222 



AMINO ACIDS, PEPTIDES AND PROTEINS 



When alpha imino acids, such as proline, are reacted with ninhydrin, a yellow pigment 

 is usually obtained. In this reaction carbon dioxide is also formed and the pigment is 

 believed to have structure IV. 



N 



REACTIONS WITH AMINO ACID SIDE CHAINS 



Specific tests, which in some cases are quantitative, have been developed for several 

 amino acids. These depend on differences in the structure of the side chains for their 

 selectivity and can be applied to peptides and proteins. Thus, the guanidino group of 

 arginine permits the colorimetric determination of this compound by reaction with sodium 

 hypochlorite and alpha naphthol in alkaline solution (61). Histidine and tyrosine are con- 

 verted to azo dyes by treatment with diazotized sulfanilic acid (62). Tyrosine also gives 

 a positive Millon test, a red precipitate with mercurous nitrate in nitric acid, a reaction 

 which is specific for phenols containing a free ortho position (63). Cysteine, due to the 

 presence of a sulfhydryl group, gives a transient purple violet color with sodium nitro- 

 prusside, Na2[ Fe(CN)5(NO)] in the presence of base (64). Tryptophan yields various 

 colored products on reaction with various aldehydes in the presence of sulfuric acid (65). 



On treatment with a 1% solution of tert-butyl hypochlorite in cyclohexane, aeration 

 in a stream of air and spraying with an aqueous solution of potassium iodide and starch, 

 peptides, acyl amino acids and proteins develop blue spots against a white baclcground 

 (66). This test is applicable to any amino derivative that contains a N-H bond. It should 

 be added that most of the reactions described above have been applied to paper chroma- 

 tography. 



SPECIFIC REACTIONS OF PROTEINS 



The specific tests listed in the above section for amino acids and peptides are of 

 course also given by many proteins. Sometimes it is found that these reactive side 

 chains are more accessible after a protein has been denatured. In addition to these re- 

 actions there are a few that are more or less diagnostic for proteins. The biuret test is 

 given by proteins and other substances that contain two amide groups either joined directly 

 or through a single atom of nitrogen or carbon (67). The reagent consists of copper sul- 

 fate in concentrated sodium hydroxide, and a positive test is indicated by a pink or purple 

 color. The action of many agents in converting soluble proteins to insoluble products is 

 also useful. General precipitants are heavy metal ions (mercuric chloride, silver nitrate, 

 lead acetate), "alkaloidal reagents" (picric acid, phosphotungstic acid, tannic acid), and 

 concentrated salt solutions (ammonium sulfate, sodium chloride and sodium sulfate). 



Molecular weight determinations are of extreme importance in work with proteins. 

 Many different methods have been developed. Osmotic pressure measurements can be 

 used but since they give number average molecular weights a small percentage of a low 

 molecular weight impurity produces a rather large error. Light scattering procedures 

 and ultracentrifugation, methods that yield weight average molecular weights, are used 



