AEBSF and PMSF are both irreversible protease inhibitors of serine proteases. With them both inhibiting serine proteases, you may be curious about their differences. What makes them different? Why do those differences matter? And when is it better to choose AEBSF or PMSF?
AEBSF is a serine protease inhibitor stable in aqueous solutions. It is ideal when longer inhibition is needed. However, AEBSF can modify off-target residues (tyrosine, lysine and protein N-terminus). PMSF is less stable in aqueous solutions but is popular due to its rapid inhibition and low cost.
There is also a difference in each inhibitor’s toxicity. PMSF requires more careful handling due to its toxicity while AEBSF is considered to be a safer alternative.
To quickly summarize the differences, AEBSF is stable in water and less toxic. PMSF hydrolyzes quickly, works rapidly, is less costly, but it is more toxic. In this article, we’ll break down the differences between AEBSF and PMSF even more, helping you understand the basics of protease inhibitors, more details about the differences between AEBSF and PMSF and some of the advantages and disadvantages of each.
Article Table of Contents
Reversible vs. irreversible protease inhibitors
Deeper differences between AEBSF and PMSF
Basics of protease inhibitors
Before we cover the deeper differences in AEBSF and PMSF, it might be good to refresh ourselves on the basics of protease inhibitors. And if you don’t need this quick overview, skip on ahead.
Protease inhibitors prevent, or inhibit, protease activity. Proteases are enzymes that break down a protein, and they do this by hydrolyzing peptide bonds.
Figure 1. Within a cell are proteins (pink) and
proteases (blue scissors). Upon cell lysis, cell contents spill out and
proteins are exposed to proteases. Protease inhibitors and inhibitor cocktails
(green squares) bind reversibly or irreversibly to proteases, protecting
proteins from degradation.
For more information about proteases and protease inhibitors, we have an article about protease inhibitor cocktails, which has a section that goes into more detail.
When you are purifying a protein or trying to study a protein, or working with proteins, proteases would damage your sample by cutting it into smaller pieces. Therefore, protease inhibitors play an important role by preventing proteases from acting on your proteins of interest.
Types of protease inhibitors
There are a few different types of protease inhibitors, and their classification is based on their action and the types of proteases they inhibit.
- Serine protease inhibitors target serine residues on the active site of a protease.
- Cysteine protease inhibitors target cysteine residues on a protease’s active site.
- Aspartic protease inhibitors inhibit aspartic proteases.
- Metalloprotease inhibitors target metal ions that are needed for protease activity.
Reversible vs. irreversible protease inhibitors
The different types of proteases listed above can either being a reversible protease inhibitor or an irreversible protease inhibitor.
Reversible protease inhibitors noncovalently bind to proteases without modifying their active sites, and they can dissociate from proteases. What this means is that reversible protease inhibitors temporarily inhibit proteases and are therefore used when a researcher only needs temporary inhibition.
Irreversible protease inhibitors are just the opposite. They bind covalently, modify the active site, and they permanently inactivate proteases (Figure 2). Researchers will use these when they need to completely inactivate a protease.
Figure 2. Proteins with protease inhibitors
covalently bound. On the left is trypsin (gray) with PMSF (magenta) and on the
right is a hydrolase from gut bacteria (gray) with AEBSF (magenta) (Hameleers
et al, 2021; Schmidt et al, 2003).
Let’s look at an example of a popular protease used in molecular biology: proteinase k . Proteinase K is a serine protease. While heat can inactivate proteinase K, it doesn’t fully inactivate it. So, what kind of protease inhibitor would you choose? Since proteinase K is a serine protease, you could inactivate proteinase K with a serine protease inhibitor, such as our two subjects of attention: AEBSF or PMSF. Both protease inhibitors are irreversible. So using them would permanently inactivate proteinase K ( Martin, 2016 ).
Figure 3. Proteinase K structure.
Deeper differences between AEBSF and PMSF
As you’re learning, both AEBSF and PMSF are irreversible (permanent) protease inhibitors of serine proteases. However, AEBSF and PMSF differ in their stability, toxicity and effectiveness. Each of these differences can make one or the other more suitable for different experiments or even just different personal needs in your lab.
Stability in water
One of the immediate differences between AEBSF and PMSF is their stability in aqueous solutions (water). AEBSF is known for its stability in water whereas PMSF degrades in water (hydrolyzes). The reason for AEBSF’s stability in water may be due to its chemical structure, which resists hydrolysis in water better.
Because of this characteristic, PMSF would seem like an unsuitable protease inhibitor, but it still has a lot of benefits. One of these benefits is that it acts rapidly. So, when you are wanting something that will quickly inhibit proteases, PMSF offers that advantage.
