VOL. 12 (1953) 



MECHANISM OF ENZYME ACTION. LV 



61 



IB' IB' IB' H''iB 



IB IBI IB! HiiBJ 



HI HI B B'-H' 

 I \WMi i^li i^Bi iBHi 



IB! IB! IB! IBIS 



HI IBI IBI IBNBI 

 I iIBi i^Bi iBBi i^Bi 



Bl IB! IBI IBIil 

 I I^Hl i^Bi i^B> i^"' 



Fig- 3 



Below pH 2.3 trypsin becomes increasingly unstable, the rate of inactivation fol- 

 lowing a monomolecular course^". In series C 

 of Figure 3 are presented the sedimentation 

 patterns of trypsin after 24 hour digestion in 

 a 0.1 HCl, o.i NaCl buffer. The faster sedi- 

 menting components which have appeared are 

 due to the aggregation of denatured protein, 

 not interfered with by any proteolysis. A 

 similar behaviour was also observed in the 

 denaturation of other proteins. At pH 8, in 

 the absence of calcium, trypsin is also quite 

 unstable and in series D of Figure 3 are 

 recorded the sedimentation diagrams of 

 trypsin after 24 hour digestion at pH 8 in 

 0.1 M borax buffer. Instead of the heavier 

 components, observable upon acid inactiva- 

 tion, some lighter ones appear which are 

 products of the self-digestion of trypsin. It 

 is important to note that the presence of 

 either the aggregation products or of the 

 breakdown products does not change in a 

 significant way the sedimentation rate of the 

 remaining unchanged enzyme. 

 It was reported^ that between the two extreme pH regions trypsin is subject to 

 aggregation with a maximum occurring at pH 5. This is in agreement with our results 

 as evidenced from the values recorded in Table V. Depending on the conditions of the 

 experimental runs at pH 5, sedimentation constants ranging from 2.4 S to over 3.5 5 

 can be obtained. The sedimenting boundary remains however at all times rather sym- 

 metrical and there is no appearance of two components as is the case in acid or alkaline 

 digestion. A typical series of sedimentation patterns is presented under E in Fig. 3. 

 Whereas this aggregation was shown to be concentration dependent^ it seems to have 

 been overlooked that the aggregation is also strongly temperature dependent, the 

 aggregation decreasing with temperature. This is evident from Fig. 4 and is a further 

 example of the importance of temperature in 

 all aggregation phenomena^. 



The aggregation occurs in the pH range 

 where the effect of calcium on the electro- 

 phoretic patterns of trypsin was observed. It 

 is natural that we have also investigated the 

 effect of calcium on the sedimentation beha- 

 viour of trypsin.The presence of 0.05 M. CaClg 

 completely prevents the aggregation of tryp- 

 sin as can be recognized in Fig. 4. The concen- 

 tration of CaCla used was chosen on the basis 

 of the results of the electrophoretic analyses, 

 these having indicated that a concentration of o.oi M is not sufficient for full charac 

 terization of its effect. A typical pattern is reproduced in Series F, Fig. 3. 



References p. 66. 



Fie 



18 



4.n- 

 o- 



20 



22 



24 



26 



28 °C 



In absence of calcium, pH 5.2. 

 In presence of calcium, pH 5.2. 



