A Specific Quantitative Colorimetric Assay for L-Asparagine

• Published in: Analytical biochemistry Vol. 211; no. 2; pp. 242 - 249
• Main Authors: Sheng, S.J., Kraft, J.J., Schuster, S.M.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.06.1993; Elsevier
• Subjects: Aminoacids, peptides. Hormones. Neuropeptides; Analytical, structural and metabolic biochemistry; Asparaginase - metabolism; Asparagine - analysis; Aspartate-Ammonia Ligase - metabolism; Biological and medical sciences; Colorimetry - methods; Ethanol; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Ninhydrin; Proteins; Recombinant Proteins - metabolism; Sensitivity and Specificity; Spectrophotometry; Spectrophotometry, Ultraviolet; Temperature; Carbon-nitrogen ligases; Enzyme; Environmental factor; Colorimetry; Optimization; Aspartate-ammonia ligase; Analysis method; Enzymatic activity; Ligases; Concentration effect; Asparagine; L stereoisomer; Hydrolases; Asparaginase; Quantitative analysis
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1993.1264

When an aqueous L-asparagine solution was mixed with a dilute ethanolic ninhydrin solution and incubated at temperatures lower than 37°C, the resulting mixture exhibited an ultraviolet (uv)-visible absorption spectrum with the maximum absorption at 340-350 nm. In contrast, the mixtures of several other amino acids with ninhydrin yielded Ruhemann purple (S. Ruhemann, 1910, J. Chem. Soc. 97, 1438-1449, 1910; S. Ruhemann, 1910, J. Chem. Soc. 97, 2025-2031) and the corresponding uv-visible spectra had absorption maxima at 405 and 570 nm. The effects of several factors including the reaction temperature, the ninhydrin concentration, and pH on the asparagine-ninhydrin reaction were investigated to optimize the specificity and sensitivity. As a result, a simple and specific colorimetric asparagine assay was developed. Using the assay protocol, the absorption of the asparagine-ninhydrin mixtures at 340 nm had a linear relationship with the asparagine concentration in the range of 50 μM to 50 mM, even in the presence of a high background of other amino acids. The application of this assay could be easily extended to more complex enzymatic reaction systems. When the enzyme activities of L-asparagine synthetases from different species and commercial L-asparaginase were measured with both the ninhydrin colorimetric procedure and the HPLC amino acid analysis, comparable results were obtained. While the chemistry of this novel asparagine-ninhydrin reaction is not fully understood, the colorimetric asparagine assay reported herein is of great practical value because it is specific, sensitive, simple, and extremely inexpensive.