AMINO ACIDS, PEPTIDES AND PROTEINS 2 19 



An aqueous extract of 26 gallons was prepared from 175 pounds of fresh green beans. 

 The extract was concentrated to six liters and a one liter fraction was adsorbed on an 

 eight hundred gram column of cation exchanger in the hydrogen form. Washing of the 

 column with distilled water removed the sugars and other non-ionic material. The col- 

 umn was then treated with seven liters of 2N-hydrochloric acid. This removed eight 

 contaminating amino acids but resulted in the retention of pipecolic acid, gamma-amino 

 butyric acid and the basic amino acids on the column. The latter compounds were eluted 

 with six liters of 0. 5 N and two liters of 1 N ammonium hydroxide. Examination of the 

 eluate by paper chromatography located the fractions that contained the bulk of the pipe- 

 colic acid. These were combined, the solvent evaporated and the pipecolic acid crys- 

 tallized as the hydrochloride. A total of 60 mg. of chromatographically pure, crystalline 

 material was obtained. Later modifications resulted in an improved procedure which 

 permitted the isolation of 13. 4 g L-pipecolic acid from about 150 pounds of green beans 

 (41). 



The isolation of gamma aminobutyric acid from potato tubers illustrates a very ele- 

 gant method of separating a non-alpha amino acid (42). The alpha amino acids form 

 stable chelates with divalent cations, whereas the non-alpha acids do not. This difference 

 can be utilized to effect separation of non-alpha amino acids from the alpha amino acids 

 by using a column consisting of alumina and copper carbonate. With this technique the 

 alpha amino acids are retained on the column under conditions which cause elution of the 

 non-alpha amino acids. 



Quantitative amino acid determinations are most conveniently carried out by column 

 procedure utilizing chromatography with ion exchange resins (43). Completely automatic 

 recording analyzers are available commercially. Another inovation is the use of volatile 

 buffers as eluting solvents for ion-exchange columns (44). With these solvents the amino 

 acids are frequently obtained in a very high state of purity after evaporation of the sol- 

 vents. Mention should also be made of procedures which combine ion exchange resins 

 with filter paper partition chromatography (45). Other methods that can be used for sep- 

 aration or isolation of amino acids are paper electrophoresis (46) and vapor phase chro- 

 matography. In the latter procedure the amino acids are first converted to the N-acyl 

 esters and a procedure has been described for the separation of 18 amino acids that re- 

 quires only 90 minutes for acetylation and esterification and less than 60 minutes for 

 resolution (47). 



The isolation of peptides can usually be accomplished by the same techniques as those 

 used for amino acids. Countercurrent distribution (48) and membrane diffusion (49) ap- 

 pear to be particularly valuable for those compounds. 



PROTEINS 



Tremendous variations exist in the degree of difficulty encountered in the purification 

 of proteins. Some can be obtained in a homogeneous, crystalline state by exceedingly 

 simple procedures. For example, extraction of jack bean meal with aqueous acetone, 

 filtration, and storage of the filtrate near 0° yields a precipitate which after several re- 

 crystallizations consists of pure urease. On the other hand, cases are known where the 

 most persistent efforts of highly skilled operators have been fruitless. However, no 

 matter what procedure is finally adopted, it is of paramount importance that conditions 

 be employed which do not lead to denaturation of the protein. Generally it is advisable to 

 work at low temperatures and to avoid extreme pH ranges. It is also helpful to keep the 

 protein concentration as high as possible. 



Proteins can frequently be fractionated by control of the ionic strength of the fnedium 

 through the use of salts. For example, a crystalline globulin fraction has been obtained 

 from squash seeds by extraction of the ground seeds with 10% sodium chloride at 40° (50). 



