An improved colorimetric method for quantitation of heme using tetramethylbenzidine as substrate
An improved colorimetric method for quantitation of heme using tetramethylbenzidine as substrate
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DOI：
10.1016/j.ab.2005.06.022
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发表时间：
2005-09-15
影响因子：
2.9
通讯作者：
Kamei, K
中科院分区：
文献类型：
--
作者：
Huy, NT;Dai, TXT;Kamei, K
血红素是一种具有多种生物学功能的重要分子，常从血红蛋白、细胞色素、血色素、过氧化氢酶和过氧化氢酶等降解的血红蛋白中释放出来。血红素的定量已用于疟疾的检测[1]和血红素相互作用的研究[2，3]。目前的血红素定量分析需要昂贵的仪器，如荧光分光光度计[4]，或使用有毒物质，如吡啶[5]。此外，还发展了几种使用NaOH[6]、十二烷基硫酸钠[7]或Triton-甲醇[8]的比色方法。在这些比色法中，Triton-甲醇被认为是最灵敏的方法。基于3，3！，5，5！-四甲基联苯胺(TMB)1被血红素的假过氧化物酶活性氧化[9]，本文描述了一种使用1-StepTM Turbo TMBELISA底物(Turbo-TMB，皮尔斯编号34022)的简单、高灵敏度的比色分析方法。我们证明了Turbo-TMB法是一种比Triton-甲醇法更灵敏、更易于操作的方法。亚铁血红素是从西格玛购买的。将氯化高铁血红素溶解于20 mM氢氧化钠中，以15000rpm的转速离心10min，除去剩余的氯化高铁血红素晶体，制成新鲜的原料液。用mMD58,400在100 mM氢氧化钠[6]中从385 nm处的吸光度估计血红素浓度，并用20 mM氢氧化钠稀释至1 mM。所有吸收光谱和吸光度在日立U-3300双光束分光光度计(日本东京)上记录，使用1.0厘米光路石英试管。用含1%牛血清白蛋白的20 mM磷酸盐缓冲液(pH 7.4)和20 mM HEPES缓冲液(pH 7.4)测定血红素浓度。Turbo-TMB是一种完整的溶液，含有TMB和稳定的过氧化氢，在使用前保持在4摄氏度并置于室温下。将血红素(100L，终浓度为4~130 nm)与Turbo-TMB(400L)混合，室温孵育30min。加入2M硫酸(500L)停止反应，测定反应混合物在450 nm处的吸光度。实验室提供的其他过氧化物酶底物，如2，2！-azino-bis-3-ethylbenzothiazoline-6-sulfonic酸和邻苯二胺也进行了测试，它们的灵敏度低于Turbo-TMB。将血红素溶液(100L，最终浓度范围为33~2100 nm)与Triton-甲醇(2.5%Triton在甲醇中)混合，在室温下孵育10min，然后如前所述在397 nm处记录吸光度[8]。
Heme, an essential molecule with various biological functions, is often released from degraded hemoprotein including hemoglobin, cytochrome, hemopexin, peroxidase, and catalase. Quantitation of heme has been used in detection of malaria [1] and studies of heme interaction [2, 3]. Current quantitative assays for heme require expensive instrumentation such as a spectrofluorometer [4], or the use of toxic substances such as pyridine [5]. In addition, several colorimetric methods have been developed using NaOH [6], sodium dodecyl sulfate [7] or Triton–methanol [8]. Among these colorimetric methods, Triton–methanol has been reported as the most sensitive method. Based on oxidation of 3, 3!, 5, 5!-tetramethylbenzidine (TMB) 1 by the pseudoperoxidase activity of heme [9], we describe here a simple colorimetric assay with a high sensitivity using 1-StepTM Turbo TMBELISA substrate (Turbo-TMB, Pierce No. 34022). We demonstrated that the Turbo-TMB assay is an easily performable method with a higher sensitivity than that of the Triton–methanol method. Heme was purchased from Sigma. A stock heme solution was prepared fresh by dissolving hemin chloride in 20mM NaOH and then centrifuging for 10min at 15,000 rpm to remove remaining hemin crystal. Heme concentrations were estimated from the absorbance at 385 nm using mMD58, 400 in 100mM NaOH [6], and adjusted to 1mM by diluting with 20mM NaOH. All absorption spectra and absorbance were recorded on a HitachiU-3300 double-beam spectrophotometer (Tokyo, Japan) using 1.0-cm light-path quarts cuvettes. Solutions of heme in 20mM phosphate buffer (pH 7.4) and 20mM Hepes buffer containing 1% bovine serum albumin (pH 7.4) were used to evaluate the heme concentration. Turbo-TMB, a complete solution that contains TMB and a stabilized hydrogen peroxide, was kept at 4 C and brought to room temperature before use. Heme (100l, final concentrations ranged from 4 to 130nM in serial 1: 2 dilutions) and Turbo-TMB (400l) were mixed and incubated for 30min at room temperature. The reaction was stopped by adding 2M sulfuric acid (500l), and absorbance of the reaction mixture at 450nm was measured. Other peroxidase substrates available in the laboratory such as 2, 2!-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid and o-phenylenediamine have also been tested and they gave smaller sensitivities than Turbo-TMB. Heme solutions (100l, final concentrations ranged from 33 to 2100nM in serial 1: 2 dilutions) and Triton–methanol (2.5% Triton in methanol) were mixed and incubated at room temperature for 10min, then the absorbance values were recorded at 397nm as described previously [8].