Peptides dansylation

Figure 4 (a) Analysis of dansyl-Ile-Thr(0 Bu -Pro-Gln-Trp-LysCBocFWang-linker-resin, giving peptide dansyl-Ile-Thr-Pro-Gln-Trp-Lys (MH+, 1006.0). The peak at 772.6 represents a small amount of undansylated material, (b) Analysis of Fmoc-Cys(Trt)-Lys(Boc)-Ile-HMPB-linker-resin, giving Fmoc-Cys-Lys-Ile (MH+, 585.5), Fmoc-Cys(Trt)-Lys-Ile (MH+, 849.8), and Fmoc-Cys-Lys-Ile disulfide [(M-S-S-M)H+, 1168.3], (c)Analysis of the disulfide of Fmoc-Cys-Asn-Cys-Lys(Boc)-Ile-FIMPB-linker-resin giving the disulfide of Fmoc-Cys-Asn-Cys-Lys-Ile (MH+, 801.0). (Reprinted from Ref. 64.)... 

Preparation of mixed peptide fibrils was similar for both of these labelled peptides 1% (by weight) was incubated at 37 °C with the other, unlabelled peptide in 10% CH3CN/H20atpH2.ForTTRio i9with l%dansyl-TTRio5 n5, a blue shift and dansyl anisotropy increase were observed, indicating the inclusion of dansyl-TTRios-i 15 into fibrils. CD spectroscopy proved that the stmcture was primarily p-sheet. 

Repeated addition of MDC to Q11 did occur, but the dominant product was Q11 with a single MDC. The fraction of Qll with higher numbers of attached MDC decreased for increasing MDC number. Separately, a lysine peptide that contained the bioactive RGD [73] sequence ( -dansyl-GLKGGRGDS-Am) was successfully TGase crosslinked with self-assembled Qll five distinct Qll-dansyl RGD were detected by mass spectrometry. 

Capillary zone electrophoresis, an up-to-date high resolution separation method useful for proteins and peptides, has been shown to be a useful method for determining electrophoretic mobilities and diffusion coefficients of proteins [3], Diffusion coefficients can be measured from peak widths of analyte bands. The validity of the method was demonstrated by measuring the diffusion coefficients for dansylated amino acids and myoglobin. 

The amino acid analyser using fluorescamine as the detecting reagent has been used to measure 250 picomoles of individual amino acids routinely [262], and dansyl derivatives have been detected fluorometrically at the 10 15 M level [260]. Where the amounts of amino acid are high enough, the fluorescamine method, with no concentration step, can be recommended for its simplicity. At lower concentrations, the dansyl method, with an extraction of the fluorescent derivatives into a non-polar solvent, should be more sensitive and less subject to interferences. For proteins and peptides, the fluorescamine method seems to be the most sensitive available method. 

Amines are another important group of analytes. Mellbin and Smith [72] compared three different fluorescent reagents, dansyl chloride, 4-chloro-7-nitrobenzo-1,2,5-oxadiazole, and o-phthaldialdehyde, for derivatization of alkylamines. The dansyl tag was found to be the most effective. Hamachi et al. [73] described the application of an HPLC-POCL method for determination of a fluorescent derivative of the synthetic peptide ebiratide. Another comparative study was done by Kwakman et al. [74], where naphthalene-2,3-dialdehyde and anthracene-2,3-dial-dehyde were evaluated as precolumn labeling agents for primary amines. The anthracene-2,3-dialdehyde derivatives were not stable, especially in the presence of hydrogen peroxide, and the POCL detection of these derivatives was therefore... 

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

Dansyl chloride (dimethylaminonaphthalene-5-sulphonyl chloride) will react with free amino groups in alkaline solution (pH 9.5-10.5) to form strongly fluorescent derivatives (Figure 10.14). This method can also be used in combination with chromatographic procedures for amino acid identification in a similar manner to the FDNB reagent but shows an approximately 100-fold increase in sensitivity. This makes it applicable to less than 1 nmol of material and more amenable for use with very small amounts of amino acids liberated after hydrolysis of peptides. The dansyl amino acids are also very resistant to hydrolysis and they can be located easily after chromatographic separation by viewing under an ultraviolet lamp see Procedure 10.1. 

