A specific quantitative colorimetric assay for L-asparagine.
When an aqueous L-asparagine solution was mixed with a dilute ethanolic ninhydrin solution and incubated at temperatures lower than 37 degrees 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 microM 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.
Sheng
S
S
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville 32610.
Kraft
J J
JJ
Schuster
S M
SM
D001215:Asparaginase / Q000378:metabolism; D001216:Asparagine / Q000032:analysis*; D001217:Aspartate-Ammonia Ligase / Q000378:metabolism; D003124:Colorimetry / Q000379:methods; D000431:Ethanol; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009555:Ninhydrin; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D011994:Recombinant Proteins; D000431:Ethanol; D001216:Asparagine; D001215:Asparaginase; D001217:Aspartate-Ammonia Ligase; D009555:Ninhydrin