2 J^g AMINO ACroS , PEPTIDES AND PROTEINS 



The contributions to the final structure of a protein made by the covalent linkages 

 are frequently termed the primary structure; and although they are of course extremely 

 important, they do not by themselves fix the conformation of a protein in solution. To 

 describe the structure of a protein completely one must also take other bonding forces 

 into account. These are referred to as the secondary and tertiary structure. Informa- 

 tion about these forces comes from such physical chemical measurements as X-ray de- 

 fraction patterns, optical rotatory dispersion measurements, spectral data and deuterium- 

 hydrogen exchange experiments. Proline, by virtue of its cyclic imino group, also plays 

 a role in the determination of the final structure of proteins. 



Hydrogen bonds between the amide links are believed to make major contributions to 

 the secondary structure of proteins. One manifestation of these forces is the presence 

 of helical structures in certain proteins. A spiral arrangement that accommodates pep- 

 tide chains in a strain-free form and which is believed to play an important role in the 

 conformation of proteins is the so-called alpha helix (36). This configuration is a right 

 handed helix for the L-amino acids, held together by hydrogen bonds between the NH 

 group of one amide and the 0=C group of another amide link four units up the chain. 

 There are 3. 6 residues per turn and the pitch is 5. 4 Angstroms. The hydrogen bonds are 

 in the direction of the fiber axis, and the side chains of the constituent amino acids are 

 at the outer periphery of the helix. The amount of helical content of different proteins 

 varies quite extensively and is maintained intact through the disulfide bridges and a favor- 

 able tertiary structure. The tertiary structure is due to still weaker interactions; Van 

 der Waals forces, solvent -protein interactions and hydrophobic bonds seem to play a 

 predominant role in these stabilization forces. 



Correlation of the results from many different types of structural data obtained for 

 the enzyme ribonuclease has permitted the construction of a tentative three-dimensional 

 model for this protein as it might exist in aqueous solution (37). The construction of a 

 three-dimensional working model of an enzyme, even if in the final analysis it should 

 prove incorrect, demonstrates very effectively the great advances that have been made. 



ISOLATION 



AMINO ACIDS AND PEPTIDES 



Before the advent of ion exchange and partition chromatography most amino acids 

 were isolated from specific protein hydrolysates or from the amino acids present in the 

 tissues of particular plants or animals. Thus asparagine, the first amino acid to be iso- 

 lated from nature was obtained by concentration of asparagus juice (1). Aspartic acid can 

 be prepared from asparagine by acid hydrolysis. Glutamic acid is obtained from acid 

 hydrolysates of wheat gluten. Isoleucine was isolated from sugar beet molasses. Phenyl- 

 alanine and arginine were isolated from etiolated lupine seedlings. Arginine can also be 

 isolated as the relatively insoluble mono or diflavianate (38). The other common amino 

 acids can in most cases be conveniently isolated from animal sources. 



The isolation of the newer plant amino acids generally requires more sophisticated 

 techniques, since these compounds are present in the cell sap together with a large num- 

 ber of other cell constituents and frequently occur only in small amounts. Almost every 

 case presents unique problems that have to be solved on an individual basis. However, 

 ion exchange processes in one form or another are often extremely useful. These pro- 

 cedures are particularly desirable for the concentration of amino acids, the removal of 

 neutral contaminants and the subdividsion of the amino acids into neutral, acidic, and 

 basic fraction (39). After preliminary concentration and purification steps have been 

 taken, it may be necessary to separate the desired amino acids from a number of closely 

 related compounds. For this purpose chromatographic methods are frequently used. A 

 typical example that illustrates these procedures is the isolation of pipecolic acid from 

 green beans (40). 



