Recovery of biologically active enzymes after HPLC separation

The mass and activity recovery of eight different enzymes (two monomeric, six oligomeric) with molecular masses between 25,000 and 240,000 daltons were tested after HPLC separation on three different HPLC instruments (two with stainless steel and one with titanium flow paths). Most of the tested proteins are known to be sensitive to heavy metal ions. Eight wide pore, ion-exchange columns, two size-exclusion columns and two hydrophobic-interaction columns were used. Both stainless steel and glass column hardware were used in all three separation modes. The elution times were between 8 and 12 minutes. In almost all cases, the activity recovery was between 90% and 100% compared with a control sample incubated in the chromatographic elution buffer for the same time at the same temperature. A severe activity loss (about 30%) was observed with only one ion-exchange column and one enzyme. Neither the column hardware nor the material of the HPLC equipment had any negative effect on the activity recovery of the enzymes tested.