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] >>> >> >>
