1012 APPENDIX. 



human blood a precipitin is produced in the animal's blood 

 which causes a precipitate when mixed with human blood or 

 with that of some of the higher monkeys, but gives no reaction 

 with the blood of other mammals. The reaction may be used, 

 therefore, in a measure to test the blood-relationship of different 

 animals.* It has been suggested that the reaction may also be 

 of practical importance in medicolegal cases, in determining whether 

 a given blood-stain is or is not human blood. For such a pur- 

 pose a human antiserum is first produced by injecting human 

 serum into a rabbit. The serum of the rabbit is then mixed with 

 an extract of the suspected blood-stain made with salt solution; 

 if a precipitate forms it proves that the blood stain is human blood 

 provided the possibility of its being monkey's blood is excluded. 

 Concerning the nature of the precipitins, little is known. They 

 combine quantitatively with the protein precipitated and they 

 are inactivated (hematosera) by a temperature of 70 C. Their 

 reactions are not sufficiently specific to be used as a means of de- 

 tecting or distinguishing closely related proteins. 

 II. The Color Reactions of Proteins. 



I. The biuret reaction. The protein solution is made strongly alkaline 

 with caustic soda or potash and a few drops of a dilute solution of 

 copper sulphate are added carefully so as to avoid an excess. A 

 purple color is obtained. Some proteins (peptones) give a red 

 purple, others a blue purple. If only a blue color, without any 

 mixture of red, is obtained, no protein is present. At present 

 this reaction gives the best single test for protein. It obtains 



its name from the fact that it is given by biuret HN<Q^Tr-, a 



compound that may be formed by heating urea. Two molecules 

 of urea give orf a molecule of ammonia and form biuret. 



2. The ninhydrin reaction. Proteins and some of their split products 



give a blue reaction with ninhydrin (triketohydrindene hydrate). 

 This reaction is especially useful in testing for the amino-acids or 

 peptids when the hydrolysis of the protein has gone so far that a 

 biuret reaction is no longer given. One to two drops of the reagent 

 are added to 1 c.c. of the suspected liquid, and the solution is heated 

 to boiling. On cooling, a blue color develops if a-amino-acids or their 

 derivatives are present. 



3. The Millon reaction. The protein solution is boiled with Millon's 



reagent. The solution or the precipitate, if one is formed, takes 

 on a reddish color, which varies in intensity with different proteins. 

 Millon's reagent consists of a solution of mercuric nitrate in nitric 

 acid containing some mercurous nitrate. This reaction is supposed 

 to be given by the tyrosin (oxy-aromatic) grouping in the protein 

 molecule, and fails, therefore, with those proteins in which tyrosin 

 is not present. 



4. Tyrosin reagent. Folin and Denis f report a new color reaction for the 



detection of phenol compounds which may bd used for the quantita- 

 tive determination of tyrosin. The reagent consists of sodium tungs- 

 tate, 10 per cent.; phosphomolybdic acid, 2 per cent.; and phosphoric 

 acid, 10 per cent. After addition of the reagent the blue color is 

 developed by further addition of an excess of a saturated solution of 

 sodium carbonate. 



5. The xanthoproteic reaction. Nitric acid is added to strong acid 



reaction and the solution is then boiled. After cooling, ammonia 

 is added. The ammonia causes the development of a deep-yellow 

 color if protein is present. This reaction is supposed to be due 

 to the presence in the molecule of the groupings belonging to the 

 aromatic series. 



* For many interesting experiments and the literature, seeNuttall, "Blood 

 Immunity and Relationship," Cambridge, 1904. 



t Folin and Denis, "Journal of Biological Chemistry," 12, 240, 1912. 



