It is active in the presence of metal ions, SDS, urea, chelating agents (e.g. EDTA), sulfhydryl reagents and trypsin or chymotrypsin inhibitors. It is stable over a wide pH range (4-12.5), with optimal activity at pH 6.5-9.5. Activity can be stimulated by addition of denaturing agents, (SDS < 2.0% w/v and urea < 4 M). The optimal temperature for the enzyme is 65°C, it is 12 times more active at 65°C than at 25°C. Rapid denaturation occurs at temperatures above 65°C. While autolysis occurs increasingly at alkaline pH, proteinase K is not completely inactivated by autolysis. Applications: Proteinase K is used to rapidly inactivate nucleases, such as DNases and RNases, when isolating RNA and high-molecular-weight DNA from tissues and cell lines. It can also be used to remove nucleases in the preparation of tissue sections for in situ hybridization. Performance and quality testing: Endodeoxyribonuclease and exodeoxyribonuclease assays, liquid form tested for absence of RNase activity. Specific activity: > 20 Units/mg, where one unit produces 1 μmol of Folin-positive amino acid in 1 minute at 37°C.