Alkaline copper reagents

Phenylalanine reacts with ninhydrin in the presence of a dipeptide (usually glycyl-L-leucine or L-leucyl-L-alanine) to form a fluorescent product. The fluorescence is enhanced and stabilized by the addition of an alkaline copper reagent to adjust the pH to 5.8 and the resulting fluorescence is measured at 515 nm after excitation at 365 nm see Procedure 10.2. 

The blank is prepared with I ml of distilled water, adding low-alkalinity copper reagent and arsenomolybdate reagent as described in step 4. 

Anhydrous sodium sulfate is added to the combined alkaline copper reagent to suppress back-oxidation by air. 

The Somogyi-Nelson assay (see Alternate Protocol 1) requires 30 min for the preparation of the low-alkalinity copper reagent and the same amount of time for the preparation of the arsenomolybdate reagent. An additional 24 hr should be considered for the 37°C incubation when preparing the arsenomolybdate reagent. Therefore, a total of 26 hr is necessary from the beginning of the experiments to obtaining the results. 

Paleg, L.G. 1959. Citric acid interference in the estimation of reducing sugars with alkaline copper reagents. Anal. Chem. 31 1902-1904. 

Many color reagents are applicable to the oligosaccharides. Some of those used are ammoniacal silver nitrate,60 resorcinol-hydrochloric acid for inulin hydrolyzates,137 acidic benzidine for maltodextrins,138 Bromo-phenol Blue or p-anisidine-hydrochloric acid for galactosiduronic acids,130 and alkaline copper reagent with phosphomolybdic acid for dextrins.71... 

Separately prepare 100 ml of fresh alkaline copper reagent by mixing together in order ... 

Add 6 ml of this alkaline copper reagent to each of the 10 tubes and mix immediately after the addition to avoid precipitation. 

Separately prepare 100 ml of fresh alkaline copper reagent by mixing, in order, 1 ml of 1% CuSC>4 5H20 and 1 ml of 2% sodium tartrate into 98 ml of 2% Na2C03 in 1 N NaOH. 

Procedure The alkaline copper reagent was prepared just prior to use by mixing 1 vol. of copper sulphate solution with 1 vol. of sodium potassium tartrate solution followed by 98 vol. of the sodium carbon-ate/sodium hydroxide solution. Alkaline copper solution (5 ml) is added to 100 pA of sample, containing between 20-100 pig of protein, diluted with 900 pil of water. After 10 min 0.5 ml of diluted Folin and Ciocalteus reagent is added. The absorbance at 750 nm is read after 30 min and within 90 min from the final addition. 

By contrast, ascorbate gives a positive result when urine is tested with alkaline copper reagents for reducing compounds, and a result that can falsely be interpreted as indicating glucosuria. 

The acid hydrolysis of CD was investigated based on the measurement of reducing power with an alkaline copper reagent [62]. The kinetics was found to be complex (Table 16.3). 

Figure 4.6 Measurement of glucose using alkaline copper reagents and glucose oxidase.

As discussed in section 4.2.1.4, glucose in plasma and urine can be determined in two ways, using an alkaline copper reagent or using glucose oxidase linked to a dyestuff. Both methods were used to determine urine glucose in six people ... 

Glucose is very soluble in water, the solution being about one-third as sweet as sucrose of the same concentration. It is dextrorotatory, [a]jj = + 52-2°. Glucose is a typical aldose sugar in all its reactions. It reduces alkaline copper reagents, forms a characteristic osazone with phenylhy azine, and is readily fermented by yeasts and bacteria. 

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