Peptides, ninhydrin reaction

Differences in the materials employed for the layers can also become evident when chemical reactions are performed on them. Thus, Macherey-Nagel report that the detection of amino acids and peptides by reaction with ninhydrin is less sensitive on layers containing luminescent or phosphorescent indicators compared to adsorbents which do not contain any indicator [7]. 

VK Sarin, SBH Kent, JP Tam, RB Merrifield. Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal Biochem 117, 147, 1981. 

Deprotection. The BOC group is easily removed under quite mildly acidic conditions, a feature which underlines its value in selective deprotection in peptide synthesis. Typically, treatment at room temperature for 30-60 minutes with a 1 m solution of hydrogen chloride in acetic acid, or with neat trifluoroacetic acid, is used. It is of interest that after t.l.c. of BOC-amino acids, brief exposure of the plates to hydrogen chloride fumes enables the ninhydrin reaction to be used to detect the presence of the liberated free amino acids. 

Friedman, M. (2004). Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences. ]. Agric. Food Chem. 52, 385-406. 

Fmoc-cleavage were monitored by Ninhydrin reaction [4] and TLC. The integrity of the protected peptides was proven by ID and 2D iH-NMR spectroscopy [19], The correct masses were established by FAB-MS. 

The ninhydrin reaction for resin bound peptides gives ambiguous results probably due to inaccessibility of the N-terminal amino group. Therefore a small fraction of the peptide was cleaved from the resin. After concentration by an N2 stream the ninhydrin-reaction or monitoring by TLC or HPLC were performed. 

This ninhydrin reaction is employed in the quantitative determmation of amino acids. Proteins and peptides that have free amino group(s) (in the side chain) will also react and give colour with ninhydrin. 

The ninhydrin reaction is not restricted to a-amino acids, however. It is well known that imino acids, notably proline, but also for example pipecolic acid, in solution produce a different color with an absorption maximum at 440 nm. Primary aliphatic amines react likewise with ninhydrin to give Ruhe-man s purple but the color yield is lower than for a-amino acids. Peptides, amino acids with secondary amino groups, mainly N-methylamino acids, and secondary amines also react with ninhydrin, although often more slowly and with smaller yield than the a-amino acids. Tertiary amines and all aromatic amines do not react with ninhydrin. Ammonia itself reacts to give a color with ninhydrin, although with a rather low yield. 

As a summary of the discussion of the ninhydrin reaction it may be commented that it is a very useful reaction, simple to operate both qualitatively and quantitatively. But, on the other hand, the reaction is not at all specific, ninhydrin giving colors with a large variety of amino groups in amino acids, amines, and peptides. Further, the reaction will not take place under strongly acid or strongly basic conditions. 

Peptides may he detected best using the methods found suitable in PC. The popidar ninhydrin reaction is not always sufficiently sensitive with higher peptides it fails utterly with cyclic peptides unless these contain free amino groups in side chains. More generally available and more sensitive (limit of detection about 0.1 (xg) is the N-halogenation of Reindel and Hoppe [73], modified as follows [8, 149] ... 

Separation of the Licheniformins. Analyzable quantities of three licheniformins can be separated by paper chromatography, using as solvent, a mixture of collidine + lutidine -t- 2N aqueous ammonia, 1 1 2 by volume. The peptides can be revealed on the paper either by the ninhydrin reaction or by antibiotic activity tests, manifested when the paper is placed in contact with an agar culture of sensitive bacteria such as Staphylococcus aureus or Mycobacterium phlei. 

Figure 13 shows the ninhydrin reactions of the eluted fractions of the tiypsin digest and the conditions of chromatography. As might have been anticipated, the heights of the peaks give no indication of the quantity, complexity, or size of the peptide. 

It is known that not all reactions proceed in the same manner on all adsorbent layers because the material in the layer may promote or retard the reaction. Thus, Ganshirt [209] was able to show that caffeine and codeine phosphate could be detected on aluminium oxide by chlorination and treatment with benzidine, but that there was no reaction with the same reagent on silica gel. Again the detection of amino acids and peptides by ninhydrin is more sensitive on pure cellulose than it is on layers containing fluorescence indicators [210]. The NBP reagent (. v.) cannot be employed on Nano-Sil-Ci8-100-UV2S4 plates because the whole of the plate background becomes colored. 

Because the amount of time required for a given amino acid to elute from a standard column is reproducible, the identities of the amino acids in a peptide can be determined. The amount of each amino acid in the sample is determined by measuring the intensity of the purple color resulting from its reaction with ninhydrin. Figure 26.3 shows the results of amino acid analysis of a standard equimolar mixture of 17 a-amino acids. Typically, amino acid analysis requires about 100 picomoles (2-3 /xg) of sample for a protein containing about 200 residues. 

Ninhydrin (1,2,3-indantrione, 100 mg dissolved in 50 ml of methanol) transforms all samples containing molecules with NHj groups, such as amino acids, peptides, or amines, into red- or purple-colored products. To perform a reaction, at least 5 to 10 min heating at 120°C is necessary. 

The peptide/polypeptide product is usually hydrolysed by incubation with 6 mol l-1 HC1 at elevated temperatures (110 °C), under vacuum, for extended periods (12-24 h). The constituent amino acids are separated from each other by ion-exchange chromatography and identified by comparison with standard amino acid preparations. Reaction with ninhydrin allows subsequent quantification of each amino acid present. 

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