Sheng S, Kraft J J, Schuster S M
Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville 32610.
Anal Biochem. 1993 Jun;211(2):242-9. 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 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.
当将L-天冬酰胺水溶液与稀乙醇茚三酮溶液混合，并在低于37摄氏度的温度下孵育时，所得混合物呈现出紫外（uv）-可见吸收光谱，最大吸收峰在340 - 350纳米处。相比之下，其他几种氨基酸与茚三酮的混合物产生了鲁赫曼紫（S. 鲁赫曼，1910年，《化学学会杂志》97卷，1438 - 1449页，1910年；S. 鲁赫曼，1910年，《化学学会杂志》97卷，2025 - 2031页），相应的紫外-可见光谱在405和570纳米处有最大吸收峰。研究了反应温度、茚三酮浓度和pH值等几个因素对天冬酰胺-茚三酮反应的影响，以优化特异性和灵敏度。结果，开发了一种简单且特异的比色法天冬酰胺测定方法。使用该测定方案，天冬酰胺-茚三酮混合物在340纳米处的吸光度与50微摩尔至50毫摩尔范围内的天冬酰胺浓度呈线性关系，即使在存在高背景其他氨基酸的情况下也是如此。该测定方法的应用可以很容易地扩展到更复杂的酶反应体系。当用茚三酮比色法和HPLC氨基酸分析法测量来自不同物种的L-天冬酰胺合成酶和商业L-天冬酰胺酶的酶活性时，得到了可比的结果。虽然这种新型天冬酰胺-茚三酮反应的化学原理尚未完全理解，但本文报道的比色法天冬酰胺测定方法具有很大的实用价值，因为它特异、灵敏、简单且极其便宜。
