Ox. reagent for proteins

Robert Solomon Bioc rgs at mole.bio.cam.ac.uk
Thu Dec 15 10:31:23 EST 1994


Lynne_A_WHITEHEAD at UMAIL.UMD.EDU (lw75) writes:
>
>I was wondering if anyone knows of a way to form disulfide bonds in a
>protein in vitro.

<snip>

>First, does anyone know why or what keeps
>disulfides from being formed in the cytoplasm (pH?, ect.)? and are there any
>oxidizing reagents (or anything) that can be used to produce the disulfide
>bonds (in vitro) in my proteins once it's been isolated?

The cyto is kept strongly reducing by high levels of GSH, amongst other
things.

As for oxidising proteins,

Have just been looking at the lit. on this, and found the following
references of particular interest.  I will group them according to
the reagent used...

Cu2+..
BBA _743_ , 331 - ...
JMolBiol _225_ , 1013 - ...
I found the method in the JMB paper quite useful - add CuSo4 to 0.1 mM
in buffer at RT, end the reaction with excess EDTA (I then desalt, as
well).

Potassium Ferricyanide..
JBiolChem _265_ , 17341 - ...
involves adding the reagent to buffered protein at a final conc. of
10mM.  Did not work as well as Cu2+ for me, but don't let that put
you off having a go.

Mixtures of glutathione oxidised and reduced..
see any Creighton paper re refolding of BPTI........

DTNB = Ellman's reagent = 5,5'-dithiobis(2-nitrobenzoate)..
usually used to assay free sulphydryls (e.g. see Shaun Black's recent
posts to this group, giving a protocol for that).  However, sometimes
promotes disulphide formation, as in..
BiochemJournal _294_ , 63 - ...
ibid           _290_ , 279 - ...
ibid           _173_ , 701 - ...
JBiolChem _266_ , 20305 - ...

O2..
i.e. get rid of the DTT/B-ME and let the thing air oxidise.  Bubble
air or O2 (for the brave) through the protein soln to hurry things
up.  (this works for me, but slowly and incompletely).
BiochemJournal _238_ , 683 - ...

>Or will this type
>of manipulation of the protein dammage it or not  produce the correct 3D
>configuration as it would be formed in vivo?

Well, if you have multiple cysteines, the possibility may exist for the
wrong crosslinks to form, but it may work - suck it and see, as my boss
used to say.  Not too sure of the long term effects of Cu or Fe on
proteins, but metal-mediated oxidation (e.g. of tyr residues) is not
unknown, so I think it's best to get rid of them once (if) they've
done the job.  Good luck, and apologies for the incomplete references,
which are also not comprehensive of the whole literature on this topic.

Rob Solomon
Lost in Cyberspace

And for those in Milton Keynes, UK, a joke for the festive season
(stolen from someone's .sig)....

And then there was the dyslexic devil-worshipper, who sold his soul
to Santa..





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