2-Mercaptoethanol reagent

Mercaptoethanol reagent 380 Mercury cations 144,311 Mercury lamps 20, 22 ff emission lines 23, 24 -, high pressure 22 ff -, technical data 23 Mercury(I) nitrate reagent 337 Mercury(II) salt reagent 340 Mesaconic acid 61 Mesoporphyrin 101, 102 Metal cations 310—312,398 Metal complexes 248, 398 Methanol, dipole moment 97 Methine dyestuffs 360 4-Methoxyaniline see Anisidine 4-Methoxybenzaldehyde see Anisaldehyde Methoxybenzaldehyde derivatives 72 Methoxycinnamic acid 277... 

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

The Npys group can be. cleaved reductively with BU3P, H2O or mercaptoethanol. It is stable to CF3COOH (24 h), 4 M HCl/dioxane (24 h), and HF (1 h). The related reagent, 2-pyridinesulfenyl chloride, has also been proposed as a useful reagent for the deprotection of the 5-trityl, 5-diphenylmethyl, 5-acetamidomethyl, 5-/-butyl, and S-r-butylsulfenyl groups, but this reagent is very susceptible to hydrolysis. ... 

A number of drawbacks in the application of the 0PA/2-ME reagent system include the instability of the fluorescent isoindole derivative (5-7) the use of the noisome reagent 2-mercaptoethanol the low and solvent-dependent fluorescence efficiencies (8,9) of the isoindole and—perhaps the most limiting—the effective restriction of the OPA assay to primary aliphatic amines and to amino acids. 

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. 

Fluoraldehyde (Pierce Huorescence Reagent, contents 0.8 mg mL highly purified fluoraldehyde phfhaldehyde crystals, Brij-35 and mercaptoethanol in specially formulated borate buffer Hexane, 99%... 

Merino-Merino et al. [32] used the OPA reagent (o-phthaldehyde condensed with 2-mercaptoethanol) to separate penicillamine enantiomers after their derivatization. Racemic and (/q-penicillamine were dissolved in aqueous 0.5 M NaOH, and treated with the derivatizing solution (methanolic o-phthaldehyde and 2-mercaptoethanol in 0.4 M potassium borate buffer solution of pH 10). The reaction mixture was set aside for 2 min at room temperture, whereupon a portion of solution was analyzed by HPLC. The method used a Cyclobond column (25 cm x 4.6 mm) maintained at 5 °C, a mobile phase of ethanol/1% triethylammonium acetate (1 1 pH 4.5) eluted at... 

Dithiothreitol (DTT) and dithioerythritol (DTE) are the trans and cis isomers of the compound 2,3-dihydroxy-1,4-dithiolbutane. The reducing potential of these versatile reagents was first described by Cleland in 1964. Due to their low redox potential (—0.33 V) they are able to reduce virtually all accessible biological disulfides and maintain free thiols in solution despite the presence of oxygen. The compounds are fully water-soluble with very little of the offensive odor of the 2-mercaptoethanol they were meant to replace. Since Cleland s original report, literally thousands of references have cited the use of mainly DTT for the reduction of cystine and other forms of disulfides. 

Add 3 ml of the detergent Brij-35 (as a 30 percent solution) and 2 ml of 2-mercaptoethanol to 950 ml of Fluoraldehyde Reagent Diluent (all reagents from Thermo Fisher). 

Cleland (1964) showed that DTT and DTE are superior reagents in reducing disulfide bonds in proteins (see previous discussion, this section). DTT and DTE have low oxidation-reduction potential and are capable of reducing protein disulfides at concentrations far below that required with 2-mercaptoethanol. However, even these reagents have to be used in approximately 20-fold molar excess in order to get close to 100 percent reduction of a protein. 

After a carrier protein has been activated with sulfo-SMCC, it is often useful to measure the degree of maleimide incorporation prior to coupling an expensive hapten. Ellman s reagent may be used in an indirect method to assess the level of maleimide activity of sulfo-SMCC-activated proteins and other carriers. First, a sulfhydryl-containing compound such as 2-mercaptoethanol or cysteine is reacted in excess with the activated protein. The amount of unreacted sulfhydryls remaining in solution is then determined using the Ellman s reaction (Chapter 1, Section 4.1). Comparison of the response of the sample to a blank reaction using... 

Phthalazinone, 19 332 o-Phthaldialdehyde-2-mercaptoethanol, chiral derivatizing reagent, 6 76t Phthalein dyes, 19 304-305 Phthalic acid, 19 332... 

Derivatization of primary amino acids with o-phthalaldehyde (OPA) is simple and the poor reproducibility due to the instability of the reaction product can be improved by automation and the use of alternative thiols, e.g. ethanthiol in place of the 2-mercaptoethanol originally used. An alternative fluorimetric method using 9-fluoroenylmethylchloroformate (FMOC-CL) requires the removal of excess unreacted reagent prior to column chromatography. This procedure is more difficult to automate fully and results are less reproducible. However, sensitivity is comparable with the OPA method with detection at the low picomole or femtomole level, and it has the added advantage that both primary and secondary amino acids can be determined. 

The cells are lysed in a buffer containing strong chaotropic reagents such as guanidine thiocyanate and 2-mercaptoethanol, which completely denatures any ribonuclease present. The supernatant is then placed on a cushion of CsCl (5.7 mol l-1) and centrifuged at 100000 g for 18 h. The RNA passes through the CsCl and is pelleted, while the DNA and protein remain in the aqueous solution. The RNA pellet is dissolved in buffer and concentrated by precipitation in cold ethanol. 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

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