Tyrosine by a Putrefactive Anaerobe. 



49 



brain, salmon, milk and suspended casein mediums not containing 

 fermentable carbohydrates, i.e., monosaccharides in excess. The 

 early stages of incubation are marked by clouding and vigorous 

 gas production. The crystals begin to appear in 4-6 days at 

 37 0 C. along with a visible liquefaction of the protein as well as 

 odorous evidence of putrefaction. Meat and brain mediums are 

 blackened presumably owing to the precipitation of iron sulfide 

 by the action of sulfuretted hydrogen upon the iron freed by 

 proteolysis. Milk, salmon and casein mediums are not blackened, 

 although sulfuretted hydrogen is produced, except upon the addi- 

 tion of iron ions, as by the inclusion of an iron nail. These facts 

 fail to support a suggestion that the blackening of certain proteins 

 by putrefactive anaerobes parallels the supposed action of a 

 tyrosinase in transforming tyrosin into melanin in the animal 

 body. 



The recovery of the crystals in a pure form involves the removal 

 of the water-soluble constituents by washing the partially digested 

 culture mediums with cold water, extraction of the crystals by 

 boiling water, with or without the addition of ammonia, followed 

 by hot filtration to remove undissolved proteins, concentration 

 of the filtrate by boiling, crystallization from the concentrated 

 filtrate by cooling, removal of the crystals by cold filtration and 

 repeated clarification with animal charcoal in boiling water alter- 

 nated with crystallization in the cooled filtrate. Impurities 

 soluble in cold water are removed by washing at each cold filtra- 

 tion and the crystals are dried after rinsing with 95 per cent, 

 alcohol followed by ether. 



Quantitative methods of extraction are yet to be devised ; there 

 is considerable loss at each step excepting in the treatment with 

 ether. The sample displayed, about 0.8 gram, represents the 

 purified product from several liters of culture. 



The crystals are identified as tyrosine by their physical and 

 chemical properties. To the naked eye they appear as snowy 

 white flakes with a silken sheen, the individual needles barely 

 visible. These flakes may be readily suspended in alcohol or 

 distilled water; in the latter particularly the characteristic crystals 

 may be seen with a hand lens as colorless double-pointed needles. 

 When allowed to crystallize slowly from somewhat dilute hot 



