Ninhydrin-Based Assay

This method is based on the quantitation of total amino acids following acid hydrolysis of proteins present in tissue samples. Microtiter plates are used in this assay. Tissue samples (10 pg) are first hydrolyzed in 500 pL of 6 M HC1 at 100 °C for 24 h to liberate ammonium. The samples are then lyophilized (chilled and evaporated), and the residue, containing ammonium chloride, is dissolved in a known volume of water. 

Ninhydrin reagent, containing ninhydrin, ethylene glycol, acetate buffer, and stannous chloride suspension, initially a pale red color, is added to 1-10 pg of protein hydrolysate in a flat-bottom microtiter plate. During the 10-min incubation at 100 °C, ammonia reacts with the ninhydrin reagent to produce diketohydrindylidene-diketohydrindamine (Eq. 1.1). 

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Starcher, B. (2001). A Ninhydrin-based Assay to Quantitate the Total Protein Content of Tissue Samples, Anal. 

Figure 1.4. Comparison of results of ninhydrin and Bradford methods for total protein concentration in tissue samples. Proteins in both assays are the same.8 [Reprinted, with permission, from B. Starcher, Anal. Hiochem. 292, 2001, 125-129. A Nirhydrin-Based Assay to Quantitate the Total Protein Content of Tissue Samples. Copyright 2001 by Academic Press.]...

Pregnalatto and Sabino200 incorporated glycerine into the ninhydrin solution and reported an improved sensitivity and reproducibility enabling neomycin concentrations as low as 4 yg/ml to be determined. An automated ninhydrin assay has been described by Kaptionak, Biernacka and Pazdera O based on a modified Moore and Stein methodl l. 

Ninhydrin Assay for Adsorbed Proteins. Measurements were made by a colorimetric procedure based on the reaction of ninhydrin with amino acids (25). The films were hydrolyzed in 5 ml of 2.5N NaOH for 2 hrs in capped plastic tubes in a boiling water bath. Then 1.5 ml of glacial acetic acid was added and mixed next I ml of ninhydrin reagent was added and mixed. [The reagent was three times more concentrated in ninhydrin, SnCb, and citrate than prescribed by Moore and Stein (25)]. The tubes were capped and boiled 20 mins more. The solution was clarified by centrifugation, and the absorbance read immediately at 570 nm on a Beckman DB spectrophotometer. If necessary, the sample was diluted with 50-50 2-propanol-water. Calibration curves (absorbance vs. fig of protein) were constructed in the 0-30 and 0-100 fig range with known amounts of each type of protein subjected to this same analysis procedure. 

The three most common assays for total protein2 are the Lowry (enhanced copper), Smith (bicinchoninic acid, BCA), and Bradford (Coomassie Blue) methods. All are colorimetric methods, and are based on the generation of absorbing species in proportion to the quantity of protein present in the sample. The ninhydrin assay is a recently reported promising method. 

The average composition of copolymers can be determined most readily for the case where the monomeric units can be isolated and identified by suitable scission or degradation reactions. This is the usual method for the elucidation of protein structure. The proteins are hydrolyzed by acids and / or bases in an automated apparatus. The resulting amino acids are chromato-graphically separated and assayed quantitatively via the color reaction with ninhydrine in so-called amino acid analyzers. 

This assay is a modified ninhydrin test to determine the degree of deacetylation in PIA. The reagent TNBS has been shown to react specifically with free amino groups to give trinitrophenyl (TNP) derivatives that can be measured with a UV spectrophotometer at 335 nm (10). Chemically, complete deacetylafion of PIA can be achieved by treatment with strong acid or base for several hours. 

Table 14.9. Average amino acid content of base wines made from different Champagne grape varieties (Assayed on sulfenic resin and detected using ninhydrin) (Results in mg/1) (Desportes et ah, 2000)...

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