\-OL. 12 (1953) MECHANISM OF ENZYME ACTION. LV 5/ 



I 



trypsin appears to be a homogeneous protein due to the interaction of the two trypsin 

 components. This interaction causes an aggregation which was evidenced by ultra- 

 centrifugal measurements^. The nature of these reversible aggregates is indicated by the 

 experiments to be described, a temperature rise to only 30° C preventing their formation. 

 Calcium ions also prevent the interaction and thus give rise to a normal sedimentation 

 behaviour^. 



EXPERIMENTAL 

 Enzyme preparation 



In this study a number of different samples were used. Commercial crystalline trypsin 

 (Worthington Biochemical Laboratory, Freehold, N.J.) which contains about 50% of MgSO^ was 

 employed in some experiments where the presence of this salt did not appear to be critical or could 

 be taken into account. Most experiments were performed, however, with salt-free preparations of the 

 enzyme either using the commercial salt-free product, prepared by dialysis of crystalline trypsin at 

 pH 2.3 and freeze-dr3'ing, or alcohol precipitated trypsin, prepared according to our previously 

 described procedure^. Due to the fact that magnesium ions seem to have no specific effect on the 

 electrophoretic or ultracentrifugal behaviour of trypsin, the three preparations were fully identical 

 in their behaviour. Similarly, no differences in properties were observed between successive batches 

 of the various preparations. 



Electrophoretic analyses 



A Perkins Elmer Model No. 38 apparatus was used as in the previous studies^. The experiments 

 were carried out in a pH range of reasonable stability of the enzyme and all the samples were 

 equilibrated against the buffer by overnight dialysis in the cold on a slowly rotating shaker. An aliquot 

 of a stock solution of the buffer was mixed with a fixed volume of solution, containing the desired 

 amount of the various neutral salts. In all experiments the final composition of the buffer is reported. 



Ultracentrifugal determinations 



A Spinco analytical centrifuge, mcdel E, was used in the sedimentation studies on the enzyme. 

 The trypsin samples were dissolved in the appropriate buffers always immediately prior to the actual 

 run except in those experiments where the effect of prolonged digestion was under investigation. In 

 this case, aliquots of the same solution were analyzed at different times. 



Usually five exposures at 32 min intervals were taken in each experimental run, using the 1.2 cm 

 cell. In a few instances the 0.4 cm cell was also utilized. The sedimentation constants were calculated 

 by measuring the displacement of the sedimenting boundary between two successive exposures. All 

 the sedimentation constants are reported as Sjo^.^,^ namely, corrected for the temperature and density 

 of the medium according to Svedberg and Pedersen^ as well as the values of Landolt and Born- 

 stein. The temperature of the rotor was measured before and after each run and the average of these 

 two readings is reported as the temperature of the experiment. By appropriate application of 

 refrigeration it was possible to maintain the temperature during the run within less than i ° C, in 

 most of the experiments. In a few cases where the difference exceeded the above limits, an individual 

 correction for each time interval was applied, assuming a linear change in temperature. 



pH measurements 



All pH values were determ ined with a Cambridge Instrument pH meter, research model, using 

 potassium acid phthalate as reference buffer. 



Activity of the enzyme 



As in previous communications the activity was determined by measuring the absorption at 

 2S0 mfx of a hemoglobin digest. i 



RESULTS 



Electrophoresis 



It is between pH 4 and 5 that the shift in the dissociation curves of trypsin caused 

 by the addition of calcium ions is the most evident^. This is why we have searched in the 

 same pH region for a specific effect of calcium ions on the electrophoretic mobility of the 

 enzyme. Preliminary experiments have shown that, rather than giving rise only to a 



References p. 66. 



