The Ultimate Guide to Proteinase K Activity & Concentration (u/mL)

From time to time, we have received questions about whether knowing proteinase K activity (u/mL) was important or not. The scenario is often this: You’re using an old protocol that calls for 1 µL of proteinase K, but is that really the right amount? After all, different companies sell proteinase K with different activities.

Since this topic has come up a few times, we wanted to straighten a few things out:

What is Enzyme Activity, Specific Activity and Enzyme Concentration?

We have a great sheet on the topic, but I’ll pull out some important points.

• Enzyme Activity: Enzyme activity is the number of moles or mg, etc. of substrate modified by an enzyme in a given time frame. For example, a single unit of enzyme X will digest 1 µmol of a substrate in 1 minute. However, reagent suppliers usually have to modify this definition proportionally for labeling ease; otherwise the numbers could get a little too tedious.
• Specific Activity: Specific activity is related to enzyme purity. It is the amount of substrate modified by a particular quantity of protein in an enzyme preparation per unit of time. The higher the enzyme purity, the higher the measured value of specific activity.
• Enzyme Concentration: Your enzyme concentration is simply the number of units of activity per volume.

Putting it together:

Let’s say, hypothetically, you’re instructed to add 1 µL of an enzyme. If your concentration, in terms of units, is 20 U/µL, then by adding 1 µL, you’re working with 20 units. Yes, it seems like I’m pointing out the obvious, but that’s because considering these things are not always so obvious.

Now you need to run this experiment again but are out of your reagent. So you borrow more from another lab, and it’s a different brand with, perhaps, a different concentration. If that reagent you’re borrowing has a concentration of 5 U/µL, then when you add 1 µL, you won’t be adding the same amount – you’re now working with only 5 units.

Evaluating Proteinase K:

If you’re working with a 20 mg/mL stock solution of proteinase K, you still want to evaluate the product for activity. For example, using proteinase K with an activity of ≥30 U/mg means you will have 600 units/mL. However if you’re using proteinase K with an activity of ≥20 U/mg, then you will have 400 units/mL.

Unfortunately, following a protocol isn’t as easy as following a Hamburger Helper recipe. Instead, you have to remember to question yourself when doing a certain procedure if even a very slight variable is introduced.