Enzymatic proteolysis can be arrested or minimized through the use of socalled cocktails containing combinations of serine protease, cysteine protease, aspartic protease, and metalloprotease inhibitors. The following combinations (or cocktails) have proved to be highly effective in the isolation of intact proteins, and some have are so popular that they are commercially available. General Protease Cocktails typically contain AEBSF, E-64, bestatin, leupeptin, aprotinin, and sodium EDTA. The latter is included only if the protein or enzyme being purified does not require a divalent metal cofactor for structural integrity. Fungal and Yeast Protease Cocktails typically contain AEBSF, pepstatin A, E-64, and 1,10- phenanthroline. Mammalian Cell Protease Inhibitor Cocktails typically contain AEBSF, pepstatin A, E-64, bestatin, leupeptin, and aprotinin. (Other metal ion chelators, such as 0.5 mM EGTA, may be added to suppress the activity of calcium ion-dependent proteases such as calpain. Again, one must determine whether the protein or enzyme being purified does not require a divalent metal cofactor for structural intgrity or biological activity. Bacterial Extract Protease Cocktails typically contain AEBSF), pepstatin A, E-64, bestatin, and sodium EDTA. For convenience, the inhibitory properties of these reagents are summarized as follows. AEBSF: This broadspectrum serine protease inhibitor, which is stable in water for one month when stored at –20 °C, has an inhibitory range 0.1-1.0 mM. AEBSF also inhibits cysteine proteases, such as papain. Antipain: This inhibitor is effective against serine proteases (e.g., plasmin, thrombin and trypsin, but not chymotrypsin, as well as some cysteine proteases, such as calpain and papain. Its inhibitory range is 1-100 μM, with IC50 = 0.15 mg/mL for papain, 0.25 mg/mL for trypsin, 1.2 mg/mL for cathepsin A). Prepare at 10 mM in water. Aprotinin: This water-soluble inhibitor, which is stable for months, when stored at 4 °C, blocks and many serine proteases at 0.3 μM (or equimolar with proteinase), but is without effect on thrombin or bloodclotting factor Xa. Bestatin: This aminopeptidase inhibitor, which has an inhibitory range of 1-10 μM, is prepared as a 1 mM stock solution in methanol and stable for 1 month, when stored at –20 °C. Chymostatin: This chymotrypsin-like serine protease inhibitor blocks chymase and cathepsins A,B,D and G, as well as some cysteine proteases (including papain). Its inhibitory range is 10–100 μM; IC50 = 0.1 mg/mL for chymotrypsin). Stock solutions are prepared at 10 mM in DMSO. DFP: This highly effective broad-spectrum serine-protease inhibitor is also a highly toxic nerve poison, and a well-ventilated fume hood and heavy rubber gloves should be used to avoid inhalation or contact with skin. For safety, DFP should be prepared as a 200 mM solution in anhydrous isopropanol or propylene glycol and then diluted 60-100x into cell extract. In aqueous solution, DFP rapidly decomposes (t1/2 = 20 min in water). E-64: This thiol protease inhibitor, which is effective in the 1-10 μM concentration range, is prepared as a 1 mM stock solution in water and is stable 1-2 weeks at –20 °C. EDTA: This broad-spectrum metalloproteinase inhibitor (effective range = 1-10 mM) nonspecifically inhibits many metalloenzymes, especially those with metal ion Kd values above 1 mM. N-Ethylmaleimide: This chemical modifying agent inhibits most thiol-proteases by reacting irreversibly with essential active-site thiol groups. NEM is water-soluble at >10mg/ml, and must be prepared freshly. Leupeptin: This inhibitor blocks trypsin-like serine proteases, such as trypsin, chymotrypsin, chymase, pepsin and thrombin. Leupeptin inhibits selected cysteine proteases (calpain, cathepsin B, H & L and papain). The inhibitory range is 10-100 μM, with the following IC50 values: 2 mg/mL for trypsin; 8 mg/mL for plasmin; and 0.5 mg/mL for cathepsin B. When prepared at 10 mM in water, leupeptin is stable 6 months when stored at –20 °C. Pepstatin A: This inhibitor blocks aspartic proteases, such as renin, chymosin and pepsin (inhibits at 1 μM). Prepare as 1mM stock solution in methanol or DMSO. 1,10-Phenanthroline: This broad-spectrum metallo-proteinase inhibitor (with an inhibitory range of 1– 10 mM) is prepared at 200mM in methanol or DMSO. This reagent nonspecifically inhibits many metalloenzymes, especially zinc- and manganese-dependent enzymes and those with metal ion Kd values above 1 mM. PMSF: This broad-spectrum serine protease inhibitor, which is soluble in DMSO and stable for 1 month at –20 °C, has an inhibitory range of 0.1– 1.0 mM. PMSF also inhibits cysteine proteases (including papain), which are reactivated by 1 mM 2-mercaptoethanol or dithiothreitol. Phosphoramidon: This agent potently inhibits metalloendoproteinases, including thermolysin and elastases at 1–10 μM, but only weakly inhibits collagenase. When prepared at 1 mM in water, phosphoramidon solutions are stable for 1 month at –20 °C. TLCK: This haloketone-containing reagent inhibits trypsin-like serine proteases at 10-100 μM. TLCK solutions must be freshly prepared at 10 mM in 1 mM HCl and then diluted 100x into cell extracts. TPCK: This haloketone-containing reagent inhibits chymotrypsinlike serine proteases at 10-100 μM. TPCK solutions must be freshly prepared at 10 mM in 1 mM HCl and then diluted 100x into cell extracts. See also AEBSF; Antipain; Aprotinin; Bestatin; Chymostatin; E-64; NEthylmaleimide; Leupeptin; Pepstatin A; 1,10-Phenanthroline; PMSF; Phosphoramidon; TPCK
