Protection of enzymes by aromatic sulfonates from inactivation by acid andelevated temperatures

Selected azoaromatic sulfonate anions protect enzymes from inactivation by acid and elevated temperatures. These anionic sulfonate ligands bind to enz yme molecules by forming ion pairs between negatively charged sulfonate gro ups and positively charged protein groups as demonstrated by the binding st oichiometry determined using isothermal titration calorimetry. When the num ber of bound sulfonate anions is equal to the total positive charge of the protein, the protein-ligand complexes coprecipitate. Coprecipitation and pr otection are well correlated, but coprecipitation does not always result in protection. The coprecipitation-protection reactions are reversible. Ligan d anions can be removed with anion exchange resins, and full enzymatic acti vity recovered. Comparison of 29 azoaromatic sulfonate ligands showed that small structural differences in the ligands produce large differences ire t heir abilities to protect enzymes. Some protected enzymes were up to 1000 t imes more resistant to acid-inactivation, and their inactivation temperatur es were over 10 degrees C higher compared to nonprotected enzymes. Protecti on of six sulfhydryl proteases, namely papain, actinidin, chymopapain, brom elain, papaya protease omega, and ficin were compared. These proteases are highly homologous, have almost identical polypeptide chain fold, but differ in the numbers and locations of positive charges, which were crucial facto rs determining protection. Catalase enzyme, which is larger than papain and of a different class, was also protected by sulfonate ligands from inactiv ation by acid. (C) 1999 Elsevier Science B.V. All rights reserved.