DANS amino acids

The DNP-amino acids, after separation into individual spots on the chromatographic plate, can be eluted from the scraped off area by adding 4 ml of water to the material in a small tube. The tube is heated at 50° in a water bath for 15 minutes and centrifuged to clear the solution. The color is read against known standards at 360 nm. Direct estimation of DNP-, PTH-, and DANS-amino acids separated on the thin-layer plate can be performed by fluorescence and fluorescence quenching techniques (P8). It is also possible to convert unmodified amino acids, separated on a silica gel G chromatographic plate, into DNP-amino acids by in situ conversion as was described in Section 4.7.18. The DNP-derivatives can then be developed in the second dimension and the spots analyzed quantitatively. 

Primary and secondary amines, amino acids and phenols react In the case of long-wavelength UV light (A = 365 nm) the DANS amides fluoresce yellow-green, while amines that have reacted at a phenolic OH group have an intense yellow to yellow-orange fluorescence The detection hmit for DANS amides is ca 10 mol [86]... 

The third step is to determine the polypeptide chain end groups. If the polypeptide chains are pure, then only one N-terminal and one C-terminal group should be detected. The amino-terminal amino acid can be identified by reaction with fluorodinitrobenzene (FDNB) (fig. 3.18). Subsequent acid hydrolysis releases a colored dinitrophenol (DNP)-labeled amino-terminal amino acid, which can be identified by its characteristic migration rate on thin-layer chromatography or paper electrophoresis. A more sensitive method of end-group determination involves the use of dan-syl chloride (see Methods of Biochemical Analysis 3B). 

Pheny lthiohy dan-toin amino acids Open-tubular UV 18... 

The most commonly used inhibitor is diazoacetylnorleucine methyl ester (DAN) because it allows the determination of the label incorporation by amino acid analysis after complete hydrolysis of the protein. (Abbreviations used are DAN, diazoacetylnorleucine methyl ester EPNP, 1,2-epoxy 3-(nitrophenoxy)propane Z-, carbobenzoxy Phe(NO) >, p-nitrophenylalanyl.) The reaction proceeds as follows ... 

Aiylcaiboxylic acids, A-dan.syl amino acids 2-Arylcarboxylic acids Halogen-substituted 2-aryloxypropionic acids Amino acid derivatives... 

Nakasono S, Matsumoto N, Saiki H (1997) Electrochemical cultivation of Thiobacillus ferrooxi-dans by potential control. Bioelectrochem Bioenerg 43 61-66 Narita K, Titani K (1969) The complete amino acid sequence in baker s yeast cytochrome c. J Biochem 65 259-267... 

For a review on the preparation of derivatives prior to TLC, see Ref. 41. Derivatization is frequently used in the TLC of amino acids where certain amino acids are easily converted into the so-called DANS forms (amino acid derivatives on silica gel. Derivatives are also used when a volatile compound can be changed into a nonvolatile compound, or when the detection sensitivity of the derivative is greater than that of the original compound. Derivatization is advantageous as a clean-up procedure when it is easier to clean up the derivative than the original compound. 

One extreme view of chemical introduction of an extrinsic fluorescent probe is found in the case ofthe alanine derivative of the fluorophore 6-dimethylamino-2-acylnaphthalene (DAN) (Figure 4.23). This derivative fluorophore, given the trivial name Aladan, is incorporated into a polypeptide by solid-phase synthetic chemistry (although a molecular biology technique known as nonsense suppression is now available for the introduction of unnatural amino-acid residues into recombinant proteins). The fluorescent emission maximum (Tnax) of Aladan shifts dramatically on different solvent exposures, from 409 nm in heptane to 542 nm in water, yet at the same time remains only mildly changed by variations in pH or salt concentration. This compares to a maximum environment-mediated shift of around 40 nm for intrinsic tryptophan fluorescence. In addition, there is little spectral overlap between extrinsic Aladan fluorescence and intrinsic fluorescence from tryptophan or tyrosine. 

An example of a zinc-sensitjve peptide is shown in Hgute 3.46. hi this case, the Zinc finger amino acid sequence was modified to ccmtaui a covalently linked dan l group near the middle of the peptide. Emission spectra of... 

DANS-NH-CH-CO2H + amino acids DANSylamino acid... 

Fig. 1. Dinitrophenyl (DNP) and 5-dimethylaminonaphthalene-l-sulfonic acid (DANS) peptides and amino acids...

Double-Isotope Dan Microassay for Cerebral Amino Acids... 

Bonnet, R., P. Niviere et V. Labeyrie Caracterisation d amino-acides sulfoxides dans la gaine et les limbes 6 Allium porum. C. R. Acad. Sci., Ser. D 279, 1919 (1974). 

The free amino groups of amino acids and peptides react with l-dimethylamino-5-naphthalenesulphonyl chloride (DANS-d) jdelding intensively fluorescing poducts. 

Dan, A., S. Ghosh, and S. P. Moulik (2010). Interaction of cationic hydroxyethylcellulose (JR400) and cationic hydrophobically modified hydroxyethylcellulose (LM200) with the amino-acid based anionic amphi-phile Sodium A-Dodecanoyl Sarcosinate (SDDS) in aqueous medium. Carbohydrate Polymers 80(1) 44-52. 

Acylation of amino acids with 5-dimethylami-nonaphthalene-l-sulfonyl chloride (dansyl chloride, DANS-Cl) is of great analytical importance ... 

---