free as in unreacted sulfydryls, not free as in speech, nor free as in beer :)
I am working with an 80 kDa plasma protein with 5 cysteines, 2 of which are in a disulfide bridge.
There are a lot of things in blood, and I am not a biochemist. Do sulfhydryls generally stay unreacted, or is it more common that they find other things to bind to?
I don't see significant densities at high molecular weight in native Western blots, implying no significant dimerization nor binding to moderate-to-large MW proteins
Hi there,
Cysteine reactivity will depend on its accessibility to solvent. If you have the purified protein run the assay with DTNB to quantify free cysteines and compare with the reactivity of the denatured protein and of the denatured and reduced protein. If free cysteine is accessible to solvent you should get a signal from the first experiment. If not, it should become more reactive with denaturation and reduction.
Hi Dominique,
Thanks for making me aware of DTNB, it may be useful for some experiments we have with a purified form of the protein, and a good place to start. I'll get some ordered for next week.
Any procedure I've seen for purification of our protein lists recovery from plasma at ~5%, which to me leaves open the possibility that the majority of the endogenous form may be chemically different.
Hi there
Free thiols including free cysteines in proteins oxidation state depend on redox potential conditions. the most important regulator being glutathione pair GSH AND GSSG (also a cysteine containing small peptide)
So the answer to your question is YES truly free under the right conditions and NOT free under other conditions.
Be aware, as soon as you extract your protein thiols start oxidise, that is why most extraction buffers contain DTT, another thiol
Hi Leonardo,
Thank you for you answer. The conditions in my experiment are plasma diluted into PBS. Where does that leave my protein?
Albumin has many disulfide bonds and one free Cys residue, if I recall correctly, and it is the most abundant plasma protein. The redox environment of plasma ought to affect the monomer:disulfide-bridged equilibrium of albumin if the free thiol is accessible, and I believe it is. Here is a paper about the effect of exercise on the free thiol content of plasma proteins, indicating that it is decreased by strenuous exertion:
https://www.jstage.jst.go.jp/article/jhs/48/5/48_5_399/_article
Hi Jeremy
As soon as you extract your protein in PBS only, as you mention, the free thiol is likely to oxydise and form a dimer with a small abundant thiol like GSH or cysteine.
However when you run your western, chances are you are running it in laemni buffer, (loads of SDS and mercaptoethanol, again another thiol) those are highly reducing conditions that will break most disulfide bonds. This is probably why you dont see dimers.
Elmans protocol ( DTNB ) may give you the answer
Hi Jeremy,
indeed, the redox state of an solvent accessible cysteine group not involved in disulfide bridges depends on the physiological condition of an organism. The free cysteine interacts with various plasma compounds. Here is one example that highlights this issue on albumin.
Article Redox State of Human Serum Albumin in Terms of Cysteine-34 i...
I also did Ellman tests on albumin, and the free cysteine concentration was always smaller than the corresponding protein concentration, although it should be expected to be 1:1. It might also depend on the protein lot you may use ...
kindly, JR
Wow, thank you everyone! I did a lot of reading yesterday to get some exposure to the forces that influence cysteine chemistry, and I really appreciate the insights and papers you sent me.
Hi Jeremy, I used to support the production of recombinant HSA and we compared our product with all (most) available human derived products. Blood derived albumin typically has only half of the 'free' sulhydryl (cysteine 32) with half complexed with free circulating cysteine. There are also disulphide dimers of HSA that are only dissociable by treatment with a sulphydryl reagent such as DTT.
Hi David,
Thank you. It sounds like your observations are roughly in line with the article Jörg posted (table 11.1, HNA1 = 40% in plasma/serum samples), which is always nice to hear!
If it's of any interest, I found this paper that puts homodimer concentration around 4% for healthy subjects (https://www.nature.com/articles/srep35987.pdf, figure 2A, sum of three white bars).
Out of curiosity, were you determining the Cys32 complexation partner with some sort of MS assay?
Sorry, it's actually 34C. Yes we measured intact masses of unmodified and various processed components using ESI-MS.
1. Inoue, M., Minamiyama, Y., Yu, H., Yamamasu, S., Inoue, K. and Sato, E. (1995) Biochemical and clinical aspects of extracellular glutathione and related thiols. In Biothiols in health and disease (Packer, L. and Cadenas, E., eds.), Marcel Dekker, New York, pp. 287-304.
2. Sen, C. K. and Packer, L. (2000) Thiol homeostasis and supplements in physical exercise. Am. J. Clin. Nutr., 72, 653S-669S.
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