Activity： > = 0.5 units/mg solid.
Dissolubility： 10 mM NaAc (pH 7.5) and 5 mM CaAc: soluble.
Physical form.
Freeze-dried powder containing calcium acetate and lactose.
Biochemical / physiological behavior.
Dispase II is a neutral protease that can hydrolyze the N-terminal peptide bond of non-polar amino acid residues. It can be used to isolate a variety of tissues and cells growing in vitro. The enzyme is very mild and does not damage the cell membrane. It can also be used to prevent cells from caking in suspension cultures. The protease can hydrolyze fibronectin and type IV collagen, but can not hydrolyze laminin, type V collagen, serum albumin or transferrin. Dispase II has the specificity of cutting leucine-phenylalanine bond. Ca2+, Mg2+, Mn2+, Fe2+, Fe3+ and Al3+ could inactivate disperse enzyme I. EDTA, EGTA, Hg2+ and other heavy metals can inhibit the activity of the enzyme. The purified disperse enzyme I contains 1 g zinc atom per g-mol. If zinc is removed by chelating agents such as EDTA or EGTA, inactive enzyme proteins are produced. The enzyme was not inhibited by serum.
Application.
The enzyme from ABCONE has been used to develop an experimental method for in vitro culture of mouse embryonic breast buds. It has been used to treat rat heart fragments during the isolation of mitochondria from rat hearts. It is also used to isolate dental pulp stem cells (DPSC) by enzymatic hydrolysis. These cells have been compared with DPSC isolated by explants to analyze their stem cell and differentiation characteristics. Dispase II has been used in fluorescence activated cell sorting (FACS). It is also used to isolate the visceral yolk sac layer.
Unit definition.
Except for special instructions, at Ph7.5,37 °C, one unit of enzyme will hydrolyze casein per minute to produce a color equivalent to 1.0 μ mol (181ug) tyrosine (color provided by Folin-Ciocalteu reagent).
Analysis consideration.
The enzyme was initially soluble in 50 mM Hepes/KOH pH 7.4,150 mM NaCl for 10 mg/ml. Further dilution should be carried out in the medium most suitable for the cells to be dissociated. Neutral protease is a kind of metalloproteinase, which can be activated by divalent cations including Ca2+, Mg2+, Mn2+ and Fe2+.
Points for attention in preparation.
A reserve solution of 50 mM NaAc can be prepared by dissolving the powder in a buffer containing 10 mM NaAc (pH 7.5) and 5 U/mL. It should be stored at 4 °C.
