W. A. Davis, A. J. Daish and G. C. Sawyer 263 



the sample were first pulped in a special machine and then subjected to 

 pressure so as to obtain the sap. Brown and Morris [1893], working 

 with Troj)ceoluni leaves, found in their preliminary experiments that 

 considerable changes may occur in the sugars when the sample is 

 prepared in this way, and Kluyver [1914] states that on estimating the 

 cane sugar and reducing sugars in the sap of the same leaf he found 

 practically no cane sugar, unless the juice were heated to 100° to 

 destroy the enzymes before making the analysis. Brown and Morris 

 in 1893, and Parkin [1912], and Kluyver more recently, therefore 

 always attempted to destroy the enzymes in the leaves by quickly 

 drying these in a steam oven. Parkin gives analyses which would 

 indicate that in the case of the snowdrop this method of working brings 

 about little change in the proportion of the sugars in the leaf. On the 

 other hand, when the leaves are moderately thick, as in the mangold 

 or sugar beet, and therefore heat up slowly, an opportunity is given to 

 the enzymes to bring about considerable change in the carbohydrates 

 before they are actually destroyed ; enzyme action is especially likely 

 to occur under these conditions, because the rate of action is greatly 

 accelerated at first by the rising temperature. We shall show (see p. 3.57) 

 that certain fundamental differences between our results and those of 

 Brown and Morris and of Kluyver (for example, the entire absence of 

 maltose in all the cases we have studied and our higher values for 

 starch in the case of the Tropcpolum leaf) are probably to be explained 

 by enzyme action having taken place in the earlier experiments. 



In all investigations hitherto, the material which was analysed was 

 either pressed-out sap or an extract of the previously dried tissue. 

 To ensure the destruction of the enzymes being as instantaneous as 

 possible, we have adopted the following procedure. The freshly 

 picked leaf material (about 1 kilogram) was dropped in small quantities 

 at a time into a large volume (2 litres) of boiling alcohol, to which 1 per 

 cent, by volume (20 cc.) of 0-880 ammonia was added so as to neutrahse 

 the acids present in the leaf ; the quantity given is generally sufficient 

 for this purpose. After the extraction with alcohol is complete, the 

 solution is generally faintly alkaline to Htmus paper; if this is not the 

 case a little more ammonia should be added. By the method given it 

 would appear that the enzymes in the plant tissue are instantly 

 destroyed ; the ammonia facilitates the destruction owing to its alkaline 

 nature and its rapid diffusion into the plant cells. The nature of the 

 results we have obtained gives us confidence that no changes occur in 

 the leaf carbohydrates during this treatment or during the subsequent 



