Overview of Protease and Phosphatase Inhibition for Protein Preparation

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Protease and phosphatase inhibitors are essential components of most cell lysis and protein extraction procedures. These inhibitors block or inactivate endogenous proteolytic and phospholytic enzymes that are released from subcellular compartments during cell lysis and would otherwise degrade proteins of interest and their activation states.

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What are proteases and phosphatases?

Inhibition of protease and phosphatase activity

What are proteases (proteolytic enzymes) and phosphatases?

Proteases and phosphatases are important enzymes in a variety of biochemical pathways in living cells. Proteases are required for many cellular functions, including cellular repair and the digestion of extracellular material. Phosphatases play a key role in regulating signal transduction events in eukaryotic cells. Protein kinases transfer a phosphate from ATP to a serine, threonine, or tyrosine residue in a protein; phosphatases remove the phosphoryl group. Phosphorylation is the most common post-translational modification on proteins, with approximately 80% occurring on serine, 20% on threonine, and 0.1 to 1% on tyrosine residues.

All living organisms contain proteolytic enzymes (proteases and peptidases). In whole cells, protease and phosphatase activities are tightly regulated by compartmentalization or inhibitors to prevent indiscriminate damage to cellular proteins and to maintain proper function of signaling pathways. Cell lysis disturbs the carefully controlled cellular environment, allowing proteases and phosphatases to become unregulated. The usual consequence of this unregulated state is reduced recovery of total protein and biologically meaningless representation of protein activities (i.e., phosphorylation status).

Graphical depiction of cellular compartments that control protease and phosphatase activities, but cellular disruption leads to all proteins being accessible for degradation

Protein degradation during cell lysis. Many of the cellular proteins are kept separate from proteolytic enzymes. Disruption of cellular and tissue architecture during protein extraction distorts the in vivo state by making all proteins potentially accessible for degradation or modification by endogenous proteases and phosphatases. The resulting unregulated proteolytic activity can reduce protein yield and function. Protease and phosphatase inhibitors can be added to the lysis reagents in order to prevent degradation of extracted proteins, and to obtain the best possible protein yield and activity following cell lysis.

Inhibition of protease and phosphatase activity

Protease inhibitors are biological or chemical compounds that function by reversibly or irreversibly binding to the protease. Most known proteases belong to one of four evolutionarily distinct enzyme families based on the functional groups involved in cleavage of the peptide bond. Known phosphatases are specific for cleavage of either serine-threonine or tyrosine phosphate groups. Thus, while numerous compounds have been identified and used to inactivate or block these enzymes, no single chemical is effective for all types of proteases and phosphatases (see tables below). Rather, a mixture or inhibitor cocktail of several different inhibitor compounds are used to ensure that protein extracts do not degrade before analysis for targets of interest. Proteases inhibitors are nearly always needed, while phosphatase inhibitors are required only when phosphorylation states (activation states) are being investigated. Research experiments may necessitate the use of single inhibitors or customized mixtures, but most protein work is best served by using a suitable protease inhibitor cocktail.

Commonly used protease inhibitors

Inhibitor	MW (kDa)	Target class	Type	Solubility (solvent)	Typical working (1X) conc.
AEBSF	239.5	Serine proteases	Irreversible	200 mg/mL (H₂O)	0.2 to 1.0 mM
Aprotinin	6511.5	Serine proteases	Reversible	10 mg/mL (H₂O)	100 to 200 nM
Bestatin	308.4	Amino-peptidases	Reversible	5 mg/mL (MeOH)	1 to 10 µM
E-64	357.4	Cysteine proteases	Irreversible	20 mg/mL (1:1 EtOH:H₂O)	1 to 20 µM
EDTA	372.2	Metalloproteases (chelates cations)	Reversible	10 g/100 mL (H₂O)	2 to 10 mM
Leupeptin	475.6	Serine and cysteine proteases	Reversible	1 mg/mL (H₂O)	10 to 100 µM
Pepstatin A	685.9	Aspartic acid proteases	Reversible	1 mg/mL (MeOH)	1 to 20 µM
PMSF	174.2	Serine proteases	Reversible	18 mg/mL (MeOH)	0.1 to 1.0 mM

Commonly used phosphatase inhibitors

Inhibitor	MW (kDa)	Target class	Type	Solubility (solvent)	Typical working (1X) conc.
Sodium fluoride	42.0	Ser/Thr and acidic phosphatases	Irreversible	40 mg/mL (H₂O)	1 to 20 mM
Sodium orthovanadate	183.9	Tyr and alkaline phosphatases	Irreversible	20 mg/mL (H₂O)	1 to 100 mM
beta-Glycerophosphate (Disodium salt)	216.0	Ser/Thr phosphatases	Reversible	10 mg/mL (H₂O)	1 to 100 mM
Sodium pyrophosphate	221.9	Ser/Thr phosphatases	Irreversible	65 mg/mL (H₂O)	1 to 100 mM

Prevention of protein degradation by proteases after cell lysis using various protease inhibitor cocktails

Comparison of commercially available protease inhibitor cocktails and tablets. Pancreatic extract (50 μL, 1 μg/μL protein) or trypsin (25 μL, 0.1 units/μL) was incubated with a quenched-fluorescent, protease-cleavable substrate for cysteine (A) or serine proteases (B) in the presence or absence of commercially available protease inhibitors with EDTA-containing (blue) or EDTA-free (purple) formulations. Reactions were incubated for 2 hours at 37°C and the fluorescence determined at indicated detecting emissions. The percent protease inhibition is shown for each protease inhibitor formulation.