To work around PMSF’s stability issues in aqueous solutions, researchers will quickly add PMSF to the experimental solution before it can degrade (James, 1978).
PMSF is stable in ethanol and isopropanol, and working solutions are often prepared using one of these solvents. Just before use, the solution is diluted into an aqueous solution.
Hydrolysis in water can also be slowed by decreasing the pH (acidic) where PMSF is a little more stable (James, 1978).
Toxicity and safety
Aside from stability issues, another drawback to PMSF is its toxicity and corrosiveness. While working with PMSF, it’s important to follow your lab’s safety guidelines. In fact, some labs recommend double gloving and fully inspecting your gloves every time before use. Further, disposing of PMSF requires you to follow your lab or institution’s procedures. And working with PMSF, especially in aqueous solutions means avoiding certain types of containers.
AEBSF can cause burns and injure your eyes. However, it is considered less toxic than PMSF. Another key difference, when it comes to toxicity between AEBSF and PMSF is that upon degradation, PMSF can release byproducts that are also toxic, whereas AEBSF does not.
For some labs, because AEBSF is considered to be a little bit safer, it can sometimes be the better option.
Effectiveness
Both AEBSF and PMSF meet different experimental and laboratory needs, and both are very effective when used for appropriate situations.
For instance, because AEBSF is stable in aqueous solutions and less toxic by comparison, it’s ideal for:
- Long-term studies
- Cell culture
- Cell lysis
- Protein purification with longer activity
- Biological systems that may be more delicate
Something to note about AEBSF, however, is that while it is a serine protease inhibitor, it can also modify tyrosine, lysine and histidine residues as well as protein n-terminal amino groups (UNIMOD). However, such modifications are more frequent when using very high concentrations of AEBSF, so by using the recommended concentration, AEBSF will still inhibit proteases and limit off-target labeling of most other proteins (Narayanan & Jones, 2015). There are a handful of proteins that are particularly susceptible to off target modification by AEBSF even at recommended concentrations (Tarade et al, 2020). If you’re working with one of those proteins, then you will want to use PMSF instead.
PMSF offers the advantage of rapidly acting on proteases, and is therefore a good choice when:
- Performing short-term experiments
- Fast inhibition is necessary
PMSF is also less expensive and can be a good choice in certain high-throughput situations.
What you’ve probably discovered from this article is that both protease inhibitors are effective inhibitors of serine proteases, each with their unique advantages and disadvantages. And choosing between either of these is actually fairly easy once you’ve identified your experimental goals and your laboratory’s overall needs. In some cases, you’ll want to have both available to meet the needs of the different types of experiments you’re working on.
References
Hameleers, L., Penttinen, L., Ikonen, M., Jaillot, L., Fauré, R., Terrapon, N., Deuss, P. J., Hakulinen, N., Master, E. R., & Jurak, E. (2021). Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans. Biotechnology for biofuels, 14(1), 127. https://doi.org/10.1186/s13068-021-01976-0
Harvard Campus Services, Environmental Health and Safety. (n.d.). Lab safety guideline: PMSF. https://www.ehs.harvard.edu/sites/default/files/lab_safety_guideline_pmsf.pdf
James, G. T. (1978). Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Analytical biochemistry, 86(2), 574-579.
Martin, K. (2016, May 5). 20 answers to important proteinase K questions – plus free printable fact sheet. https://goldbio.com/articles/article/20-answers-to-important-proteinase-k-questions-plus-free-printable-fact-sheet
Narayanan, A., & Jones, L. H. (2015). Sulfonyl fluorides as privileged warheads in chemical biology. Chemical science, 6(5), 2650–2659. https://doi.org/10.1039/c5sc00408j
Purdue University. (n.d.). Standard Operating Procedure Phenylmethanesulfonyl fluoride (PMSF). https://www.purdue.edu/ehps/rem/documents/sops/sop...
Schmidt, A., Jelsch, C., Ostergaard, P., Rypniewski, W., & Lamzin, V. S. (2003). Trypsin revisited: crystallography AT (SUB) atomic resolution and quantum chemistry revealing details of catalysis. The Journal of biological chemistry, 278(44), 43357–43362. https://doi.org/10.1074/jbc.M306944200
Tarade, D., He, S., St-Germain, J., Petroff, A., Murphy, A., Raught, B., & Ohh, M. (2020). The long form of pVHL is artifactually modified by serine protease inhibitor AEBSF. Protein science : a publication of the Protein Society, 29(8), 1843–1850. https://doi.org/10.1002/pro.3898
UNIMOD. (n.d.). UNIMOD, view record [ accession #: 276 ]. Unimod. https://www.unimod.org/modifications_view.php?edit...
