Analysis Note

The enzyme can 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 and is activated by divalent cations including Ca2⁺ , Mg2⁺, Mn2⁺ and Fe2⁺.

Application

The enzyme from Sigma has been used in developing a protocol for ex vivo culture of mouse embryonic mammary buds. It has been used in the treatment of rat heart pieces during the isolation of mitochondria from rat heart. 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 explant method to analyse their stem cell and differentiation properties.Dispase^® II has been used for Fluorescence-Activated Cell Sorting (FACS). It has also been used for separating visceral yolk sac layers.

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 many tissues and cells grown in vitro. The enzyme is very gentle and does not damage cell membranes. It can also be used to prevent clumping in suspension cultures. This protease cleaves fibronectin and type IV collagen, but not laminin, type V collagen, serum albumin, or transferrin. Dispase^® II is specific for the cleavage of Leucine-Phenylalanine bonds. Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Fe³⁺ and Al³⁺ activate the enzyme. EDTA, EGTA, Hg²⁺ and other heavy metals inhibit the enzyme activity. 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, an inactive apoenzyme is obtained. The enzyme is not inhibited by serum.

Legal Information

Dispase is a registered trademark of Godo Shusei Co., Ltd.

Physical form

lyophilized powder containing calcium acetate and milk sugar

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) and 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.