A comparison of fluorescamine and naphthalene-2,3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids
A comparison of fluorescamine and naphthalene-2,3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids
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DOI：
10.1006/abio.2001.5338
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发表时间：
2001-10-15
影响因子：
2.9
通讯作者：
Grant, KB
中科院分区：
文献类型：
--
作者：
Bantan-Polak, T;Kassai, M;Grant, KB
使用适当的荧光衍生程序是相当重要的准确测定生物样品中的氨基酸和金属辅助肽水解反应。尤其关键的是，等量氨基酸的相对荧光强度（RFI）尽可能相似。虽然荧光胺和萘-2，3-二甲醛（NDA）已被证明是用于氨基酸检测的优良荧光试剂，但各种因素（如有机溶剂、缓冲液和pH）的影响从未被严格评估过相对于归一化单个氨基酸的相对荧光强度。为此，在这里，我们描述了优化的荧光胺和NDA衍生化反应，提高了基于微孔板的氨基酸检测的准确性。对于荧光胺和NDA，我们已经表明，19种氨基酸中有16种的RFI值大于70%。虽然色氨酸的测定是有问题的，但通过向NDA反应中加入β-环糊精克服了这一困难。原则上，本文报道的优化的荧光胺和NDA微孔板程序可以用作检测20种天然氨基酸中的19种的互补技术。(C)北京：科学出版社.
The use of appropriate fluorometric derivatization procedures is of considerable importance for accurate determination of amino acids in biological samples and in metal-assisted peptide hydrolysis reactions. it is especially critical for the relative fluorescence intensities (RFI) of equal amounts of amino acids to be as similar as possible. While fluorescamine and naphthalene-2,3-dicarboxaldehyde (NDA) have proven to be excellent fluorogenic reagents for amino acid detection, the effects of various factors such as organic solvent, buffer, and pH have never been rigorously evaluated with respect to normalizing the relative fluorescence intensities of individual amino acids. To this end, here we describe optimized fluorescamine and NDA derivatization reactions that enhance the accuracy of microplate-based detection of amino acids. For both fluorescamine and NDA, we have shown that the RFI values of 16 of 19 amino acids are greater than 70%. Although determination of tryptophan is problematic, this difficulty is overcome by the addition of beta -cyclodextrin to the NDA reaction. In principle, the optimized fluorescamine and NDA microplate procedures reported here can be utilized as complementary techniques for the detection of 19 of 20 naturally occurring ami-no acids. (C) 2001 Academic Press.