**4.2. Metabolic labeling with radiolabeled palmitate**

od of time. These assays are by no means outdated and some continue to be the most

Working with palmitoylated proteins is inherently difficult due to the labile nature of the thioester bond and the increased hydrophobicity of the protein or peptide due to palmitate. On the other hand, the unique physical and chemical properties of thiols, palmitoylated thi‐ ols, and the thioester bond make them particularly amenable to modification by highly spe‐

Thiol modification occurs most commonly in cells by one of two routes: disulfide exchange or alkylation. Many of the reactive groups that undergo these two reactions are relatively stable in aqueous environments; the reactions are rapid and provide high yields of thioether and disulfide bonds [88]. Thiols will also react with many amine reactive reagents including isothiocyanates and succinimidyl esters but lack a high degree of specificity, resulting in un‐ stable bonds that are much less useful for routine modification of thiols in proteins. Thiolspecific reagents and chemistry figure strongly into the design and development of novel assays for palmitoylation. Most investigators are limited somewhat to reagents that are available from a catalog but, fortunately, there are already many useful reagents available. Among the most useful are thio-reactive chemicals that are linked to another moiety (reac‐ tive or reporter) by a spacer arm of variable length and physical characteristics. Such heteroand homo-bifunctional crosslinking reagents have provided much of the foundation for recent developments in palmitoylation assays and provide a fairly rich toolbox for future as‐

Iodoacetamide conjugates are among the most commonly used tools for modifying cysteine thiols. These undergo nucleophilic substitution to form stable thioether bonds at physiologi‐ cal pH in aqueous environments. When using iodoacetamide and its conjugates, one should remember that depending on the pH of the solution, they can also react with histidine, ly‐ sine, and methionine (at pH >1.7) residues and N-terminal amines. However, when used at slightly alkaline pH in the dark and in the absence of reducing reagents, cysteine modifica‐ tion will be the exclusive reaction [88]. A good example of iodoacetamide-based probes are the isotope-coded affinity tags or ICAT [120]. These have proved particularly useful in de‐

Maleimides are also common constituents of heterobifunctional crosslinking reagents and blocking reagents that target cysteines. They are ~1000 times more specific for cysteine sulf‐ hydryls at pH 6.5-7.5, but at higher pH some cross reactivity can occur with amines. Mal‐ eimides form stable thioether bonds by adding the sulfhydryl across the double bond of

Phenylmercury derivatives react with thiols, including nitrosothiols, under conditions simi‐ lar to iodoacetamides and maleimides to form stable mercury-thiol bonds that can be re‐

**4.1. Chemistry and physical properties of palmitoyl cysteines: Reactions and probes**

appropriate way to answer specific questions.

262 Drug Discovery

*Reactions of free thiols in the cytoplasm*

say development.

the maleimide.

cific chemistry and a wide variety of thiol reactive probes.

*Chemical moieties that react with palmitoyl-cysteines*

termining the substrates of DHHC2 [37].

The most common method to identify palmitoyl proteins and to determine the residence half-life of palmitate on a specific protein or palmitate turnover (e.g. [122] for a particularly interesting example) is to metabolically label cells with radiolabled palmitate. 14C-, 3 H- and 125I-labeled palmitate have all been used, but 3 H-palmitate is most common because it is rela‐ tively inexpensive and widely available. However, using 125I -labeled palmitate provides some advantages. In practical terms, the time required for detection is considerably shorter hours instead of (often) weeks with 3 H-palmitate. The *γ-*irradiation from the 125I is also com‐ patible with phosphorimaging technology which is much more rapid and quantitative than densitometric measurements from films generated by autofluorography (as is used with tri‐ tium). The principle downside of using 125I-labeled palmitate is that it is not commercially available, and the labeling must be done by the investigator. Reviews of the methods using radiolabeled palmitate and including technical details have been published recently [123-125].
