Chromophores and You

Chances are if you work in a lab that does any sort of biochem related activities, at some point you’ll have to stick something to a protein. The two most popular points of attachment are primary amines and thiols because they’re fairly nucleophilic under conditions which won’t completely destroy your protein.

protein001

Primary amines are most often conveniently encountered in the form of lysine residues, or N-termini. In fact, most proteins are lousy with the damn things which is a problem if you want site specificity. The N-terminus tends to be more nucleophilic than other sites, but you’ll generally end up drenching your protein with the reactive compound and hope it doesn’t land anywhere that it will get in the way. Thiols show up as cysteine residues, and unlike primary amines tend to be much rarer and quite a few proteins have none at all naturally. If you know the site where you want something attached, it is fairly easy to engineer a cysteine there for site specificity.

thiol_labeling

Methods for modifying thiols tend to come down to two options: forming a thioether, or forming a disulfide. The most popular groups for the first are iodoacetamides, and maleamides. The third usually uses a thiosulfonyl. However, thioethers are pretty irreversible, and disulfides usually get reduced somewhere along the line. So how do you nail something onto a thiol but leave yourself the option of selectively removing it later?

488_phenylmercury

That’s phenylmercury, baby! Stuck to Alexa Fluor® 488. This autism campaigner’s wet dream of a chromophore is available from Invitrogen. That S-Hg bond will stand up to TCEP reduction, but can be knocked off by DTT or 0.1 M HCl.

Can’t say I have any plans to rush out and use this thing though. I’ve got much better means of killing brain cells.

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