AEBSF is an irreversible serine protease inhibitor. AEBSF has been shown to inhibit trypsin, chymotrypsin, plasmin, kallikrein and thrombin. It inhibits the active site of the enzyme through acylation. As an alternative to PMSF and DFP, AEBSF offers lower toxicity, improved solubility in water and improved stability in aqueous solutions. AEBSF has been used in cell culture in concentrations of up to 0.25 mM. Aqueous solutions are stable between pH 5-6; limited stability above pH 7.5.

Aprotinin is a monomeric globular polypeptide derived from bovine lung tissue. It consists of a chain of 58 amino acids that fold into a stable, compact tertiary structure of the 'small SS-rich" type, containing 3 disulfides, a twisted β-hairpin and a C-terminal α-helix.

Aprotinin inhibits several serine proteases, specifically trypsin, chymotrypsin, plasmin and kallikrein. Its action on kallikrein leads to the inhibition of the formation of factor XIIa. Binding is reversible with most aprotinin-protease complexes dissociating at pH >10 or < 3. Aprotinin is freely soluble in water (>10 mg/ml) as well as low ionic strength aqueous buffers.

Asunaprevir is a potent inhibitor of the enzyme serine protease NS3 in hepatitis C viruses (HCV) and is currently in phase III clinical trials. This enzyme’s C terminal functions as a helicase for host DNA allowing access for viral manipulation. Asunaprevir is used in tandem with ribavirin and other direct or indirect acting antiviral compounds results in a thorough eradication of HCV.

Benzamidine hydrochloride monohydrate is an aromatic amidine. It is used in protein extractions as a strong reversible inhibitor of serine proteases such as trypsin, thrombin, plasmin and other trypsin-like proteases. Compared to aprotinin, benzamidine is considered to be just as effective in preventing the degradation of glucogon.

E-64 (trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane) irreversibly inhibits cysteine proteases without affecting other enzymes that contain cysteine residues. E-64 is capable of inhibiting bromelain, trypsin, papain, staphopain, calpain, collagenase, ficain, cathepsin B, H and L. Inhibition of serine proteases is not seen with E-64, excepting trypsin.

E-64 is usually used in a concentration range of 1 to 10μM. It is widely used for in vivo studies due to its cell permeability, low toxicity, and potency. E-64 is being investigated for the treatment of diseases caused by cysteine protease activity. It has been observed to inhibit programmed cell death and restore antigen and mitogen activity related to normal proliferative responses in T cells obtained from HIV+ donors.

Leupeptin hemisulfate (Ac-Leu-Leu-argininal) is a reversible inhibitor of serine and cysteine endopeptidases. Leupeptin hemisulfate is capable of inhibiting plasmin, trypsin, papain, kallikrein, calpain and cathepsin B. Leupeptin is usually used in a concentration range of 10 to 100μM. Leupeptin is widely used in many protease inhibitor cocktails due to its potency, low toxicity, and range of inhibition. Leupeptin is also used in a quantitative assay to measure macroautophagic flux in mammals and is being investigated for its use in treating malaria.

Pepstatin A is a competitive, reversible aspartic protease inhibitor and inhibits proteases such as pepsin, chyomsin, renin, HIV proteases, and cathepsins D and E. Peptstatin A is highly selective and is often used in a mixture with other enzyme inhibitors. In vitro studies have shown pepstatin A is the only aspartic acid inhibitor that exhibits inhibitory effects on HIV proteases.

Pepstatin A is effective at inhibiting most affected proteases at a concentration of 1μM, but can inhibit pepsin at the picomolar range of concentrations. It is believed to function by way of a collected-substrate inhibition mechanism. Pepstatin A may also suppress differentiation of osteoclasts by inhibiting phosphorylation of MAP kinase and inhibiting NFATc1 expression.

PMSF is a serine protease inhibitor that irreversibly inhibits serine proteases by sulfonylation of the serine residue in the active site of the protease. It is widely used in protocols for the isolation of proteins.

PMSF is inactivated in aqueous solutions. The rate of inactivation increases with increasing pH and is faster at 25°C than at 4°C. The half-life of a 20mM aqueous solution of PMSF is ~35 minutes at pH 8.0. This short half-life means that aqueous solutions of PMSF can be safely discarded after they have been rendered alkaline (pH >8.6) and stored for several hours at room temperature.

Trypsin inhibitor is a serine protease inhibitor from soybeans that is specific for trypsin and trypsin-like proteases. Trypsin inhibitor from soybeans is a monomeric protein containing 181 amino acid residues in a single polypeptide chain cross-linked by two disulfide bridges. Soybean trypsin inhibitor forms a 1:1 complex with the active protease site. Inhibition may be reversed and is pH dependent. The optimal pH for trypsin binding is 8.0.