How to remove PLP cofactor from SufS and IscS (Enzyme) to make Apo confirmation to Holo confirmation.
To confirmation that all protein in the same confirmation of the Protein. i want to remove PLP from the protein Apo confirmation to Holo confirmation. after that how can i added again.
Hi Abdul,
From apo to holo form or holo to apo form - perhaps performing dialysis in the presence of PLP cofactor in dialysis buffer to obtain the holo-form or make up a dialysis buffer (1-2 L) in the absence of PLP.
The on and off rate of the interaction (enzyme:PLP) will dictate the achievement of the apo or holo form.
You can do mass spectrometry to confirm whether you have the apo/holo form following dialysis.
All the best!
I assume you are talking about pyridoxalphosphate, the active form of vit. B6.
In enzymes, this occurs covalently bound to the e-amino group of a particular Lys as LLP (see for example pdb-code 4eu1). From that bond, PLP is displaced by the amino-group of the substrate amino acid. Thus, it is probably easiest to incubate the enzyme with high concentrations of PLP, so that all sites have LLP bound, and then desalt the excess PLP away if desired. This would give you the holo-enzyme.
If you really want the apo-form, you need to incubate with the substrate amino acid and then try to remove the aldimine. Depending on of the stability of the enzyme-aldimine complex the methods required would be more or less rigorous, say, incubation with mild to strong denaturants (Hofmeister series) followed by desalting or dialysis. Perhaps you can find something in BRENDA?
Hello Abdul,
Well this is an interesting question because it seems that no all PLP-containing enzymes react in the same way with pyridoxalphosphate.
In tryptophanase (TNase), it is known that a simple incubation of a solution of TNase from 25°C to 2°C (at cold) lead to an inactivation of the enzyme with loss of pyridoxal phosphatase (See for instance: Erez et al 2002 Biochim Biophys Acta 1594(22) 335-340). So a simple incubation at 2°C trasnforms holoTNase into apoTNase ... Maybe you could simply try an incubation at cold ? Easy to try, right? Then what I know is that TNase is subjected to a conformational transformation thta makes the aldimine bond spontaneously cleavable ... Is it working the sme way with your enzyme, I dunno. But it will be quite easy to check.
Philippe
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