Gary-
I, too, am interested in the low-pH activity of Glu-C. In his chapter in Methods in Enzymology (Vol. 45, p 469 (1976)), Drapeau states that an Asp-Gly bond in lysozyme is split by Glu-C at a low but detectable rate, but that this low cleavage is not observed when the digestion is carried out in ammonium acetate buffer at pH 4.0. Does this imply that the enzyme is even more specific for glutamyl bonds at pH 4.0?
According to product literature, when digestions are done with ammonium carbonate buffer at pH 7.8, only glutamyl bonds are cleaved by Glu-C, and both aspartyl and glutamyl bonds are cleaved in ammonium phosphate buffer. I would very much like to know what happens to the asp/glu specificity at pH 4 if phosphate is present.
Vernon
Vernon A. Shoup
Regeneron Pharmaceuticals
Rensselaer, NY 12144
vernon.shoup@regpha.com
--------------------------------------
Date: 03/04/1999 12:54 PM
From: GARY.W.LANGE
Katheryn, I was quite interested in your suggestion that Glu-C (V-8) would work at a low pH. Even though this has been reported in the literature (before MS was routinely used for proteins) I have been totally and absolutely unable to show any cleavage on a protein of any sort at a pH of 4.0 (the purported lower pH optimum). After trying it on some half dozen proteins (and I think even a few peptides) I gave up trying to use it at a low pH. Have you, or any of the other members successfully used this enzyme at a low pH and obtained greater than a 5-10% cleavage rate? Don't forget, Asp/Pro cleavages don't count as they were most likely induced by the low pH. Maybe if a lot of members respond positively I'll put Glu-C back in the arsenal for low pH digestions! I'm assuming that you need to find the disulfide pairs, otherwise you would just reduce and alkylate. I guess for a tightly knotted (pr!
otein/peptide) that doesn't respond to trypsin, I would try something like subtilisn or thermolysin, both quite non-specific. Lots of time points, of course the easiest way to look at this is with a mass spec. Another useful way (probably the best choice) would be to try pepsin (at pH 2.0, very nonspecific) again looking at lots of time points and a relatively low amount of enzyme(1:100 or 1:150). If you obtain 3 or 4 useful cleavages (as shown by MS) you could then raise the pH back to 7.5 and try trypsin again. The sites that were unavailable originally might now be exposed. When trying to obtain a disulfide bonding pattern one should be a little cautious about raising the pH too high, especially if incubation is at 37 C. Depending on the peptide you can cause reshuffling of the disulfide pairs. (My pH cutoff for a 37 C digestion is 7.5. Above that I have seen reshuffling. YRMV). Once you obtain some !
new masses you can then follow up (using a small aliquot) by
rerunning on mass spec. For MALDI work I use a 10X stock of 20 mM TCEP (freshly made in water). Incubation with 2 mM TCEP breaks most bonds in peptides within 30 minutes, incubating a pH 7 or higher at 37 degrees. HPLC separation of linked peptides, followed by TCEP might make the puzzle easier to interpret. If you try Glu-C (V-8) at a high pH (7.5-7.8) and have success don't forget to follow up with a bit of Asp-N. I have found that Glu-C (at high pH's) often doesn't cleave at aspartic acids. Asp-N works fairly well, especially as a secondary enzyme when the peptide/protein has been partially digested. Good luck, sounds like fun!
Gary Lange
Monsanto Co.
gary.w.lange@monsanto.com
ph 314- 737-6602
fax 314-737-7005