Figure 10.14 The reaction of dansyl chloride with compounds containing a free amino group. At an alkaline pH, the reaction results in the formation of fluorescent derivatives of free amino acids and the N-tenninal amino acid residue of peptides.

The same group extended their work recently to the synthesis of a peptide-based device detecting Zn2+ in aqueous solutions. [441 The principle is to add a dansyl fluorophore on a Lys side chain of a synthetic Zn finger peptide. Upon complexation of Zn2+, an important conformational change occurs that brings the fluorophore into a different environment and leads to a change in the fluorescence properties. 

When synthetic peptides were used as acceptors for N-glycosylation from GlcNAc2-PP-Dol, the necessity for the tripeptide sequence Asn-X-Ser/Thr could be demonstrated. Moreover, the rate of glycosylation was affected by the chain length of the peptides. Other factors, such as hydrophobicity and steric hindrance, have different effects on the acceptor properties of the peptides, as judged by the results of (2,4-dinitrophenyl)ation and dansyl-ation of them.134... 

The primary structure of a peptide or protein is defined by the sequence of amino acids. In this experiment the procedures that are in common use to determine protein primary structure are applied to an unknown dipeptide. Amino acid composition of the peptide will be determined by acid hydrolysis followed by HPLC, CE, or paper chromatography. The identity of the NH2-terminal amino acid will be achieved by the dansyl method followed by thin-layer chromatography. 

Even more versatile than the dansyl method is the Edman method (Figure E2.4). The NH2-terminal amino acid is removed as its phenylthiohydan-toin (PTH) derivative under anhydrous acid conditions, while all other amide bonds in the peptide remain intact. The derivatized amino acid is then extracted from the reaction mixture and identified by paper, thin-layer, gas, or high-performance liquid chromatography. The intact peptide (minus the original NH2-terminal amino acid) may be isolated and recycled by reaction with phenylisothiocyanate. Since this method is nondestructive to the remaining peptide (aqueous acid hydrolysis is not required) and results in good yield, it can be used for stepwise sequential analysis of peptides. The method is now automated. 

Period 1 Part A—Begin acid hydrolysis of peptide. Part B—Complete dansyl chloride reaction and begin acid hydrolysis of dansyl peptide. Part A.2—if applicable, prepare paper chromatogram by applying standard amino acids. 

Period 2 Part B—Work up the dansyl hydrolysate and spot on the TLC plate with standard dansyl amino acids. Part A. 1—Work up peptide hydrolysate and prepare FMOC derivatives of amino acids for analysis by HPLC or CE. Part A.2-If applicable, develop paper chromatogram in solvent system. 

A related and more sensitive method makes a sulfonamide of the terminal NH2 group with a reagent called dansyl chloride. As with 2,4-dinitrofluorobenzene, the peptide must be destroyed by hydrolysis to release the N-sulfonated amino acid, which can be identified spectroscopically in microgram amounts ... 

Dansyl chloride and phenylisothiocyanate (PITC) are the derivatizating agents most used in UV detection. Dansyl chloride reacts with the primary and secondary amino groups of peptides in a basic medium (pH 9.5), forming dansylated derivatives that are very stable to hydrolysis but are photosensitive. The derivatives are detectable in UV at 254 nm and by fluorescence. Dansyl sulfonic acid is formed as a by-product of the reaction, and excess reagent reacts with the dansyl derivatives to form dansyl amide the conditions of derivatization must therefore be optimized in order to avoid the formation of such by-products to the extent possible. The conditions of the reaction with dansyl chloride and of the separation of the derivatives thus formed have been thoroughly studied (83,84). Martin et al. (85) carried out derivatization using an excess concentration of dansyl chloride of 5 -10-fold in a basic medium (lithium carbonate, pH 9.5) in darkness for 1 h. 

Dansyl chloride has been widely utilized in peptide analyses to determine the N-terminal amino acid (60,86). Mendez et al. (87) suggested that derivatization of the peptides with dansyl... 

Phenyl isothiocyanate has also been utilized to analyze small quantities of peptides or short-chain peptides (88) by carrying out sequential degradation with detection by dansylation. Using dansyl-Edman degradation, Leadbeater and Bruce-Ward (60) identified 60 tryptic peptides of/8-casein that had previously been separated by high-voltage paper electrophoresis. 

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