Thank you for such great explanation. Hopefully people won't consider me hijacking this thread. Nian
On Sun, Apr 17, 2011 at 11:08 AM, Artem Evdokimov <[email protected] > wrote: > TCEP relies on a completely different chemistry to achieve the same goal as > BME or DTT. > DTT/BME use S(-)>SS with redox potentials of -0.26 to -0.33 V (at pH 7) > whereasTCEP uses P(3+)->P(5+) oxidation with redox potential that's a lot > higher (I don't know of a reference with a stated redox potential for this > system) because TCEP readily reduces oxidized forms of both DTT and BME in > solution. TCEP also does not readly break S-Hg bonds unlike DTT or BME. > > TCEP does not readily react with oxygen in solution (both DTT and BME react > rapidly) and has long shelf life as buffered 1M solution at pH 5.6-6.3. > > TCEP has two non-trivial disadvantages that are for the most part not > relevant for the purposes of crystallography: one is that it does not work > very well in high Phosphate concentration, and the other is that it hinders > the reaction of thiols with haloacetamides and suchlike (because it itself > can react with haloacetamides). If you're labeling with haloacetamides you > may want to use tri- tertbutul phosphine instead (it's much less soluble > than TCEP, but 1 mM solution in water can be made). > TCEP does not permeate biological membranes and therefore has been used > to reduce thiols outside the cell while keeping intracellular ones intact. > Due to its size and charge, it also is quite selective which protein > disulphides are readily reduced - ones on or near protein surface are > reduced quickly whereas buried or shielded ones are often not reduced at all > w/o the use of a chaotrope. That's rather useful to us as it often allows us > to reduce the unwanted inter-molecular disulphides (bad: aggregates) while > at the same time preserve the valuable intra-molecular ones (good: > structure) > > Artem > On Sat, Apr 16, 2011 at 11:46 PM, Nian Huang <[email protected]> wrote: > >> Dear Horacio, >> How does TECEP compare to BME or DTT? People claim it is better, but I >> want some crystallographers' opinion? >> >> Nian >> >> On Sat, Apr 16, 2011 at 4:24 PM, Horacio Botti <[email protected]>wrote: >> >>> Dear Mike >>> >>> BME readily autooxidizes (need for metal traces and dissolved O2). Is >>> yours a metalloprotein? Is your buffer contaminated with metals? Those >>> situations would make the case a bit different. If not, unless your BME >>> stock is already oxidized, blocking of the accesible thiols with BME should >>> take some time. If you treat your protein for 40 min with fresh BME you >>> should not observe thiol blocking. If you let the preparation to stay for >>> several days, even at 4-6 °C you may observe the blocking that you may be >>> observing. >>> >>> If you want to prevent Cys blocking you can also change to DTT (it is a >>> dithiol, does not readily form mixed disulfides) and use it with caution >>> (for thiol reduction it is advisable to use stoichiometric DTT (with respect >>> to the number of Cys you need to reduce) and 10 fold excess of BME, look for >>> their redox potentials). Take care of not "over-reducing" your protein if >>> internal disulfide bonds are expected. Once reduced I suggest you to remove >>> any reducing agent and store the protein at -80 °C. >>> >>> External Cys can be easily oxidized, they are highly expossed to metals >>> and oxidants (H2O2, BME disulfides, etc). Diffusion is for sure much faster >>> than SS bond formation, although some cys react at almost >>> diffusion-controlled rates with oxidants (is yours a thiol'dependen t >>> peroxidase?) You can take a look at the following reference (advertising): >>> >>> 2011. Factors Affecting Protein Thiol Reactivity and Specificity in >>> Peroxide Reduction. Chem Res Toxicol. >>> >>> Metals can contaminate bad quality materials (water, salts, buffers, >>> etc), take care of that too. If you need to control the redox state of your >>> protein you should use DTNB (Ellman´s reagent), or DTDPy, to measure >>> accesible reduced thiol groups. >>> >>> Good luck! >>> >>> Horacio >>> >>> >>> >>> >>> Quoting Kendall Nettles <[email protected]>: >>> >>> We see BME adducts in all of our estrogen receptor structures, though we >>>> don't always put them in the models. Sometimes we only see one or two >>>> atoms >>>> of the adduct, and in others it is completely ordered. We only see it on >>>> the solvent accessible cysteines. We do it on purpose. We used to treat >>>> the >>>> protein with iodoacetic acid to generate uniform modification of the >>>> cysteines, but then we realized we could get then same homogeneity with >>>> 20-50mM BME. >>>> >>>> Kendall Nettles >>>> >>>> On Apr 15, 2011, at 4:09 PM, "Michael Thompson" <[email protected]> >>>> wrote: >>>> >>>> Hi All, >>>>> >>>>> I was wondering if anyone knew whether or not it is possible for >>>>> reducing agents with thiol groups, such as DTT or beta-mercaptoethanol >>>>> (BME), to form covalent S-S bonds with Cys residues, particularly >>>>> solvent-exposed Cys? I have some puzzling biochemical results, and in the >>>>> absence of a structure (thus far), I was wondering if this might be >>>>> something to try to control for. I have never heard of this happening (or >>>>> seen a structure where there was density for this type of adduct), but I >>>>> can't really think of a good reason for why this wouldn't happen. >>>>> Especially for something like BME, where the molecule is very much like >>>>> the >>>>> Cys sidechain and seems to me like it should have similar reactivity. The >>>>> only thing I can think of is if there is a kinetic effect taking place. >>>>> Perhaps the rate of diffusion of these small molecules is much faster >>>>> that >>>>> the formation of the S-S bond? >>>>> >>>>> Does anyone know whether or not this is possible, and why it does or >>>>> does not happen? >>>>> >>>>> Thanks, >>>>> >>>>> Mike >>>>> >>>>> >>>>> >>>>> >>>>> -- >>>>> Michael C. Thompson >>>>> >>>>> Graduate Student >>>>> >>>>> Biochemistry & Molecular Biology Division >>>>> >>>>> Department of Chemistry & Biochemistry >>>>> >>>>> University of California, Los Angeles >>>>> >>>>> [email protected] >>>>> >>>> >>>> >> >
