Dear all,
My aim is to determine the number of zinc atoms bound to a protein. I came across a technique called PAR/PMPS assay which is quite accurate in determining the zinc-protein stoichiometry when compared to its crystal structure.
Most papers use PAR with PMPS but only a very few papers used PAR with DTNB (e.g. Okeley, N. et al., 2003). However, I prefer using DTNB over PMPS as PMPS is a mercuric compound (health hazard issues).
So...
1. What is the advantage of using PMPS over DTNB?
2. What is the mechanism of PMPS in reacting with -SH groups?
3. Can DTNB be used to displace the zinc that is/are bound to cysteines?
From my reading, the mechanism of DTNB is to react with free cysteines but I do not know the actual mechanism of PMPS.
I would really appreciate any suggestions and comments.
Thank you very much for your time,
Luke
*DTNB: 5,5′-Dithiobis(2-nitrobenzoic acid)
*PMPS: 4-(Hydroxymercuri)benzoic acid
*PAR: 4-(2-pyridylazo)resorcinol
Dear Pongpawan,
I work with cysteine-containing zinc proteins.
Regarding your questions:
1 and 2). Mercury compounds including Hg(II) displace Zn(II) from zinc proteins. Once it is displaced you may measure it using PAR assay, beacuse released Zn(II) easily binds to PAR. DTNB is a disulphide reagent and it reacts with cysteines bound-to-Zn(II). Once cysteines are oxidized Zn(II) disassociates from protein and you may use PAR to quantify Zn(II). In the second approach reaction is relatively slow, depending on Zn(II)-to-protein affinity and structure of the protein and zinc environment. However, based on my experience and hundreds of zinc motifs studied so fat, it is good assay, however you need to ensure that all Cys are oxidized (please measure it in kinetic mode using your spectrophotometer) and make sure that you use proper molar coefficient of PAR. Below is a link to my paper on Zn(II) and other metal ions complexes with PAR reagent. It contains molar coffeicinet for various conditions.
Article Molar absorption coefficients and stability constants of met...
3) As above, answer is yes. In case of metallothioneins that we study, the oxidation takes about 10-20 minutes when you mix DTNB with PAR. When we use the same assay for zinc fingers or zinc finger-like proteins oxidation can takes even more than 1h.
Note, that we usually use two parallel assays, one for quantification of thiols oxidized by DTNB, another to quantify Zn(II) ions. The molar ratio is very informative and allows you to confirm proper protein perpetration and so on ( you know it well).
The first assay is: DTNB in the presence of low concentrations of EDTA. DTNB oxidizes thiols, while EDTA promotes Zn(II) binding. As a result reaction is reactively fast. To calculate thiol concnetration in our sample we use revised molar absorption coefficient 14,150 cm -1 M-1.
The second assay is PAR in the presence of DTNB (link above). PAR acts as EDTA because it is metal ion chelator. Everything is described in my following papers:
Article Dual Nanomolar and Picomolar Zn(II) Binding Properties of Me...
https://www.ncbi.nlm.nih.gov/pubmed/?term=22922308
if youe have any additonal question please let me know.
Good luck,
Artur
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