I know that trypsin is not a metalloprotease, but I was always told that it is inhibited by very high EDTA concentrations (>0.5M). Is this true? I need to perform a protein digestion on biomineralized proteins. I am using 0.5M EDTA to decalcify then (and no I cannot use an acid for other reasons) and I want to know if I must remove the EDTA by filtration before starting the digestion. I know that proteinase K works fine even in 0.5M EDTA, but I can't find anything about trypsin. All the reported uses of EDTA with trypsin are very low concentration, ~5mM. Does anyone know what happens to trypsin when you increase the EDTA concentration 100x?
Both Mg2+/Ca2+ INHIBIT TRYPSIN. EDTA is a Calcium chelator which will "mop" up the remaining divalent cations so that Trysin can do its action.
In my opinion, having too much EDTA will not affect trypsin action for digestion. Though it is unnecessarily extra concentration of EDTA but it should not hurt.
The ionic strength of 0.5 M EDTA (usually the Na4 salt) is very high (equivalent to 5 M NaCl). That might cause inhibition of enzyme activity. I don't know if this is true for trypsin.
The literature in which to find this type of information is bound to be quite old. I found this paper:
http://jgp.rupress.org/content/jgp/44/6/1103.full.pdf
It is a little hard to understand because of the choice of units. In Figure 1, which is for NaCl, the x -axis is the square root of the ionic strength. The y-axis is an expression of the rate at each ionic strength relative to the control rate, in the form of the log of the ratio +1 (so a value of 1 means that there is no effect of ionic strength, and a value of 2 means a 10-fold enhanced activity). Trypsin is the lower curve, and you can see that the activity decreases as the ionic strength increases above a certain point, which is about 0.2. Figure 3 shows similar modest effects for other common salts. Interestingly, ionic strength had the opposite effect on chymotrypsin. This makes sense to me from the perspective of the substrates of these two enzymes. Trypsin recognized basic side chains (Lys, Arg), so ions may compete with the substrate or reduce dissociation of the cationic substrate from its anion. Chymotrypsin recognizes aromatic side chains. High ionic strength would favor binding of the hydrophobic substrate.
Binding of calcium ion to a loop on bovine pancreatic trypsin stabilizes the enzyme but is thought not to otherwise affect the catalytic rate. Here is a link to the abstract. Chapter Structural Calcium (Trypsin, Subtilisin)
(Sorry, but ResearchGate keeps substituting this link that does not have the 3D structure. It does lead to the same book chapter.) EDTA will, of course, chelate the calcium ions. Working with such a high concentration of EDTA, in addition to the concerns about ionic strength, I would also suggest making sure that your solution is at the pH that you need.- Badges
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