DNP-Amino acid

Table 3-1. Values of enantiomeric resolution of DNP-amino acids in a running electrolyte containing the three fractions 1, 2, and 3 of the cyclo(Arg-Lys-X-Pro-X-(3 Ala) sublibrary separated by preparative HPLC.

The reaction of 2,4-dinitrofluorobenzene (DNFB) (Sanger s reagent [10]) with amino acids is another useful technique which is often employed for the analysis of N-terminal amino acids by TLC and column chromatography after derivatization. The reaction involved in product formation is shown in Fig.4.6. The separated derivatives are determined by measuring the quenching of fluorescence on TLC plates or by UV analysis after column chromatography. The generalized absorption curves of dinitrophenyl (DNP)-amino acids in acidic and alkaline solutions are shown in Fig. 4.7. 

Method. DNP-amino acids are usually formed in alkaline solution at pH 9-10 in the presence of as little light as possible. The amino acid residue is usually dissolved in 1 ml... 

In many cases it is advantageous to remove the hydrolyzed reagent, DNP-OH, from the reaction mixture in order to prevent its interference during chromatography with amino acid derivatives which are soluble in diethyl ether. This may be accomplished by dissolving the crude DNP derivatives in 91% sulfuric acid and extracting the DNP-OH with benzene. The acid solution is then diluted at 0 °C to 30% sulfuric acid and extracted with 10% tert.-pentanol in benzene for recovery of the DNP-amino acids. DNP-OH may also be removed by sublimation [ 11 ], or by column chromatography on silica gel [ 12] or alumina [13]. 

TLC separation of DNP-amino acids may be carried out on cellulose [14] or on polyamide layers [15,16] with several types of solvent systems. A two-dimensional separation of DNP-amino acids on cellulose with toluene—2-chloroethanol—pyridine—5 N ammonia (5 3 1.5 6.5) and saturated ammonium sulfate-water-sodium dodecyl sulfate (25 ml 175 ml 0.144 g) is shown in Fig.4.8. A similar separation on polyamide layers is... 

Fig. 4.10. Separation of water-soluble DNP-amino acids (A) and diethyl ether soluble DNP-amino acids (B).

DNP-Amino acids which are soluble in diethyl ether can be separated [19-23] in a number of solvent systems (Table 4.1). The column chromatographic separation of many diethyl ether- and water-soluble DNP-amino acids has been accomplished by Beyer and Schenk [24,25]. They used a column packing material which consisted of nylon powder, and eluted with citrate buffer (pH 3.0) at 30 °C and at a flow-rate of 0.5 ml/min for water-soluble DNP-amino acids. The diethyl ether-soluble amino acid derivatives were eluted with phosphate buffer (pH 8) at 30 °C and at a flow-rate of 0.5 ml/min. The water-soluble compounds were monitored at 313 nm, while 366 nm was used for the diethyl ether-soluble derivatives. The separation of both types of DNP-amino acids with this system is shown in Fig.4.10. 

Dansylated phenylalanine Dansylated phenylalanine DNP amino acids... 

Day 2 Thin Layer Chromatography of the DNP-Amino Acid and Paper Chromatography of the Acid-Hydrolyzed Dipeptide... 

Add 2 ml of distilled water and 2 ml of ether. Swirl for 10 sec, allow the phases to separate, and transfer the ether phase to a clean 16-by-125-mm glass test tube. Repeat this ether extraction two more times and combine the ether phases in the same glass test tube. This final ether extraction is done to separate the N-terminal DNP-amino acid from the other amino acid (or DNP-amino acid) in the sample. Remember that the N-terminal DNP-amino acid carries no charge at low pH (ether phase), while the C-terminal amino acid has a charged a-amino group at low pH (aqueous phase). 

Evaporate the ether extracts (containing your N-terminal DNP-amino acid) to dryness under N2 as before. 

Carefully spot 5 pi of the DNP-amino acid standards (provided by the instructor) along the origin of the plate. To prevent diffusion of the sample, it is a good idea to spot 1 pi of the sample, allow it to dry, spot another -pl aliquot, allow it to dry, and so forth. 

Dissolve the dried DNP-amino acid (N-terminal) in 100 pi of acetone. 

Using the same technique as described in step 6, carefully spot a 5-/xl aliquot of your sample along the same origin as the DNP-amino acid standards. If you did not recover a sufficient amount of the sample in the last ether extraction, you may also want to spot a 1 ()-pl sample next to this. The sample spot should be yellow enough in color that you will be able to identify it once the plate is developed. 

From the Rf value of your unknown sample compared to the Rf values of the DNP-amino acid standards, what is the identity of the N-terminal amino acid in your unknown dipeptide Discuss any uncertainty that may be involved in your identification, as well as how these uncertainties may be eliminated through modifications in the experiment. 

DNP-amino acid standards (for thin-layer chromatography)—Dissolve 1 mg of each standard in 1.5 ml of... 

Fig. 16. A-Terminal amino acid residues detected in human. serum albumin after treatment with varying amounts of NBS. Yields of DNP-amino acids were corrected for losses on hydrolysis. From Peters (1959).

The entry artifact refers to a spot, in the Levy two-dimensional chromatogram of DNP-amino acids, occurring in the region of phenylalanine and whose identity has not been established. 

Woodin (1956). Values corrected for destruction of DNP-amino acids during hydrolysis. 

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