138 THE PROTEINS AND PROTEASES [ch. 



the amino-acids then accumulate and can be detected. Of all the amino- 

 acids the one which is most readily identified is tryptophane. If the autolyzed 

 product is boiled, acidified and filtered to remove the remaining proteins, 

 and, to the filtrate, bromine is added, drop by drop, the formation of a 

 pink or purple colour will indicate the presence of free tryptophane, and 

 hence it may be assumed that protein-hydrolysis has taken place. 

 Probably the formation of amino-acids in autolysis is a universal property 

 of plant tissues, for tryptophane has been detected on autolysis of many 

 different parts of plants. Examples are the germinating seeds of the 

 Bean (Vicia Faha), Scarlet Runner (Phaseolus inultiflorus), Pea {Pisum 

 sativum), Lupin (Lupinus kirsutus) and the Maize (Zea Mays) : and in 

 ungerminated seeds of the above, though less readily. It is also said to 

 be formed on autolysis of leaves of Spinach {Sjnnacia), Cabbage (Brassica), 

 Nasturtium (Tropaeolum tnajus), Scarlet Geranium {Pelargonium zonale). 

 Dahlia (Dahlia variabilis) and others : also of fruits of Melon (Cucumis 

 Melo), Cucumber (Cucumis sativus), Banana (Musa sapientum), Tomato 

 (Lycopersicum esculentum) and others: of bulbs of the Tulip (Tulipa), 

 Hyacinth (Hyacinthus orientalis) and underground roots of Turnip 

 (Brassica), Carrot (Daucus Carota) and Beet (Beta vulgaris) (Vines, 

 16-24; Blood, 3; Dean, 5, 6). 



E.vpt. 137. The formation of tri/ptophane on autolysis of resting seeds. Grind up 

 in a coffee-mill 15 gms. of Mustard {Brassica alba) seed. Transfer to a flask, and add 

 100 c.c. of distilled water and about 2 c.c. of toluol. Plug the mouth of the flask 

 ■with cotton- wool and put in an incubator for 3 days. Then filter off' the liquid, boil 

 the filtrate and add a few drops of acetic acid. Filter off" any precipitate formed, 

 cool the filtrate and add bromine water slowly and carefully drop by drop, shaking 

 well after each drop. A pink or purple colour denotes the presence of tryptophane. 

 Excess of bromine will destroy the colour. Then shake up with a little amyl 

 alcohol. The purple colour will be extracted by the amyl alcohol which will rise to the 

 top of the water solution. A control experiment should be made using 10 gms. of 

 seed which has been well boiled with water in an evaporating dish. 



It has been assumed that the formation of amino-acids from proteins 

 on autolysis is the outcome of two processes, the hydrolysis of proteins 

 to peptones by pepsins, and the hydrolysis of peptones to amino-acids 

 by erepsins. 



The next point to be considered is the possibility of detecting these 

 two classes of enzymes separately. If either the pulp, or water extract, 

 of various plant tissues be added to peptone solution and allowed to 

 incubate at 38° C, tryptophane can be readily detected after a day or 

 two. This has been found to be true for the tissues of many seeds, 

 seedlings, roots, stems, leaves and fruits (such as those already mentioned 



