412 



CHAPTER 32 



What bearing have these results upon the 

 genera] hypothesis o\ one gene-one primary 

 phenotypic effect? Although the general hy- 

 pothesis is unaffected, the specific hypothesis 

 to test it — one enzyme-one gene — should be 

 made more comprehensive and should be 

 stated as one polypeptide-one gene, meaning 

 that the eomposition of a polypeptide chain 

 is completely determined by one gene. Ac- 

 cording to the general hypothesis, then, the 

 primary effect of at least some genes is to 

 specify completely the amino acid content 

 of a polypeptide. If the one polypeptide- 

 one gene hypothesis is correct, we expect 

 every polypeptide chain in every protein — 

 including proteins that are not enzymes — to 

 be completely specified by the primary and 

 solitary action of a single gene. 



Biochemical Genetics of Hemoglobin 



In man, hemoglobin 4 is a protein with a 

 molecular weight of about 66,700. In the 

 horse (and probably in man) the shape of 

 the molecule is spheroidal; its dimensions 

 are 55 by 55 by 70 A; and it is composed 

 of two dimers. Each dimer is composed of 

 two identical polypeptide chains and the 

 polypeptides in the two dimers are usually 

 different. Each of the four monomeric 

 chains contains about 140 amino acids and 

 has a molecular weight of about 1 7,000. 

 The chains partly coil to form what are 

 called right-handed helices, and different 

 chains are coiled about each other in a reg- 

 ular way. An iron-containing heme group 

 fits into a pocket on the outer surface of the 

 coil of each chain. In the whole hemoglobin 

 molecule, therefore, there are four heme 

 groups — one for each of the chains — and a 

 total of about 560 amino acids. Since the 

 heme groups are not involved in the varia- 

 tions to be considered, we shall henceforth 



4 Based upon the work of V. M. Ingram, L. Paul- 

 ing. H. A. Itano, H. Lehrmann. J. V. Neel. M. F. 

 Perutz, and others. 



be concerned only with the protein, or globin, 

 part of the molecule. 



Hemoglobin isolated from normal adults 

 contains three components: A (or A,), A L ., 

 and A ;! . The A component, called hemo- 

 globin A (Hb-A) comprises about 90% 

 of the total hemoglobin and the A 2 compo- 

 nent (Hb-Aj) about 2.5%. The remain- 

 ing percentage of about 7.5 is due to the 

 A. t component, probably Hb-A that has be- 

 come chemically altered during aging of the 

 red blood corpuscles. 



Hemoglobin A. In vitro, Hb-A can be 

 dissociated into the two kinds of homo- 

 dimer. and can be reassociated to reform 

 the Hb-A tetramer."' Since the monomers 

 are called « A and /i\ the reversible reaction 

 can be written « A p£ ^± a A + /3 2 v . The 

 globin part of the molecule can also be par- 

 tially digested with trypsin, which specif- 

 ically cleaves the peptide bonds between the 

 carboxyl group of lysine or arginine and the 

 amino group of other amino acids. This 

 digestion produces 28 smaller polypeptides, 

 or peptides, in duplicate (since there are 

 two chains of each type), plus an undigested 

 core composed of about 25% of the orig- 

 inal globin. The 28 peptides can be sepa- 

 rated from each other since, on filter paper, 

 they migrate at different rates when the 

 digest containing them is subjected to an 

 electrical field and various solvents. This 

 treatment results in separate spots — "finger- 

 prints" — on the filter paper for each of 

 the peptides (Figure 32-5); each peptide 

 (fingerprint) is given a different number 

 and then analyzed for amino acid content. 

 Peptide 4, for example, normally contains 

 eight amino acids in the following sequence: 

 Val-His-Leu-Thr-Pro-G/M-Glu-Lys. . . . 6 

 The core of globin can be digested with 

 chymotrypsin and fingerprints obtained of 



5 See also G. Guidotti, W. Konigsbcrg. and L. C. 



Craig (1963). 



,; These abbreviations are explained in Figure 



32-4. 



