The Lewis B. and Dorothy Cullman Cancer Chemoprotection Center, Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
Cancer Epidemiol Biomarkers Prev. 2011 Jul;20(7):1516-23. doi: 10.1158/1055-9965.EPI-11-0279. Epub 2011 May 20.
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine with keto-enol tautomerase activity, rises rapidly in response to inflammation and is elevated in many chronic diseases. Isothiocyanates, such as sulforaphane from broccoli, are very potent inactivators of MIF tautomerase activity. A simple rapid method for determining this activity in tissues and body fluids may therefore be valuable for assessing severity of inflammation and efficacy of intervention.
Existing spectrophotometric assays of MIF, based on conversion of methyl L-dopachrome to methyl 5,6-dihydroxyindole-2-carboxylate and associated loss of absorption at 475 nm, lack sensitivity. Assay sensitivity and efficiency were markedly improved by reducing the nonenzymatic rate, by lowering pH to 6.2, replacing phosphate (which catalyzes the reaction) with Bis-Tris buffer, and converting to a microtiter plate format.
A structure-potency study of MIF tautomerase inactivation by isothiocyanates showed that sulforaphane, benzyl, n-hexyl, and phenethyl isothiocyanates were especially potent. MIF tautomerase could be readily quantified in human urine concentrated by ultrafiltration. This activity comprised: (i) a heat-labile, sulforaphane-inactivated macromolecular fraction (presumably MIF) that was concentrated during ultrafiltration; (ii) a flow-through fraction, with constant activity during filtration, that was heat stable and insensitive to sulforaphane. Administration of the sulforaphane precursor glucoraphanin to human volunteers almost completely abolished urinary tautomerase activity, which recovered over many hours.
A simple, rapid, quantitative MIF tautomerase assay has been developed as a potential biomarker for assessing inflammatory severity and effectiveness of intervention.
An improved assay for measuring MIF tautomerase activity and its applications are described.
巨噬细胞移动抑制因子（MIF）是一种具有酮-烯醇互变异构酶活性的促炎细胞因子，它能迅速响应炎症反应而升高，并且在许多慢性疾病中升高。异硫氰酸盐，如西兰花中的萝卜硫素，是 MIF 互变异构酶活性的非常有效的抑制剂。因此，一种在组织和体液中测定这种活性的简单快速方法，可能对于评估炎症的严重程度和干预的效果是有价值的。
现有的基于甲基 L-多巴色素转化为甲基 5,6-二羟基吲哚-2-羧酸酯以及 475nm 处吸光度降低的 MIF 比色法，缺乏灵敏度。通过降低非酶反应速率、将 pH 降低至 6.2、用 Bis-Tris 缓冲液替代磷酸盐（促进反应）以及转化为微量滴定板格式，显著提高了测定的灵敏度和效率。
对异硫氰酸盐对 MIF 互变异构酶失活的构效关系研究表明，萝卜硫素、苄基、正己基和苯乙基异硫氰酸盐特别有效。人尿经超滤浓缩后，MIF 互变异构酶很容易被定量。这种活性包括：（i）一种热不稳定、受萝卜硫素失活的大分子部分（推测为 MIF），在超滤过程中被浓缩；（ii）一种滤过液部分，在过滤过程中具有恒定的活性，该部分热稳定且对萝卜硫素不敏感。给人类志愿者施用萝卜硫素前体萝卜硫苷，几乎完全消除了尿中的互变异构酶活性，这种活性在数小时内恢复。
已经开发出一种简单、快速、定量的 MIF 互变异构酶测定法，作为评估炎症严重程度和干预效果的潜在生物标志物。
描述了一种改进的用于测量 MIF 互变异构酶活性及其应用的方法。
