Analysis Note
The enzyme c be initially dissolved in 50 mM Hepes/KOH pH 7.4, 150 mM NaCl at 10 mg/ml. Further dilutions should be made in the cell culture media most appropriate for the cells being dissociated. Dispase is a metalloproteinase d is activated by divalent cations including Ca2+ , Mg2+, Mn2+ d Fe2+.
Preparation Note
A 50 U/mL stock solution may be prepared by dissolving the powder in a buffer containing 10 mM NaAc (pH 7.5) d 5 mM CaAc. It should be stored at 4 °C.
Unit Definition
One unit will hydrolyze casein to produce color equivalent to 1.0 μmole (181 μg) of tyrosine per min at pH 7.5 at 37 °C (color by Folin-Ciocalteu reagent), unless otherwise indicated.
Physical form
lyophilized powder containing calcium acetate d milk sugar
Application
The enzyme has been used in developing a protocol for ex vivo culture of mouse embryonic mammary buds.[4] It has been used in the treatment of rat heart pieces during the isolation of mitochondria from rat heart.[5] It has also been used for the isolation of dental pulp stem cells (DPSCs) by enzymatic hydrolysis. These cells have been compared with DPSCs isolated by explt method to alyse their stem cell d differentiation properties.[1]
Dispase II has been used for Fluorescence-Activated Cell Sorting (FACS).[7] It has also been used for separating visceral yolk sac layers.[2]
Biochem/physiol Actions

Dispase II is a neutral protease that hydrolyzes the N-terminal peptide bonds of non-polar amino acid residues. It may be used for separating my tissues d cells grown in vitro. The enzyme is very gentle d does not damage cell membres. It c also be used to prevent clumping in suspension cultures. This protease cleaves fibronectin d type IV collagen, but not laminin, type V collagen, serum albumin, or trsferrin.[3] Dispase II is specific for the cleavage of Leucine-Phenylaline bonds. Ca2+, Mg2+, Mn2+, Fe2+, Fe3+ d Al3+ activate the enzyme. EDTA, EGTA, Hg2+ d other heavy metals inhibit the enzyme activity.[6] The enzyme contains 1g-atom of zinc per g-mol of purified enzyme. If this zinc component is removed by chelating agents such as EDTA or EGTA, inactive apoenzyme is obtained. The enzyme is not inhibited by serum.

Solubility：10 mM NaAc (pH 7.5) d 5 mM CaAc: soluble