TNBS reagent

Monitoring of solubility and dissolution Gravimetric analysis, a protein assay method (involving color reaction with bicinchoninic acid) and UV absorbance measurements at 214 nm Chemical analysis using TNBS reagent Fluorescence spectrophotometry... 

Procedure Remove a resin sample and wash with ethanol-HOAc (3 X 2 mL) and ethanol (3X2 mL). Transfer the resin to a small centrifuge tube with 1 mL ethanol. Add 1 drop of the TNBS reagent, mix, and let stand at room temperature for 10 min. Centrifuge if necessary to collect the beads. Free amino groups result in a red-orange color of the beads. Completely negative beads have a steel-gray color. The TNBS test has approximately the same sensitivity as the Kaiser test. 

Figure 1.79 The reaction of Ellman s reagent with a sulfhydryl group releases the chromogenic TNB anion, which can be quantified by its absorbance at 412nm.

It has been known for a long time that polynitroaromatic compounds produce colored products in contact with aUcafis [1]. These color reactions have been extensively used for the identification of nitroaromatic explosives. In the Janowski reaction [7], a solution of the polynitroaromatic compound (di- or trinitroaromatic) in acetone is treated with concentrated aqueous KOH solution. 1,3,5-Trinitrobenzene (TNB) and 2,4,6-trinitrotoluene (TNT), treated with 30% aqueous KOH, produced violet-red and red colors, respectively. Many variations of the Janowski reaction were reported, using KOH or NaOH in aqueous or ethanoHc solutions as reagents, and dissolving the explosives in acetone, ethanol or acetone-ethanol mixture [3,8]. The reaction was used both for spot tests and for spraying TLC plates [9]. 

Tetracyanoethylene (TONE), 1,3,5-trinitrobenzene (TNB), ra-dinitro-benzene, nitrobenzene, naphthalene, and anthracene were all laboratory chemical grade reagents and were purified by standard procedures. Pery-lene from Rutgerswerke-Aktiengesellschaft was used as received. Benzene (AR) was stored over active silica-alumina and filtered before use. Solutions of the adsorbates in benzene were 10 XM for TONE and TNB and 5 X 10 3M for naphthalene, anthracene, and perylene. 

The ninhydrin reaction (see Basic Protocol 1), the TNBS reaction (see Alternate Protocol 1), the fluorescamine reaction (see Alternate Protocol 2), and formol titration (see Alternate Protocol 3) all evaluate released amino groups by comparing the amounts of free amino groups before and after hydrolysis. The first three methods are spectro-photometric techniques, whereas the fourth is a potentiometric technique. The first and second are chromogenic techniques, whereas the third is fluorometric. These techniques are usually performed as time-course experiments. As the hydrolysis reaction proceeds, aliquots (samples) of the reaction are taken periodically and treated with a test reagent. Products of this reaction are proportional to the amount of free amino groups at each time point. 

Trinitrobenzene sulfonic acid (TNBS) is known to react with protein lysine residues, forming colored trinitrophenylated (TNP) derivatives with absorption maxima at 345 mp, (46). When E. coli pyrophosphatase was incubated with a 400-fold molar excess of the reagent, there was progressive decay of enzymic activity accompanied by increasing absorbance of the solution at 345 mp. (Fig. 5). Half the enzymic activity had disappeared by the time 16% of the protein lysines had been modified eventually (6 hr) all 98 enzyme lysines reacted (Table III and Fig. 5). Isolation and hydrolysis of the product showed total conversion of all lysine to e-TNP-lysine without formation of any other... 

A succinylated casein derivative that has nearly all its amines blocked can be used as a substrate in protease assays (Hatakeyama etal., 1992). As the casein is degraded by a protease, free amines are created from a-chain cleavage and release of a-amino groups. The creation of amines can be monitored by an amine detection reagent such as trinitrobenzene sulfonic acid (TNBS Section 4.3). The procedure forms the basis for a highly sensitive assay for protease activity. 

Blocking of amine groups on proteins also has been used to create a sensitive reagent for measuring protease activity (Hatakeyama etal., 1992). With nearly all the primary amines of casein blocked, an amine detection reagent such as trinitrobenzene sulfonic acid (TNBS) will only minimally react with the protein and form its typical orange derivative. As proteases cleave the protein, however, primary a-amines are created from cleavage of the a-chain peptide bonds, and TNBS can react with them. The more protease activity present, the more color is formed. 

Ellman s reagent, 5,5 -dithio-bis-(2-nitrobenzoic acid), or DTNB, is a compound useful for the quantitative determination of sulfhydryls in solution (Ellman, 1958,1959). The disulfide of Ellman s reagent readily undergoes disulfide exchange with a free sulfhydryl to form a mixed disulfide and release of one molecule of the chromogenic substance 5-sulfido-2-nitrobenzoate, also called 5-thio-2-nitrobenzoic acid (TNB). The intense yellow color produced by the TNB anion can be measured by its absor-... 

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