Solutions

Protease Solutions


Thermolysin: According to Sigma. T7902 can be dissolved to about 0.5 mg/ml in 10 mM calcium acetate, 5 mM sodium acetate, pH 7.5. They didn't give info on how long it was stable. Assume it is stable for at least 3 months frozen at -70. Don't freeze/thaw repeatedly. So, aliquot into small amounts.

Protease Inhibitor Solution


BAL (500x): Jan 1999
pH 10ml PBS to pH 6.5. Dissolve the following in 1ml of that PBS.
  • 5 mg leupeptin (10.51 mM)
  • 3 mg antipain (4.96 mM)
  • 10 mg benzamidine (63.86 mM)
  • 25 mg AEBSF (104.30 mM)
  • 1 mg phorphoramidon (1.84 mM)
Aliquot 20 µl of above to each of 50 tubes labeled BAL. Store -70C. When using, thaw and use. Discard any remaining, i.e., do not refreeze.

0.5M EDTA pH 8.0 (1 Liter):
This takes a LONG time to go into solution. Have to add approximately 20g NaOH (about 1/2 mole) if using disodium dihydrate EDTA.
EDTA has fw of 372.24
If want 0.5 moles/liter x 1 liter = 0.5 moles
0.5 moles x 372.24g/mole = 186g to make 1 L

Sources:
  • Leupeptin: L-2884 Sigma, fw 475.6; stock conc can be 10.5 mM
  • Antipain: A-6191 Sigma; fw 604.7; stock conc 5 mM
  • Benzamidine: fw 156.6; stock conc 64 mM
  • AEBSF: A-8456 Sigma; fw 239.7
  • Phosphoramidon: R-7385; fw 543.5; stock conc 1.8 mM

What they do:
  • AEBSF: Use at final of 0.4-4 mM (Boehringer); 0.1-0.25 mM (Sigma) MW=239.5. Soluble in water to 50mg/ml.
  • Rhamno pyranosyl ... (Phosphoramidon): Use at 4-330 µg/ml (5-570 µM). MW=579.6. Soluble to 20 mg/ml in water for stocks.
  • Leupeptin: inhibits serine & cysteine proteases like trypsin, papain, cathepsin B. Stable for >6 months -20 in water or buffer
  • Antipain: inhibits papain and trypsin
  • Benzamidine:
  • AEBSF: irreversibly inhibits serine proteases: trypsin, chymotrypsin, plasmin, kallikrein, thrombin -- use at final concentration of 0.4-4mM
  • Phosphoramidon: metallo-endoproteases, thermolysin, collagenase

Next time: Maybe use AEBSF and leave out leupeptin and antipain?

