Stimulation of Proteinase K Action by Denaturing Agents: Application to the Isolation of Nucleic Acids and the Degradation of ‘Masked’ Proteins

Abstract

Hydrolysis of serum albumin by proteinase K was strongly (> 7-fold) stimulated by urea and dodecylsulfate in a dose-dependent manner. With an oligopeptide as substrate, however, proteinase K was inactivated by dodecylsulfate. This indicates that the apparent activation of proteinase K by urea and dodecylsulfate is caused primarily by denaturation of the protein substrates.Although dodecylsulfate inhibited ribonuclease activity in the test-tube completely, it could not prevent RNA degradation during isolation of polysomal RNA, to which ribonuclease had been added, because of the reversible nature of the dodecylsulfate inhibition. Complete protection of RNA, however, was achieved by a combination of dodecylsulfate and proteinase K.The combined action of the detergent and proteinase K was also effective in degrading "masked" proteins in a poly(adenosine diphosphoribose) preparation which could not be attacked by the proteinase alone.Dodecylsulfate and other protein-denaturing agents are often used with the intention of inhibiting enzymic degradation of nucleic acids during isolation. Although it is generally assumed that these agents inactivate nucleolytic enzymes irreversibly, we were unable to prevent degradation of RNA during isolation from ribonuclease-containing samples by sodium dodecylsulfate. Therefore, we had introduced proteinase K (a mold enzyme from Tritirachium album Limber [l]) for the isolation of undegraded mRNA and ribosomal RNA [2 -41. The procedure was based on the simultaneous proteolytic digestion of (endogenous) ribonuclease and of ribosomal proteins. In the meantime the method has also been successfully applied to the isolation of various mRNA species and the preparation of superlong DNA [5-71. In one of these papers [7] a slight stimulation of proteinase K by sodium dodecylsulfate was reported. This paper demonstrates that a dose-dependent stimulation of proteinase K by dodecylsulfate and urea is only

The John Charnley Award: An Accurate and Sensitive Method to Separate, Display, and Characterize Wear Debris: Part 1: Polyethylene Particles

“…Digestion of lubricant proteins with proteinase K in the presence of urea and calcium was chosen because denaturation of proteins due to urea-dependent cleavage of hydrogen bonds leads to more complete proteolytic digestion [23] without the complications of detergents. Inclusion of calcium during digestion partially protected the proteinase K from autodigestion in urea [2].…”

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

“…In the absence of such information, it is prudent to use ribonuclease inhibitors or denaturing methods (10,19) in the isolation of mRNA from wheat endosperm.…”

Encoded Hydrogel Microparticles for Sensitive and Multiplex microRNA Detection Directly from Raw Cell Lysates

“…Also, it has been known that PK efficiently digests and inactivates RNase and DNase in the presence of SDS. 38 There were not significant differences in fluorescence among three different concentrations of PK at 2% (w/v) SDS, but 200 μg/mL PK resulted in the highest target signal ( Figure 2B). To determine whether adding RNase inhibitor would increase the fluorescent signal, 0, 0.2, or 20 U of RNase inhibitor were added to the reaction (0, 4, and 400 U/mL in final reaction volume, respectively).…”