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I have a pretty basic biochemistry question but am having trouble finding the answer to it:

Normally, DNFB (dinitrofluorobenzene, or 1-fluoro-2,4-dinitrobenzene to be exact) reacts with just the amino terminus of amino acids, creating dinitrophenyl (DNP) derivatives. However, with lysine, DNFB can react in two places (at the amino terminus and to the epsilon amino group). The DNP derivative of lysine is referred to as "epsilon-DNP-lysine".

Does "epsilon-DNP-lysine" refer to a product where the DNP is bound only to the epsilon amino group (but not to the amino terminus), or does it refer to a product where DNP is bound simultaneously to the epsilon amino group and to the amino terminus?

Also, can the same thing happen with other amino acids that have an amino group in the side chain? i.e. can there be epsilon-DNP-arginine, for instance, or is this behavior unique to lysine?

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    $\begingroup$ Yes! my mistake. $\endgroup$ – mshum Feb 16 '16 at 13:43
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Does "epsilon-DNP-lysine" refer to a product where the DNP is bound only to the epsilon amino group (but not to the amino terminus), or does it refer to a product where DNP is bound simultaneously to the epsilon amino group and to the amino terminus?

Good question. ε-DNP-lysine usually refers to a lysine in which the dinitrophenyl substituent is bound only to the ε atom, i.e., to the side-chain nitrogen.

The reason is that in lysine-focused derivatization methods, the reaction with DNP can be done prior to protein hydrolysis. Thus, during DNP derivatization, the vast majority of lysine residues will have no free α-amino terminus because they are part of polypeptide chains. Only the ε-amino groups are free to react with DNFB. Derivatization forms DNP-ylated proteins where the peptide bonds are intact. Later steps in the procedure hydrolyze all the peptide bonds. During this step, free α-amino groups are created. But no DNFB is used in these later steps.

I found a paper from 1963 which describes the derivatization procedure in detail.

...Care was taken to deposit the sample at the bottom of the flask. Ten milliliters of bicarbonate solution were added, care being taken that no meal adhered to the side of the flask. The contents were then thoroughly mixed by gentle swirling, and the suspension was permitted to stand for 10 minutes. A solution of 0.3 ml. of DNFB in 10 ml. of ethanol was then added and the contents of the flask were thoroughly mixed by gentle swirling. The side of the flask was rinsed with 3 ml. of absolute ethanol and then the contents were shaken in subdued light for two hours on a shaker with a wrist-like motion. Previous work (10) has established that 2 hours reaction time is sufficient for substantially complete reaction between DNFB arid the free epsilon amino groups of lysine. alcohol and most of the water were removed by evaporation under an air stream, and the residue was extracted with four 50-ml. portions of anhydrous, peroxide-free diethyl ether. The ether in each case was removed from the residue by decantation and the residue in the flask was dried at ambient temperature by aeration.

The ether washings will remove the vast majority of unreacted DNFB.

... Two hundred milliliters of constant-boiling aqueous HCl nere added to the flask, and the resulting mixture was boiled overnight at the reflux temperature, cooled and then filtered through a sintered glass funnel directly into a 250-ml. volumetric flask. The filtrate, and washings from the filtrate, were made up to 250 mi. with distilled water. An aliquot (2.0 to 4.0 ml.) of the hydrolyzate containing from 0.1 to 0.3 mg. of epsilon-dinitrophenyllysine...

Now for your other question:

Also, can the same thing happen with other amino acids that have an amino group in the side chain? i.e. can there be epsilon-DNP-arginine, for instance, or is this behavior unique to lysine?

DNFB is not reactive with arginine. The arginine side-chain is best viewed as a guanidine, not as an amino group. Guanidine is considerably less nucleophilic than lysine.

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  • $\begingroup$ And likewise for the amides present in glutamine and asparagine, even though they may formally be $\ce{NH2}$-including. $\endgroup$ – Jan Nov 8 '16 at 16:43

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