I am trying to digest proteins by proteinase in blood samples for RNA purification. Suggested approach is to use SDS around 1% to denature proteins for better enzyme accessibility. would that impact the enzyme activity itself or it remains still active?
Hello Shahab Boroun,
The Proteinase K activity increases with temperature (optimal temperature for activity ranges between 50-65 ˚C). The higher temperatures help with protein unfolding, easing the ability for Proteinase K to breakdown those proteins. SDS is one of the Proteinase K activators that make enzyme more stable and more active if the buffers contain SDS or Urea.
On the other hand, the activity of Proteinase K is inhibited if heated at high temperature (at 95°C for 10 minutes). However, heating Proteinase K does not fully inactivate the enzyme. There will always be a small amount of activity remaining through this method.
Be clear that the addition of 1% SDS would not have any adverse impact the enzyme activity, instead it will make Proteinase K more stable and active.
Best
Hello Shahab Boroun
Proteinase K is very useful in the isolation of highly native, undamaged RNA, particularly in the presence of 0.5 – 1% SDS as RNases are rapidly inactivated by the enzyme. Moreover, the enzyme’s activity towards native proteins is stimulated by denaturants such as SDS. Addition of SDS helps proteinase K in its action as SDS will unfold proteins and make them more accessible to proteinase K action.
Best Wishes.
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