🚀 go-pugleaf

juicymixx@mailinator.com wrote:
> 
> Hello,
> 
> This is a pretty simple question; however I can't seem to find an
> answer to it.   I have a rather small protein (60 amino acids) in which
> I've mutated in a single cysteine for labeling purposes.   There are no
> other cysteines in the protein.   Additionally the protein is normally
> unstructured in solution (that is in 30uM phosphate buffer at 25C,
> there is no significant structure).   Recently we've noted that after
> purifying the protein (but before labeling it) we appear to have some
> dimer formation which isn't seen in the wild type protein.
> 
> I was just wondering what the equilibrium constant (reaction rate,
> etc.) for intermolecular disulfide formation from 'free' cysteine would
> be (or better yet, cysteine in a polymer/peptide/protein).   Someone
> has probably already done this experiment, however I'm not sure of
> where to look for this number...

Work under inert gas, add some modest reducing agent.  Cysteine to
cystine is entirely reversible under mild conditions if not rpevented
beforehand,

http://en.wikipedia.org/wiki/Dithioerythritol.  

At high concentrations it induces cleavage of disulfide bonds.  At low
concentrations it prevents oxidation of SH groups during
polyacrylamide gel electrophoresis.


-- 
Uncle Al
http://www.mazepath.com/uncleal/
 (Toxic URL! Unsafe for children and most mammals)
http://www.mazepath.com/uncleal/lajos.htm#a2

DK wrote:
> In article <1168370284.242721.50740@o58g2000hsb.googlegroups.com>, juicymixx@mailinator.com wrote:
> >
> >Hello,
> >
> >This is a pretty simple question; however I can't seem to find an
> >answer to it.   I have a rather small protein (60 amino acids) in which
> >I've mutated in a single cysteine for labeling purposes.   There are no
> >other cysteines in the protein.   Additionally the protein is normally
> >unstructured in solution (that is in 30uM phosphate buffer at 25C,
> >there is no significant structure).   Recently we've noted that after
> >purifying the protein (but before labeling it) we appear to have some
> >dimer formation which isn't seen in the wild type protein.
> >
> >I was just wondering what the equilibrium constant (reaction rate,
> >etc.) for intermolecular disulfide formation from 'free' cysteine would
> >be (or better yet, cysteine in a polymer/peptide/protein).   Someone
> >has probably already done this experiment, however I'm not sure of
> >where to look for this number...
>
> There is no single number to answer your question. "It depends".
> Lots of factors will influence propensity of your cysteins dimerizing:
> their precise pKa (depends on envirnoment; put a Lys next to it and
> it will be A LOT more reactive), degree of solvent exposure and
> steric accessibility, amound of O2 and pH of your buffer,
> the amount of traces of metals, etc, ect. Just work as fat as you can,
> perform labeling and, if the experiment demands it, purify monomer
> away from dimer on gel-filtration or by affinity chromatography
> against the probe.
>
> DK


Thanks for the reply.   I understand that the local environment around
the CYS makes a difference (as well as the bulk environment).   Is
there any literature (or a journal article) that expands on this?
Someone must have tried to measure the reaction rate under some certain
condition...?

Thanks again.

"Bob" <bbx107.XYZ@excite.XYZ.com> wrote in message
news:a49bq2lve6k3skf7thuqronoo2csh4hq3a@4ax.com...
>
[Bob]
> If you have it at fairly high concentration in an oxidizing
> environment (air), and the Cys are exposed, one would expect that.
> Solutions include keeping the conc low, avoiding oxidation -- and
> reducing the dimers back to monomers, say with mercaptoethanol.
>
[hanson]
Interesting. Are you basically telling here that the oxidized/dimer of
Cysteine, now Cystine: HOOC(NH2)C-CH2-S-S-CH2-C(NH2)COOH
together with 2 HS-CH2-CH2-OH disproportionates/redoxes into
 2 HS-CH2-C(NH2)COOH  and HO-CH2-CH2-S-S-CH2-CH2-OH.

