I extracted protein from a roasted food sample using alkaline extraction followed by acid precipitation. Polyphenols were always coextracted since they were covalently bound with proteins during roasting and the formation of covalent bonds were irreversible. How can I know how abundant covalently-bound and noncovalently-bound polyphenols are in my protein extracts? I would greatly appreciate it if you could provide any suggestion or guidance.
If I use methanol and acetic acid to extract polyphenol and wash protein extracts. Are the polyphenols in washing solution all non-covalently bound? And are the unremoved polyphenols in protein extracts covalently-bound? Can I detect the TPC values in both washing solution and protein extracts to indicate the abundance of noncovalently bound and covalently-bound polyphenols?
To proof that a small molecule is covalently bound to a polymer is very challenging. I was in similar situation when I tried to bind polyphenols covalently to chitosan. Generally, the polyphenols in your washing solution were either non-covalently bound or the covalent bond was cleaved during washing. The unremoved polyphenols are not necessarily bound covalently, as non-covalent interactions can still be too strong.
You could try to depolymerise your protein (e.g. by enzymatic hydrolysis) and then analyse the fragments (e.g. by LC-MS). If you find a fragment with the polyphenol bound covalently, you have a proof.
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