Thiol specific labeling

Amino acids and even peptides can be incorporated directly during PNA synthesis using either t-Boc or Fmoc synthesis methods. The most common amino acid additions are lysine and cysteine. Lysine can provide additional aqueous solubility to PNA and carboxy-terminal lysine with orthogonal protection may also be used for doubly labeled PNA preparations. Cysteine allows the attachment of thiol-specific labels and further derivatized thiol-or maleimide-linked reagents. 

The stability of the reagent to the assay conditions should also be ascertained. The instability of azides and diazo compounds towards thiols was mentioned earlier, and it would be prudent to ensure that all new reagents whether they contain familiar photoactivatable groups or not are stable to the prevailing chemical and enzymatic conditions. In an attempt to label the cAMP receptor of D. discoideum, Wallace and Frazier (1979b) found that 8-N rcAMP was converted to NrAMP by the phosphodiesterase of a crude membrane preparation. The NrAMP specifically labeled actin and not the cAMP receptor. 

As no systematic study has been made of the concentration of a thiol required to scavenge various reactive species a study of the concentration dependence should be made in each case to ensure that maximal prevention of non-specific labeling is obtained. It should not be assumed that the efficacy of a thiol scavenger will be independent of pH as thiols have pKvalues of 9. 

The ratio of peak heights for HDS and H2S loss from 3, 3-d2- and 4, 4-d2-pentane thiols was found to differ in 12 eV El mass spectra compared with 70eV spectra [256]. An intramolecular isotope effect of 1.2 was observed in the loss of C3H7S2 in the El mass spectrum of bis-1, 3-dithiane specifically labelled with deuterium on one of the tertiary carbon atoms [762]. 

In order to obtain a heterodimeric disulfide bridge the cysteine residue of one component, either PNA or peptide, must be derivatized. 3-Nitro-2-pyridine-sulphenyl (NPys)-derivatized Cys is specifically reactive toward free thiols. Npys-labeled Cys is commercially available and with special cautions (see Note 3) can be assembled into a peptide chain like a commonly protected amino acid. 

A combination of circular dichroism, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chemical crosslinking, and analytical ultracentrifugation studies showed that both the apo- and metallated derivatives of H21(31-mer) form two-stranded a-helical coiled coils in aqueous solution. Further characterization of these derivatives by EPR spin-label experiments helped to determine its three-dimensional backbone structure. In these studies, a Cys-21 mutant of the 31-mer coiled coil, H21/C21(31-mer), was prepared and labeled with a thiol-specific nltroxide spin label (MTSL = l-oxyl-2,2,5,5-tetramethyl-A -pyrroline-3-methyl-methanethiosulfonate) at position 21 of the peptide sequence which is the site of metal substitution in the ET heterodimer. Comparison of the low-temperature, dipolar-broadened spectrum of the spin-labeled dimer with those of magnetically dilute peptide samples yielded a backbone-to-backbone distance that was nearly identical to that of the GCN4 homodimer. Based on these results, computer modeling studies provided an estimate of the metal-to-metal distance in the ET heterodimer of m-m > 25 A. The electron-transfo properties of this system are now being studied by a combination of laser flash-quench and pulse radiolysis techniques. 

C- and H-labelled D-glucose were carried out to confirm the hypothesis that the thiol acts as a nuclet hile in the glycolytic pathway and also to evaluate the potential of the process for tiie preparation of specifically labelled D-glycerols. ... 

Figure 3.3. Structure of the ICAT reagent. The reagent contains a biotin affinity tag that is used to isolate ICAT-labeled peptides. The reagent also contains a linker that exists in a heavy (where X= deuterium) or light form (X= hydrogen) and a reactive group with specificity towards the thiol groups of cysteine residues. Figure adapted from Gygi et al. (1999).

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