64 F. F. NORD, M. BIER VOL. 12 (1953) 



of the moving boundary into two well-defined peaks, the larger of the two moving with 

 the mobility of the enzyme in absence of calcium whereas the smaller moves at a slightly 

 lower rate. 



This fact cannot be understood without a detailed analysis of our ultracentrifugal 

 results. It was already noted that in the same pH range the sedimentation constant of 

 trypsin increases, the concentration-dependent aggregation being maximal at about pH 

 5^. This observation was left, however, without a rational explanation. But we have 

 noticed that the aggregation is strongly temperature dependent. It is significant that 

 by varying the three factors, e.g. concentration, temperature as well as the pH, sedi- 

 mentation constants can be obtained ranging from 2.45, the normal value, to 3.55, 

 the protein presenting a single sedimenting boundary at all times^. 



This is in contrast to the appearance of faster sedimenting boundaries upon acid 

 denaturation of the enzyme or of slower sedimenting boundaries during the self-digestion 

 at pH 8. In both cases the appearance of the faster or slower sedimenting boundaries 

 does not affect the rate of sedimentation of the remaining unchanged enzyme to a signifi- 

 cant extent. 



A continuous variation of the sedimentation constant can only be explained by the 

 existence of a reversible equilibrium between monomeric and dimeric or polymeric forms 

 of the enzyme, the time necessary for the establishment of the equilibrium being small 

 in comparison to the sedimentation rate. The rate of sedimentation will then depend 

 on the relative proportion of the various forms present and it is evident that in the case 

 of trypsin the equilibrium is shifted towards the monomeric form by an increase of 

 temperature or decrease in concentration. 



The addition of calcium ions in sufficient concentration fully prevents the aggrega- 

 tion reducing the sedimentation constant to its normal value, namely that of the 

 monomeric enzyme. It appears, therefore, that calcium, bound to the carboxyl groups 

 of the enzyme, prevents the interaction of enzyme molecules which results in the forma- 

 tion of dimeric or higher polymeric forms. On the basis of this interpretation it must also 

 be considered that the electrophoretic patterns obtained when calcium is present 

 correspond to the actual molecular distribution of the protein, namely that trypsin 

 is electrophoretically heterogeneous and contains two components. The apparently 

 homogeneous patterns obtained in the absence of calcium are but an artefact, resulting 

 from the interaction of the two components, enhanced by the low temperature at which 

 electrophoretic measurements are carried out. It is, of course, open to speculation 

 whether the interaction is limited to a combination of the two different types of trypsin 

 molecules or whether, which seems the more probable case, it is a random combination 

 of any two or more molecules present. 



As in the case of sedimentation, the electrophoretic mobility of such a system 

 existing in reversible equilibrium would be a weighed average of the mobilities of the 

 various forms coexisting at the equilibrium. The apparent identity of the mobilities of 

 the total trypsin in absence of calcium and of the major component of trypsin in the 

 presence of calcium is, therefore, purely coincidental. The average mobility of trypsin 

 when calcium is added is lower than in the absence of calciimi in accordance with the 

 smaller net charge of the calcium-trypsin. 



The properties of the two components of trypsin were further anal}zed. Besides 

 calcium ions, manganese and cadmium ions also give rise to the appearance of the 

 two characteristic components in electrophoretic analysis. The same ions, together with 



References p. 66. 