Does HSEtOH work as a reducer for all -S-S- molecules in biologial
systems?

juicym...@mailinator.com wrote:
>
> This is a pretty simple question; however I can't seem to find an
> answer to it.

Dimerization is almost a given under your conditions.

There was a time, in the paleolithic days of biochemistry, that newly
isolated proteins were routinely kept in buffers containing
mercaptoethanol (2 to 10 mM) until you knew better. Later,
dithiothreitol (Cleland's Reagent) came along, which is a stronger
reducing agent for stubborn cases, and didn't stink quite as much.

Of course, any labeling you plan to do on the cyse will be completely
derailed until you get rid of all the added thiols by dialysis.
Afterward, keep the protein under nitrogen and label as soon as
possible. 

Dangerous Bill

"Bob" <bbx107.XYZ@excite.XYZ.com> wrote in message 
news:5srdq2haa7beq0d40s2o9ipn1eobfki0br@4ax.com...
> "hanson" <hanson@quick.net> wrote:
>
>>"Bob" <bbx107.XYZ@excite.XYZ.com> wrote in message
>>news:a49bq2lve6k3skf7thuqronoo2csh4hq3a@4ax.com...
>>>
>>[Bob]
>>> If you have it at fairly high concentration in an oxidizing
>>> environment (air), and the Cys are exposed, one would expect that.
>>> Solutions include keeping the conc low, avoiding oxidation -- and
>>> reducing the dimers back to monomers, say with mercaptoethanol.
>>>
>>[hanson]
>>Interesting. Are you basically telling here that the oxidized/dimer of
>>Cysteine, now Cystine: HOOC(NH2)C-CH2-S-S-CH2-C(NH2)COOH
>>together with 2 HS-CH2-CH2-OH disproportionates/redoxes into
>> 2 HS-CH2-C(NH2)COOH  and HO-CH2-CH2-S-S-CH2-CH2-OH.
>>
[Bob]
> or heterodimers
>
> Note that the OP is talking about Cys in proteins, not free cystine.
> The oxidation is dimerizing his protein (60 subunits to 120, in his
> case). In other cases, it can create unwanted crosslinks within a
> peptide chain. Major structural effects.
>
[hanson]
>>Does HSEtOH work as a reducer for all -S-S- molecules in biologial
>>systems?
>>
[Bob]
> Yes, sort of.
>
> But a key point is that enzymes are large and BME
> (beta-mercaptoethanol) is small. So one can easily have a vast molar
> excess of BME. (You may recall that, typically, in enzyme reactions
> the conc of substrate is a few order of magnitude higher than the
> enzyme conc. Same idea.) Using it with small molecules, at similar
> conc, would be more problematic.
>
> Bill Penrose has usefully elaborated. BME is simply one commonly
> reducing agent.
>
> (But BME is doubly effective as a reducing agent. Not only does it
> reduce disulfide bonds in the proteins, but it reduces the
> concentration of people around you, thus making it easier for you to
> concentrate on your work. The second effect is sometimes
> irreversible.)
> bob
>
[hanson]
Bob, you are a good man. Thanks.
I remember the smell faintly (though not being as bad a
Et-SH), so, since I have left the lab long time ago, when
I come back home from my stints in the field (deserts,
mtns. or jungles, after a few weeks and 18 hrs flights),
BME smells like roses when compared to me... ahaha...
Thanks for the laughs, Bob,
ahahaha... ahahanson 

Bob wrote:
>... it reduces the
> concentration of people around you, thus making it easier for you to
> concentrate on your work. The second effect is sometimes
> irreversible.)

My late thesis supervisor (late, meaning 40 years ago) once said that
if he didn't smell mercaptoethanol coming into the lab in the morning,
it meant no one was working. On the other hand, it clung to the clothes
almost as nauseatingly as collidine or lutidine. 

Dangerous Bill