Cysteine reagent

The -ATPase gene sequence indicates the presence of eight cysteine residues in the molecule [37,38]. In order to ascertain the chemical state of these cysteine residues, direct chemical studies with established cysteine and cystine reagents were carried out [44]. Titrations with the cysteine reagent, dithiobisnitrobenzoate, and the cystine reagent, nitrothiosulfobenzoate, indicated the presence of six free cy-... 

Kutlan, D., Presits, P., and Molnar-Perl, I. Behavior and characteristics of amine derivatives obtained with o-phthaldialdehyde/3-mercaptopropionic acid and with o-phthaldialdehyde/Al-acetyl-L-cysteine reagents. J. Chromatogr. A 949, 235, 2002. 

Because of the different and low stability of the isoindoles obtained from the reaction of AAs with OPA/MCE reagent, alternative precolumn derivat-izations reagents, such as 3-mercaptopropionic acid (MPA) and several N-alkyl-L/o-cysteines, were proposed. The OPA/MPA and OPA/N-acetyl-L-cysteine (NAC) reagents provide more stable isoindoles compared to those formed with the OPA/MCE, and the optical resolution of enantiomeric AAs with the OPA/NAC, as well as with further N-alkyl-L/o-cysteine reagents, have opened a new area in separation of AA enantiomers. Due to robotic autosamplers, which provide excellent reproducibility even for moderately quantitative interactions, most AA analyses are performed with the OPA derivative. The unexplainable contradictions of this most popular process - relating to the particularly low stability of the OPA derivatives of six very important AAs (glycine, y-aminobutyric acid (GABA), jS-alanine,... 

Mengerink Y, Kutlan D, Toth F, Csampai A, and Molnar-Perl I (2002) Advances in the evaluation of the stability and characteristics of the amino acid/amine derivatives obtained with the o-phthaldialdehyde (OPA)/3-merca-ptopropionic acid and OPA/N-acetyl-L-cysteine reagents HPLC/MS study. Journal of Chromatography A 949 99-124. 

The deterruination of amino acids in proteins requires pretreatment by either acid or alkaline hydrolysis. However, L-tryptophan is decomposed by acid, and the racemi2ation of several amino acids takes place during alkaline hydrolysis. Moreover, it is very difficult to confirm the presence of cysteine in either case. The use of methanesulfonic acid (18) and mercaptoethanesulfonic acid (19) as the protein hydroly2ing reagent to prevent decomposition of L-tryptophan and L-cysteine is recommended. En2ymatic hydrolysis of proteins has been studied (20). 

In recent years, biochemists have developed an arsenal of reactions that are relatively specific to the side chains of particular amino acids. These reactions can be used to identify functional amino acids at the active sites of enzymes or to label proteins with appropriate reagents for further study. Cysteine residues in proteins, for example, react with one another to form disulfide species and also react with a number of reagents, including maleimides (typically A ethylmaleimide), as shown in Figure 4.11. Cysteines also react effectively... 

A method that has been the standard of choice for many years is the Lowry procedure. This method uses Cn ions along with Folin-Ciocalteau reagent, a combination of phosphomolybdic and phosphotnngstic acid complexes that react with Cn. Cn is generated from Cn by readily oxidizable protein components, such as cysteine or the phenols and indoles of tyrosine and tryptophan. Although the precise chemistry of the Lowry method remains uncertain, the Cn reaction with the Folin reagent gives intensely colored products measurable spectrophotometrically. 

The advantages of this method are a short reaction time and the nonfluorescence of the OPA reagent. Therefore, excess reagent must not be removed before the chromatography stage. Using this method, it is possible to measure tryptophan, but not secondary amino acids such as proline or hydroxyproline. Cysteine and cystine can be measured, but because of the low fluorescence of their derivatives, they must be detected using an UV system, or alternatively oxidized to cysteic acid before reaction. 

Relatively few reports have been published subsequently on the use of these reagents. Hope and coworkers99 have used sodium in liquid ammonia to cleave the benzyl sulphonyl derivatives of cysteamine, L-cysteine and L-homocysteine to prepare the corresponding sulphinic acids, as in equation (42). 

Note o-Phthaldehyde in the presence of mercaptoethanol or cysteine has already been discussed as a reagent [4]. The present monograph describes the use of o-phthal-aldehyde in the presence of sulfuric add. There are, in addition, a number of applications, which have been described, employing o-phthalaldehyde without any additives e. g. for the detection of primary arylamines, histamine, histidine and histidylpeptides [5-71. 

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

Cysteine Iodoacetamide, maleimides, Ellman s reagent, p-hydroxymercuribenzoate... 

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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