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
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
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.