Dansyl chloride protein labeling

Extrinsic fluorescence is used whenever the natural fluorescence of a macromolecule is inadequate for accurate fluorescence measurement. In this case, one can attach a fluorescent reporter group by using the reactive isocyanate or isothiocyanate derivatives of fluorescein or rhodamine, two intensely fluorescent molecules. One can covalently also label a protein s a- and e-amino groups with dansyl chloride (/.e., A,A-dimethylaminonaphtha-lenesulfonyl chloride). Another useful reagent is 8-ani-lino-l-naphthalenesulfonic acid (abbreviated ANS). This compound is bound noncovalently by hydrophobic interactions in aqueous solutions, ANS is only very fluorescent, but upon binding within an apolar environment, the quantum yield of ANS becomes about 100 times greater. 

Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3-25a). Sanger developed the reagent l-fluoro-2,4-dinitrobenzene (FDNB) for this purpose other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3-24) were subjected to this procedure. 

The N-terminal amino acid of a protein can be determined by reacting the protein with dansyl chloride or fluorodinitrobenzene prior to acid hydrolysis. The amino acid sequence of a protein can be determined by Edman degradation which sequentially removes one residue at a time from the N terminus. This uses phenyl isothiocyanate to label the N-terminal amino acid prior to its release from the protein as a cyclic phenylthiohydantoin amino acid. 

The amino-terminal (N-terminal) residue of a protein can be identified by reacting the protein with a compound that forms a stable covalent link with the free a-amino group, prior to hydrolysis with 6 M HC1. The labeled N-terminal amino acid can then be identified by comparison of its chromatographic properties with standard amino acid derivatives. Commonly used reagents for N-terminal analysis are fluorodinitrobenzene and dansyl chloride. If this technique was applied to the oligopeptide above, the N-terminal residue would be identified as Val, but the remainder of the sequence would still be unknown. Further reaction with dansyl chloride would not reveal the next residue in the sequence since the peptide is totally degraded in the acid hydrolysis step. 

Chen, R.F. 1968. DANSYL labeled proteins determination of extinction coefficient and number of bound residues with radioactive dansyl chloride. Anal. Biochem. 25, 412 116. 

Hill, R.D. and Laing, R.R., Specific reaction of dansyl chloride with one lysine residue in rennin, Biochim. Biophys. Acta 132, 188-190, 1967 Chen, R.F., Huorescent protein-dye conjugates. I. Heterogeneity of sites on serum albumin labeled by dansyl chloride. Arch. Biochem. Biophys. 128, 163-175, 1968 Chen, R.F., Dansyl-labeled protein modified with dansyl chloride activity effects and fluorescence properties. Anal Biochem. 25, 412M16, 1968 Brown, C.S. and Cunningham, L.W., Reaction of reactive sulfydryl groups of creatine kinase with dansyl chloride. Biochemistry 9, 3878-3885, 1970 Hsieh, W.T. and Matthews, K.S., Lactose repressor protein modified with dansyl chloride activity effects and fluorescence properties. 

In direct fluorescence of either labeled (dansylated) or unlabeled proteins one has to keep in mind that polyacrylamide gel itself is also a fluorescent material its fluorescence appears when excited at 280 or 340 nm. These wavelengths are very close to the excitation wavelengths of native proteins and dansyl chloride or... 

Dansyl chloride (DNS-Cl) (l-dimethylaminonaphthalene-5-sulphonyl-chloride). This reagent was originally introduced into protein chemistry for end-group analysis over twenty years ago (Gray and Hartley, 1963) and has been widely used because of the simplicity of the reaction and its ability to react with both primary and secondary amines, unlike OPA and fluram. Furthermore, in contrast to other fluorescent reagents, the dansyl derivatives are stable to acid hydrolysis, and can therefore be used in N-group labelling before hydrolysis. HPLC separations of dansyl derivatives have recently been published (Tapuhi et al., 1981). Sensitivity of detection is at the low picomole level. The sensitivity is limited because of the side-reactions which can occur with lysine and, to a lesser extent, histidine and tyrosine. 

In instances where nature has not provided an appropriate fluorophore, one can often add an extrinsic label. The earliest probes include dansyl chloride [1] and ANS (Fig. 3). Dansyl chloride can be covalently attached to macromolecules by reaction with amino groups. ANS often binds spontaneously but non-covalently to proteins and membranes, probably by hydrophobic and electrostatic interactions. The emission of both molecules is sensitive to the polarity of the surrounding environment. ANS is nearly non-fluorescent in water, but fluoresces strongly upon association with serum albumin, immunoglobulins and other proteins. A wide variety of covalent and non-covalent probes are available [2,3]. 

Fluorescein isothiocyanate (FITC) and dansyl-chloride were among the first extrinsic fluorescent labels for proteins used for immunofluorescence microscopy and polarization measurements. Fluorescein and rho-damine labels were extensively employed due to their bright emission in the visible range. These probes have drawbacks including hydrophobicity, small Stokes shifts, and sensitivity to pH and photobleaching, which led to the development of new dyes such as Alexa, Bodipy, and cyanine dye families (see Table 1). These dyes cover a broad... 

While dansyl chloiide today seems like a common fluorophore, its Introduction by Professor Weber represented a fundamental change in the paradigm of fluorescence spectroscopy. One of Professor Weber s main contributions was the introduction of molecular considerations Into fluorescence spectrosetpy. The dansyl group is solvent sensitive, and one is thus forced to consider its interactions with its local environment. Professor Wfeber recognized that proteins could be labeled with fluorophores, which in turn reveal information about the proteins and their interactions with other molecules. The probes which the Professor developed are still in widespread use, including dansyl chloride, ANS, TNS, and Prodan derivatives. 

The amino and carboxyl residues can be elucidated chemically by labeling them. Two common methods modify the N-terminal residue by the chemical addition of fluoro-2,4-dinitrobenzene or dansyl chloride. The protein is then hydrolyzed to its component amino acids. The amino acid labeled with fluoro-2, 4-dinitrobenzene is colored yellow and that with dansyl chloride is fluorescent. This enables visualization of the labeled amino acid. Comparison of further... 

This test is based on the extraction of a fluorescent marker, dansyl chloride, previously bound to the amino groups of proteins inside the full thickness of the stratum comeum [76]. Fluorescent labeling of the skin is performed 1 week prior to the application of the surfactants. When surfactants are applied at 2% of active ingredients and under occlusion, two or three applications of 30 min each are sufficient to detach and extract all or part of the fluorescent dye from the skin in the absence of visible irritation. The level of dansyl chloride extraction has been shown to correlate with the irritation potential of the surfactants as determined in separate soap chamber tests. This test demonstrates that surfactants interact with the skin surface proteins very quickly and are able to release molecules that would be linked to these proteins. The evaluation of fluorescence extraction is done visually by a trained assessor on a 0-4 scale, 0 being no fluorescence extraction and 4 being complete fluorescence extraction. 

Labeling of the skin with dansyl chloride has also been extensively used by dermatologists to investigate the turnover rate of the epidermis [77]. After labeling with the fluorescent dye, the effect of test products on the epidermis proliferation rate is measured by the kinetics of fading of the fluorescent from the skin. However, this method has to be used very carefully when surfactants or surfactant-based products are tested, as many of them are able to quickly remove the fluorescence by direct interaction with skin proteins. 

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