220 



AMINO ACIDS, PEPTIEDS AND PROTEINS 



When the filtered and centrifuged extract was diluted with four volumes of water and 

 stored at 2° C, crystalline protein precipitated. Ammonium sulfate precipitations are 

 also employed very frequently. Other techniques that occupy important places in many 

 fractionation schemes are dialysis and the use of organic solvents as precipitants. 



In these cases in which those relatively simple techniques do not prove satisfactory, 

 the protein chemist can turn to more sophisticated methods. Ultracentrifugation (51) and 

 electrophoresis (52) have proven their value in many separation problems. Chromato- 

 graphic methods based on the use of modified cellulose derivatives such as diethylamino- 

 ethyl cellulose have been shown to be extremely effective (53). Separations with these 

 columns are to a very large extent due to ion exchange interactions. The protein concen- 

 tration in the eluate can frequently be estimated by ultraviolet scanning near 280 millimi- 

 crons due to the presence of aromatic amino acids in many proteins. 



With the simpler organic solids, crystallinity can usually be taken as a safe criterion 

 for homogeneity. Unfortunately, this is not true for proteins since many crystalline prep- 

 arations can be shown to be heterogeneous according to other tests. The purity of an 

 isolate can be tested by phase solubility studies, electrophoretic separations at different 

 pH values, ultracentrifugation and chromatography. Only after application of each of 

 these tests has indicated lack of heterogeneity can it be concluded that according to these 

 criteria the isolate is homogeneous. 



CHARA CTERIZA TION 



At present the best-known method for rapid identification of the amino acids in a mix- 

 ture is paper chromatography. Usually two dimensional chromatography is needed for 

 good separations of complex mixtures. The required apparatus is inexpensive, the ma- 

 nipulations can be learned rapidly, and in most cases as little as 5-10 micrograms of a 

 given amino acid can be detected routinely. The Rf values for a large number of naturally 

 occurring and synthetic amino acids have been compiled (23, 10b), and some of these are 

 presented in Table 1. The most popular developing solvents seem to be 1-butanol/acetic 

 acid/ water mixtures and water -saturated phenol. A 0. 1% solution of ninhydrin in butanol 

 or ethanol is almost universally used as the detecting reagent. Spots show up after stand- 

 ing at room temperature for several hours or in a few minutes if the paper sheet is heated 

 to 100° C. after spraying. Thin layer chromatography on silica gel (54) and paper electro- 

 phoresis (55) are also effective methods for amino acid analysis. Further characteriza- 

 tion of the amino acids, peptides and proteins is based on chemical reaction with the 

 amino group, the specific side chains, the carboxyl groups and the amide bonds. Mole- 

 cular weight determinations are also used frequently. Many of the older procedures are 

 assuming new significance due to their application to chromatographic techniques. 



REACTION WITH THE AMINO GROUP 



Several quantitative procedures have been developed which are based on interactions 

 with the amino group. One very useful method for the estimation of total amino nitrogen 

 of proteins, peptides and free amino acids of a plant extract is a micro adaptation of the 

 Kjeldahl method (56). The nitrogen-containing material is digested with concentrated 

 sulfuric acid in the presence of catalysts yielding ammonium salts. The digestion mix- 

 ture is made alkaline and the resulting ammonia distilled into standard acid and deter- 

 mined quantitatively by titration. To obtain a rough estimate of the total percent protein, 

 etc. , the percentage nitrogen is multiplied by the factor 6. 25. 



The fact that primary amines react with nitrous acid to yield nitrogen forms the basis 

 of the manometric Van Slyke method for the estimation of amino acids (57). 



