1912.] 



Studies on Enzyme Action. 



113 



literature of the subject is very fully brought together and discussed in 

 Eeynolds Green's ' Soluble Ferments and Fermentation ' and similar works. 



No simpler case of hydrolysis by an enzyme than that of urea is known. 

 On this account we have long desired to include the interaction of urea and 

 urease in our series of studies of enzymes. At various times, we have 

 attempted to obtain the necessary enzyme for the work but the difficulties 

 we encountered have been greater than we anticipated : little progress was 

 made, therefore, until we were able to avail ourselves of the discovery made 

 by Takeuchi, in 1909,* that the Soja bean {Glycine kispida) is particularly 

 rich in urease. 



Preparation of Enzyme. — To prepare a solution of the enzyme, the Soja 

 beans are twice ground in a coffee mill and the meal extracted with petroleum 

 spirit, the extraction being repeated six times ; usually the petroleum is left 

 in contact with the meal during about 45 minutes and then removed by 

 nitration under reduced pressure. The operation is carried out in a large 

 glass separating funnel. The washed meal is spread out during a night on 

 glass plates to allow adherent petroleum to evaporate. Next morning it is 

 mixed with five times its weight of water and a little toluene. After 

 24 hours, the liquid is separated from the meal by filtration through a large 

 Buchner funnel ; from 60 to 70 per cent, of liquid is recovered. 



The solution thus prepared has a considerable degree of activity, 20 c.c. 

 mixed with an equal volume of a solution containing about half a gramme of 

 urea sufficing to hydrolyse the urea within half an hour at 20°. 



The solution contains a large amount of albuminous matter. The 

 endeavour has been made to purify the enzyme by precipitating with alcohol 

 and extracting the precipitate with water ; as the extract prepared in this 

 manner is very weak in comparison with the new extract, we have preferred 

 to use the latter. 



The fresh extract is faintly acid ; when kept, it increases in acidity and 

 a coagulum is deposited ; at the same time, its hydrolytic activity falls, the 

 fall being considerable when the precipitation sets in. The changes that 

 take place in the activity of the solution will be considered later on. 



Method of Determining Activity of Enzyme. — We have found the simplest 

 method of determining the activity of urease to be to digest the solution of 

 urea with the enzyme and subsequently neutralise the ammonia that is 

 formed by an excess of decinormal acid, then boil during two to three minutes 

 to expel carbon dioxide, finally titrating with baryta solution. The errors 

 incidental to this method do not appear to be greater than those associated 

 with more elaborate methods. 



* 'J. Coll. Agric. Imp. Univ. Tokyo,' 1909, vol. 1, pp. 1—14. 

 VOL. LXXXV. — B. I 



