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



difference in electrophoretic mobility, calcium ions cause a change in the appearance 

 of the electrophoretic pattern. Whereas in the absence of calcium trypsin presents 

 patterns characteristic of a homogeneous compound as shown by the photographs in 

 series A, Fig. i, two well-defined peaks appear upon the 

 addition of calcium. We have studied the conditions of 

 maximal separation of the two components. Two factors 

 are found to influence the separation of the two components : 

 pH and concentration of calcium. In Fig. i, series B and C, 

 the electrophoretic patterns obtained in the presence of o.oi 

 and 0.033 ^I CaCla respectively are presented. It is evident 

 that at the lower concentration of the calcium ions the sepa- » 

 ration of the two components is not yet complete. A further ^^%^^^ 

 increase of concentration of calcium ions gives rise to no HL-J2^S 

 other changes in pattern and therefore the concentration of 

 0-033 ^^ was applied in the comparison of all chlorides of 

 divalent cations. To study the effect of monovalent cations 

 a concentration of o.io M was chosen to keep the ionic 

 strength constant. Because of the relatively high require- 

 ments of calcium ion concentration, advantage could not be 

 taken of the observed better separation of electrophoretic 

 components at very low ion concentrations^' ^^. As expected 

 from the dissociation curves of trypsin, the separation of the 

 two components is best evidenced between pH 4 and 5. At 

 pH 3.5 and 5.5 the two peaks remain partially superimposed, 

 even after prolonged electrophoresis. 



In Table I are listed all the cations, applied as chlorides, 

 the effect of which on the electrophoretic patterns of trypsin ^" 



was investigated. Besides calcium, only manganese and cadmium cause a separation of 

 peaks to occur, whereas all other cations give rise to patterns characteristic of an 

 electrophoretically homogeneous protein. The electrophoretic patterns obtained in the 

 presence of manganese and cadmium are also included in Fig. i, series D and E. 



TABLE I 



EFFECT OF VARIOUS IONS ON THE ELECTROPHORETIC PATTERNS OF TRYPSIN 

 Separation of components not observed in presence of: Components separated in presence of: 



Li+, Na+, K+, NH4+, Ca++, Mn++, Cd+K 



Mg++, Ba++. Sr++, 

 Co++, Cu++, Ni++. 



All cations were added as chlorides Ionic strength o.io; pH 4.75-5 





The mobilities and relative areas of the components of trypsin in the presence of 

 various ions arc recorded in Table II. It can be seen that of the two boundaries the larger 

 one moves with a mobility characteristic of trypsin in absence of calcium ions whereas 

 the smaller boundary moves at a slightly lower rate. 



It was of importance to obtain small samples of electrophoretically separated faster 

 and slower moving components to test their respective proteolytic activity. Due to the 



References p. 66. 



