr-Pro-G/w-Glu-Lys ' 



There is also evidence that the valine is an 

 end amino acid in the /3 chain. This sequence 

 is found in normal adult hemoglobin, called 

 hemoglobin A. The core of globin can be 

 digested with chymotrypsin and fingerprints 

 obtained of its peptides. 



Persons heterozygous for the recon for 

 sickling have the "sickle cell trait," which is 

 readily detected when their red blood corpus- 

 cles are exposed to an oxygen tension very 

 much lower than normal, while persons 



"• Valine, histidine, leucine, threonine, proline, glu- 

 tamic acid, glutamic acid, lysine. 



homozygous for this mutant have "sickle cell 

 anemia," and their red cells will sickle even 

 when the oxygen tension is not so drastically 

 reduced. The hemoglobin of such people 

 has been fingerprinted and analyzed as to 

 amino acid content. The mutant homo- 

 zygote has hemoglobin apparently identical 

 with hemoglobin A, with the proven excep- 

 tion that the sixth amino acid in peptide 4 

 has valine substituted for glutamic acid (the 

 particular amino acid italicized in the pre- 

 viously given sequence) (Figure 32-3). The 

 heterozygote produces both this type of ab- 

 normal hemoglobin, called hemoglobin S, and 

 hemoglobin A. Previous study of the pleio- 

 tropism of the recon for sickling (see Chapter 

 10) showed that all its phenotypic effects are 

 traceable through a pedigree of causes to this 

 single amino acid substitution in the /3 chain. 

 This is excellent evidence that a change in the 

 specification of a single polypeptide has been 

 produced as a result of the change in the pri- 

 mary action of a cistron whose nature had 

 been changed previously by mutation. 



Another mutant is known that is located 

 on the same chromosome as the recon for 

 sickling and is probably an allele of it. This 

 produces hemoglobin C which differs from 

 hemoglobin A by replacing the same glutamic 

 acid in the chain, this time by lysine. 



Still another genetic change produces an- 

 other hemoglobin, hemoglobin G. The amino 

 acids in all the trypsin-produced peptides are 

 the same as in hemoglobin A, except that the 

 seventh one from the end in peptide 4 is 

 glycine instead of glutamic acid. In this case, 

 then, the amino acid sequence in peptide 4 is: 



Val-His-Leu-Thr-Pro-Glu-G/y-Lys 



Here, then, an amino acid in a different posi- 

 tion is changed in the /3 chains. In hemoglobin 

 E, a glutamic acid, normally found in pep- 

 tide 26, is replaced by lysine, and it is likely 

 that this is the only change in the whole mole- 

 cule. Peptide 26 is also part of the ^ chain. 

 While we have seen that there are mutants 



