A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples
A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples
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DOI：
10.1006/abio.2001.5464
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发表时间：
2002-01-15
影响因子：
2.9
通讯作者：
Trackman, PC
中科院分区：
文献类型：
--
作者：
Palamakumbura, AH;Trackman, PC
赖氨酰氧化酶催化正常成熟功能性不溶性细胞外基质生物合成中胶原蛋白和弹性蛋白交联所需的最终已知酶促步骤。此外，赖氨酰氧化酶已被确定为一种可能的肿瘤抑制因子。生物样品中赖氨酰氧化酶活性传统上最可靠地通过氚释放终点测定法进行评估，所述测定法使用放射性标记的胶原蛋白或弹性蛋白底物，涉及释放的氚化水的费力的真空蒸馏。此外，存在一种灵敏度较低的荧光法，该方法采用nonpeptidyl胺赖氨酰氧化酶底物，并用辣根过氧化物酶偶联高香草酸氧化来测量过氧化氢的产生。本研究描述了一种更灵敏的赖氨酰氧化酶活性的荧光检测，利用1，5-二氨基戊烷作为底物，并释放过氧化氢检测使用Amplex红辣根过氧化物酶偶联反应。该方法允许在37 ℃下每2 ml测定中检测40 ng酶，并且比目前可用的用于酶活性的荧光测定灵敏7.5倍。该方法消除了某些生物样品中的干扰，可成功地用于细胞培养实验中赖氨酰氧化酶活性的检测。(C)2001年，爱思唯尔科学。
Lysyl oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking in the biosynthesis of normal mature functional insoluble extracellular matrices. In addition, lysyl oxidase has been identified as a possible tumor suppressor. Lysyl oxidase activity in biological samples is traditionally and most reliably assessed by tritium release end-point assays using radiolabeled collagen or elastin substrates involving laborious vacuum distillation of the released tritiated water. In addition, a less sensitive fluorometric method exists that employs nonpeptidyl amine lysyl oxidase substrates and measures hydrogen peroxide production with horseradish peroxidase coupled to homovanillate oxidation. The present study describes a more sensitive fluorescent assay for lysyl oxidase activity that utilizes 1,5-diaminopentane as substrate, and released hydrogen peroxide is detected using Amplex red in horseradish peroxidase-coupled reactions. This method allows the detection of 40 ng of enzyme per 2 ml assay at 37degreesC and is 7.5 times more sensitive than the currently available fluorometric assay for enzyme activity. This method eliminates the interference that occurs in some biological samples and can be successfully used to detect lysyl oxidase activity in cell culture experiments. (C) 2001 Elsevier Science.