Protection of enzymes by aromatic sulfonates from inactivation by acid and elevated temperatures

Selected azoaromatic sulfonate anions protect enzymes from inactivation by acid and elevated temperatures. These anionic sulfonate ligands bind to enzyme molecules by forming ion pairs between negatively charged sulfonate groups and positively charged protein groups as demonstrated by the binding stoichiometry determined using isothermal titration calorimetry. When the number of bound sulfonate anions is equal to the total positive charge of the protein, the protein-ligand complexes coprecipitate. Coprecipitation and protection are well correlated, but coprecipitation does not always result in protection. The coprecipitation-protection reactions are reversible. Ligand anions can be removed with anion exchange resins, and full enzymatic activity recovered. Comparison of 29 azoaromatic sulfonate ligands showed that small structural differences in the ligands produce large differences ire their abilities to protect enzymes. Some protected enzymes were up to 1000 times more resistant to acid-inactivation, and their inactivation temperatures were over 10 degrees C higher compared to nonprotected enzymes. Protection of six sulfhydryl proteases, namely papain, actinidin, chymopapain, bromelain, 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 factors determining protection. Catalase enzyme, which is larger than papain and of a different class, was also protected by sulfonate ligands from inactivation by acid. (C) 1999 Elsevier Science B.V. All rights reserved.