56 BIOCHIMICA ET BIOPHYSICA ACTA VOL. 12 (lCj53) 



ON THE MECHANISM OF ENZYME ACTION. LV. 



A STUDY OF THE INTERACTION BETWEEN CALCIUM AND TRYPSIN 



by 



F. F. NORD AND M. BIER 

 Department of Organic Chemistry and Enzymology* , Fordham University, New York 58 {U.S.A.) 



The role of certain metallic cations in chemical processes occurring in living matter 

 has received considerable attention since the early days of physiology and enzymology. 

 The most characteristic aspect of these phenomena is the specificity of the ions and en- 

 zymes involved. In recent years it became increasingly evident that these biologically 

 important effects of metallic ions are but a particular case in the much broader range 

 of more or less specific interactions between proteins and other macromolecular com- 

 pounds on the one side and smaller molecules such as cations, anions, dyes, etc. on the 

 other. 



In the particular case of trypsin it has long been known that calcium augments 

 the extent of its formation from trypsinogen^. Recently there were discussed further 

 examples of the specific interaction of calcium and certain other ions with trypsin^. 

 Notably, calcium protects the enzyme against selfdigestion and it also slightly increases 

 its proteolytic activity. It was therefore suggested that calcium is an integral part of a 

 more stable trypsin molecule as it causes a shift towards the active form in the existing 

 equilibrium between the native and denaturated enzyme. It is proposed to call this form 

 calcium-trypsin . 



Most of these manifestations of the interaction of trypsin and calcium were ob- 

 served in the pH range 6 to 9, the region of the proteolytic activity of the enzyme. A 

 more direct evidence for the formation of a calcium-trypsin complex was obtained 

 through the study of the dissociation of trypsin. Calcium ions, in contrast to magnesium, 

 sodium or potassium ions, give rise to a shift in the titration curves of the enzyme in the 

 region of the dissociation of the carboxyl groups towards lower pH values. The acidity 

 of the carboxyl groups of the enzyme is evidently increased by the specific binding of 

 calcium. This is also a unique example of a difference in the effect of calcium and mag- 

 nesium ions upon the dissociation curves of a protein. 



The present communication explores the stabihty zones of trypsin over the entire 

 practicable pH scale. The effect of calcium ions on the dissociation curves prompted 

 an elcctrophoretic investigation of the enzyme in the same pH region and it resulted 

 therefrom that trypsin is electrophoretically inhomogencous. In the presence of calcium 

 two well-defined peaks appear on elcctrophoretic examination. In the absence of calcium, 



* Communication No. 277. — Read at the Symposium on Frontiers in Enzymology held at the 

 autumn meeting of the Am. Chem. Society, 1953. — For the previous paper of this series see Arch. 

 Biochem. Biophys., 45 (1953) No. i. 



References p. 66. 



