Combination of 0.05% Triton and 0.2 mg/ml heparin give the optimal refolding activities to cleave the synthetic coumarin-labelled peptide substrate, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2. Panel A: Shows the refolded ZBD activities increased in dose-dependent manner. In the absence of the refolding accessory factors, Triton X-100 and heparin. The significant reduced activities in the high-concentration (> 100 μg/ml) was observed which could be due to autolysis. Panel B: Under 37 °C incubation for 18 hours, Triton X-100 and heparin can prevent the activity loss. (All experiments were repeated at two batch of purification and refolding preparation and data collected from a representative experiments).