香豆素-3-羧酸作为化学产生和γ辐射产生的羟基自由基的检测剂。

Coumarin-3-carboxylic acid as a detector for hydroxyl radicals generated chemically and by gamma radiation.

作者信息

Manevich Y, Held K D, Biaglow J E

机构信息

Department of Radiation Oncology, Medical School, University of Pennsylvania, Philadelphia 19104, USA.

出版信息

Radiat Res. 1997 Dec;148(6):580-91.

PMID:9399704

Abstract

Coumarin-3-carboxylic acid (3-CCA) was used as a detector for hydroxyl radicals (.OH) in aqueous solution. The .OH was generated by gamma irradiation or chemically by the Cu2+-mediated oxidation of ascorbic acid (ASC). The excitation and emission spectra of 3-CCA, hydroxylated either chemically or by gamma irradiation, were nearly identical to those of an authentic 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). The pH-titration curves for the fluorescence at 450 nm (excitation at 395 nm) of 3-CCA, hydroxylated either chemically or by gamma radiation, were also identical to those of authentic 7-OHCCA (pK = 7.4). Time-resolved measurements of the fluorescence decays of radiation- or chemically hydroxylated 3-CCA, as well as those of 7-OHCCA, indicate a monoexponential fit. The fluorescence lifetime for the product of 3-CCA hydroxylation was identical to that of 7-OHCCA (approximately 4 ns). These data, together with analysis of end products by high-performance liquid chromatography, show that the major fluorescent product formed by radiation-induced or chemical hydroxylation of 3-CCA is 7-OHCCA. Fluorescence detection of 3-CCA hydroxylation allows real-time measurement of the kinetics of .OH generation. The kinetics of 3-CCA hydroxylation by gamma radiation is linear, although the kinetics of 3-CCA hydroxylation by the Cu2+-ASC reaction shows a sigmoid shape. The initial (slow) step of 3-CCA hydroxylation is sensitive to Cu2+, but the steeper (fast) step is sensitive to ASC. Analysis of the kinetics of 3-CCA hydroxylation shows a diffusion-controlled reaction with a rate constant 5.0 +/- 1.0 x 10(9) M(-1) s(-1). The scavenging of .OH by 3-CCA was approximately 14% for chemical generation with Cu2+-ASC and approximately 50% for gamma-radiation-produced .OH. The yield of 7-OHCCA under the same radiation conditions was approximately 4.4% and increased linearly with radiation dose. The 3-CCA method of detection of .OH is quantitative, sensitive, specific and therefore accurate. It has an excellent potential for use in biological systems.

摘要

香豆素 - 3 - 羧酸（3 - CCA）被用作水溶液中羟基自由基（·OH）的检测剂。·OH通过γ辐射产生，或通过铜离子（Cu²⁺）介导的抗坏血酸（ASC）氧化化学产生。经化学或γ辐射羟基化的3 - CCA的激发光谱和发射光谱与纯品7 - 羟基香豆素 - 3 - 羧酸（7 - OHCCA）的光谱几乎相同。经化学或γ辐射羟基化的3 - CCA在450 nm（395 nm激发）处荧光的pH滴定曲线也与纯品7 - OHCCA（pK = 7.4）的相同。对辐射或化学羟基化的3 - CCA以及7 - OHCCA的荧光衰减进行时间分辨测量，结果显示为单指数拟合。3 - CCA羟基化产物的荧光寿命与7 - OHCCA的相同（约4 ns）。这些数据，连同通过高效液相色谱对终产物的分析表明，3 - CCA辐射诱导或化学羟基化形成的主要荧光产物是7 - OHCCA。3 - CCA羟基化的荧光检测允许实时测量·OH产生的动力学。γ辐射使3 - CCA羟基化的动力学是线性的，尽管Cu²⁺ - ASC反应使3 - CCA羟基化的动力学呈S形。3 - CCA羟基化的初始（慢）步骤对Cu²⁺敏感，但较陡（快）的步骤对ASC敏感。3 - CCA羟基化动力学分析表明这是一个扩散控制反应，速率常数为5.0±1.0×10⁹ M⁻¹ s⁻¹。对于Cu²⁺ - ASC化学产生的·OH，3 - CCA对其清除率约为14%，对于γ辐射产生的·OH，清除率约为50%。在相同辐射条件下，7 - OHCCA的产率约为4.4%，并随辐射剂量线性增加。3 - CCA检测·OH的方法具有定量、灵敏、特异且准确的特点。它在生物系统中具有极好应用潜力。