ENZYMES. FERMENTATION 423 



(e) Hatas Method. 



It was shown by Hata in 1909 that a suspension of coagulated 

 egg-white was a very delicate substrate for estimating the activity of 

 pepsin solutions. Under the influence of the enzyme the cloudy 

 solution becomes quite clear. This method also possesses the 

 advantage that egg-white is obtainable everywhere and the suspension 

 is readily prepared. The comparison is carried out by adding varying 

 quantities of enzyme solution to the substrate and observing in which 

 tube clarification is produced by the least amount of enzyme after a 

 given time. 



The substrate is prepared by rubbing up egg-white in a basin 

 until it is of a uniform consistency. It is then slowly mixed and 

 rubbed up with water until it has been diluted five times. The solu- 

 tion is strained through muslin and heated in a water-bath at 60 for 

 20 minutes, after which it is once more strained through muslin. A 

 homogeneous suspension is thus obtained. Before use it is diluted 

 with 9 volumes of water. 



5 c.c. of the above substrate are measured out into each of a series 

 of five test tubes. To each is added 5 c.c. of 0*4 per cent, hydro- 

 chloric acid solution and then in order, 0*2, 0*4, 0*6, O'8, and I c.c. of 

 pepsin solution. The tubes are placed in a water-bath at 40 for 1 5 

 or 30 minutes. It is noted in which tube in the series the smallest 

 amount of enzyme first produces complete clarification. 



(/) Gross 1 Method. 



Gross has suggested a solution of caseinogen instead of edestin for 

 estimating peptic activity. It is prepared by dissolving I gm. of pure 

 caseinogen in 1 6 c.c. of 25 per cent, hydrochloric acid of sp. gr. 1-124 

 in a 1000 c.c. flask on a water-bath and diluting to 1000 c.c. It is pre- 

 cipitated by a 20 per cent, solution of sodium acetate. 



A series of tubes are filled with varying quantities of the pepsin 

 solution from 'I to I c.c. To each IO c.c. of the caseinogen solution 

 warmed to 40 are added. The series is placed in a bath at 38-40 for 

 15 minutes. A few drops of the sodium acetate solution are added 

 to each tube; undigested caseinogen is precipitated. The smallest 

 quantity required to digest the 10 c.c. is the .value noted. 



If I c.c. of pepsin solution be the basis of the calculation and -025 

 c.c. were sufficient, the pepsin solution corresponds to >Tr | T or 40 units. 



Other methods for estimating pepsin are those of Hammerschlag, who 

 uses Esbach's reagent to precipitate unchanged protein ; of Volhard, who 

 digests caseinogen and titrates the amount of hydrochloric acid used up in 

 combination with caseoses ; of Jacoby, who digests the protein ricin in sus- 

 pension in dilute hydrochloric acid in a similar way to Hata. 



