junej. i9i8 Destntction of Tetanus Antitoxin 481 



quick and accurate results. Thus in experiment 6 the following were 

 obtained with 2 c. c. of the mixtures containing 0.4 c. c. of antitoxin X : 

 Mixture A, 43.3, 44.8 mgm.; mixture C, 43.8, 44, 44.8, 45.4 mgm.; 

 mixture B, 9.8, 10.2, 10.2, 10.5 mgm.; mixture D, 10.4, 10.5 mgm. In 

 this experiment an error was made by the addition of trypsin to mixture 

 B instead of C, resulting in two pairs of identical mixtures, one of which 

 contained only the antitoxin, the other trypsin-sodium-hydroxid in 

 addition. 



The most difficult step in the determination of coagulable protein is 

 the complete flocculation of the protein. The above-mentioned close 

 duplicates probably will not be obtained by the inexperienced or those 

 who do not realize the effect of small quantities of acid or alkali in this 

 determination. This point is well illustrated by the following data 

 obtained in experiment 20 (see Table III) : To 2 c. c. of mixture C con- 

 taining 0.4 c. c. of antitoxin 420F., 7 c. c. of water were added, and the 

 protein was flocculated with o.io c. c. N/50 acetic acid. The total 

 coagulable protein obtained was 19.4 and 20 mgm. The average of these 

 two, calculated to i c. c. of antitoxin, gave 49 mgm., the result given in 

 Table III. Two days later the determinations were repeated, but neither 

 acid nor alkali was used for flocculation. Obtained 22.5 and 23.3 m^-m., 

 which, calculated as before, gave 57.2 mgm. total coagulable protein per 



1 c. c. of antitoxin. So small a difference as o. i c. c. of N/^o acetic acid 

 made an appreciable difference in the result — that is, 8 mgm. 



It will be noticed that the figure for mixture C, 57.2 mgm., is appre- 

 ciably lower than that for mixture A, 63.5 mgm., as if 9 per cent of the 

 protein had been digested past the coagulable stage. Mixture C con- 

 tained pepsin only, and this in all probability exerted no digestive action 

 in the neutral solution. In general it was found that the addition of 

 pepsin or trypsin to a mixture lowered the amount of protein coagulated 

 when the coagulation was attempted immediately after the addition. 

 Thus, in experiment 3, eight 2-c. c. portions of mixture B were pipetted 

 into as many centrifuge tubes. Four were coagulated in the usual way 

 with 0.9 c. c. of N/3 acetic acid. There were obtained 22.1, 22.2, 24.0, 

 and 24.2 mgm. of coagulable protein. To the other four tubes weighed 

 portions of trypsin 4 were added, varying from 27 to 72 mgm., and the 

 coagulation carried out as before without allowing any time for the 

 trypsin to act proteolytically. The coagulable protein obtained amounted 

 to 19.0, 19.5,20.2, and 20.3 mgm. Plainly the trypsin lowered the amount 

 of coagulable protein by 3.4 mgm. A correction was not made, because 



2 c. c. of the digestion mixture contained but 8 mgm. of trypsin, and the 

 correction probably varied at different stages of digestion. This effect 

 is regarded as the probable explanation of the apparent digestion in 

 mixtures C, experiments 20 to 22. 



To calculate the amount of coagulable protein digested past the 

 coagulable stage, in experiment 22 for example, from the analytic data 