CHELATORS EDTA and EGTA chelate divalent cations. Log stability constant are given in table: Ca Mg Mn Fe Co Ni Cu Zn EDTA 10.6 8.7 14.0 14.2 16.0 18.6 18.8 16.4 EGTA 11.0 5.2 12.1 11.8 12.3 11.8 17.7 12.9 So, EDTA chelates both Mg and Ca; EGTA chelates Calcium efficiently but leaves Mg if you have considerable Mg around. Source: Data for Biochemical Research, Dawson. Quoted to me by Sigma rep. You can make a solution of EGTA by putting 10g in 90ml 0.1M NaOH, or 1g in 9 ml 0.1M NaOH. To make either EGTA or EDTA from the salt forms you need to add base (more than youÕd expect). And you need to be patient, it will take a while to go in solution. EGTA fw is 380.4. Dissolving 1.9g in 15ml would give 0.33M solution. According to the BOEHRINGER MANHEIM Corporation 1998 Biochemicals Catalog
(To print table change Page Setup to LANDSCAPE.)
INHIBITOR CHARACTERISTICS SPECIFICITY SUGG. STARTING CONC. NOTES STOCK
Antipain dihydrochloride Mr = 677.63 Inhibits papain, trypsin, and cathepsin A and B. Plasmin is inhibited to a small extent. 50µg/ml; 1U of papain is inhibited to 49% of its original activity by 0.9µg of antipain. (EWs Final Conc 0.5µg/ml) More specific for papain and trypsin than leupeptin. The inhibitory potency of antipain is approx. 100-fold higher than that of elastatinal. 5mg/ml (10,000x), aqueous - not ethanol or DMF (dimethylformamide)
Aprotinin
Activity: approx. 200 trypsin inhibitor units/mg lyophilizate at 25oC with benzoyl-L-arginine ethyl ester as substrate .
Purity: electrophoretically homogeneous.
Note: avoid repeated freezing and thawing and exposure to strong alkaline solutions (inactive at pH > 12.8)		 Serine protease inhibitor not acting on thrombin or factor X. Inhibits plasmin, kallikrin, trypsin and chymotrypsin with high activity. 0.06-2µg/ml
(EWs Final Conc 5µg/ml)		 Used for termination of tissue disappregation and subcultivation procedures as well as inhibition of protease activity in serum-free cell culture. Sold as liquid
Bestatin Purity:>95% (HPLC)
Mr = 344.8		 Aminopeptidases like aminopeptidase B, leucine aminopeptidase, tripeptide aminopeptidase and aminopeptidases on the surface of mammalina cells. Does not inhibit carboxypeptidases. 40µl/ml Shows antitumor properties, enhances blastogenesis and lymphocyte proliferation in vitro and establishes a delayed-type hypersinsitivity in vivo  
Benzamidine Aqueous        
Chymostatin Mr = 607.71 Specific inhibitor of a, b, gamma, and delta?-chymotrypsin.
Note: soluble in glacial acetic acid and DMSO		 6-60µg/ml.1U chymotrypsin is inhibited to 49% of the original activity by 1.8µg of chymostatin.
(EWs Final Conc 0.5µg/ml)		 Mixture of chymostatins a, b, and c Stock is made in DMSO, maybe aqueous
DFP     (EWs Final Conc 1mM)   Stock is made in DMSO or acetone
EDTA Mr = 372.24 Chelator; inhibits metalloproteases 0.2-0.5mg/ml The disodium salt of EDTA will not go into solution until the pH is adjusted to approx. 8.0 by addition of e.g. NaOH  
EGTA Mr = 380.35 Chelator; specifically inhibits Ca2+-dependent proteases   Hardly soluble in water and all organic solvents. Slightly soluble in DMF and DMSO.x  
E-64 Purity:>95% (HPLC)
Mr = 357.4		 Inhibits papain and other cysteine proteases like cathepsin B and L 0.5-10µl/ml    
Leupeptin (synthetic) Mr = 475.6 Inhibits serine and thiol proteases like trypsin, plasmin, proteinase K, kallikrein, papain, thrombin, and cathepsin A and B. Not affected are a, b, gamma, and delta?-chymotrypsins, pepsin, cathepsin D, elastase, renin and thermolysin 0.5µg/ml
(EWs Final Conc. 0.5µg/ml)		   5mg/ml (10,000x)
Pefabloc®SC
 Mr = 239.5 Specific, potent and irreversible inhibitor of serine proteases. The inhibitory activity is comparable to PMSF or DFP, however, it is non-toxic. 0.1-1.0mg/ml Ideal substitute for PMSF and DFP due to markedly lower toxicity. Shows higher stability at physiological pH-values and higher effectivity to inhibit a broad rangeof serine proteases. Used to completely inactivate proteinase K. Represents in contrast to PMSF an excellent blocker of thrombin activity in serum or plasma.  
Pepstatin (from Streptomyces species) Mr = 685.9 Inhibits potently the HIV protease and other aspartic proteases like pepsin, renin, cathepsin D, chymosin and many microbial acid proteases 0.7µg/ml Insoluble in water  
PMSF
 Purity: chromatographically homogeneous
Mr = 174.4
Note: to be added freshly at every purification step		 Inhibits serine proteases like chymotrypsin, trypsin and thrombin as well as the cysteine protease papain (reversible by DTT treatment). Does not inhibit metalloproteases, most cysteine proteases and aspartic proteases 17-170µg/ml
(EWs Final Conc 1mM)		 Under certain conditions, PMSF may have a halflife as short as 35min. High salt conditions (3.0M), often used to precipitate proteins, may also reverse the inhibition of PMSF. Pefabloc®SC is recommended as a non-toxic water-soluble and water-stable alternative.  
Phosphoramidon
 Purity:>95% (HPLC)
Mr = 543.5		 Metallo-endopeptidases, especifically thermolysine. 4-330µl/ml      


Abbreviations:
EDTA = (Ethylenedinitrilo)tetraacetic acid disodium salt
E-64 = N-[N-(L-3-Trans-carboxirane-2-carbonyl)-L-leucyl]-agmatine
Pefabloc®SC = AEBSF
4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride
PMSF = phenylmethylsulfonyl fluoride
Phosphoramidon = N-(a-Rhamnopyranosyloxyhydroxyphosphinyl)- L-leucyl-L-tryptophan disodium salt
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Last update Sept 18, 2000
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