Separation of chymosin and pepsin in calf rennet by dye-ligand affinity chromatography.
When calf rennet containing approximately 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorbed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain greater than 97% pure chymosin. The fraction that passed through unadsorbed was found to contain greater than 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzyme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.
D000327:Adsorption; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D012085:Chymosin / Q000032:analysis* / Q000302:isolation & purification*; D004396:Coloring Agents; D019855:Ethylene Glycol; D005026:Ethylene Glycols; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D010434:Pepsin A / Q000302:isolation & purification*; D012685:Sepharose / Q000031:analogs & derivatives*; D012965:Sodium Chloride; D013431:Sulfates / Q000494:pharmacology
D004396:Coloring Agents; D005026:Ethylene Glycols; D013431:Sulfates; C017842:cibacron blue-sepharose; C012036:sodium sulfate; D012965:Sodium Chloride; D012685:Sepharose; C025350:rennet; D010434:Pepsin A; D012085:Chymosin; D019855:Ethylene Glycol