这项研究得到了捷克科学基金会 (GACR, 格兰特 17-12816S) 的支持。

所有动物实验都是按照《赫尔辛基宣言》 (311/1997, 捷克共和国农业部) 的规定进行的, 这是根据《关于保护和使用实验动物的条例》进行的。
1. 肝微粒体和胞浆组分的分离
<ol><li>用简单的微分离心法 (10.5万 x g) 制备大鼠肝脏亚细胞分 (微粒富含 P450 酶或富含硝酸或可溶性过氧化物的 cytosols)。 注: 颗粒和上清液分别作为微粒和 cytosols。<ol>
<li>用50毫米三盐酸缓冲液将肝脏样品 (1-10 克) 冲洗两次, pH 值7.4 包含150毫米氯化钾缓冲 (缓冲 1) (10 倍以上的体积比组织的重量,即10-100 毫升), 并把组织切成小块 (大约 2 x 2 毫米大小)。</li>
		<li>融汇该缓冲器 (> 3 体积/重量毫升/克) 存在于4摄氏度5分钟内的组织, 并通过过滤纸将组织中残留的非匀质件丢弃。离心匀浆在 600 x g为10分钟在4°c 和转移上清到另一个离心管。</li>
		<li>重新融汇在缓冲 1 (1 毫升每1克组织) 的颗粒, 重复步骤 1.1.3, 并丢弃颗粒。离心机汇集上清液在 1.5万x g 为20分钟在4°c。将上清液转移到另一离心管。</li>
		<li>离心机在 10.5万 x g上清液60分钟4摄氏度。收集上清 (细胞质) 和存储在整除数 (1-10 毫升) 在-80 摄氏度。使用布拉德福德26描述的方法表征细胞质的蛋白质含量。</li>
		<li>在100毫米磷酸钠缓冲液中重新悬浮颗粒, pH 值为 7.4 (> 2 体积/重量毫升/克), 离心机在 10.5万 x g处为60摄氏度。放弃上清。重新融汇颗粒 (微粒) 在50毫米三盐酸缓冲, pH 7.4 包含150毫米氯化钾和20% 甘油 (< 5 体积/重量毫升/克) 在均质体在4°c。存储微粒在 0.5-1 毫升整除数在-80 摄氏度。使用布拉德福德26描述的方法表征蛋白质含量的微粒。</li>
		<li>测定微粒中细胞色素 P450 的浓度。 注: P450 酶在微粒中的浓度是由大村和佐藤27所描述的, 用于测定 P450 与一氧化碳 (CO) 的复合物的吸收。一氧化碳是一种有毒化合物, 必须小心处理和罩着。</li>
	</ol>
</li>
</ol>2. 在酶系统存在的情况下, 用 DNA 孵化测试化学品 (致癌物/药物)
<ol><li>在含有细胞色素 P450 氧化酶系统的情况下, 用 DNA 培养测试化学品 (致癌物/药物)
	<ol>
<li>准备孵化混合物, 其最终体积为0.75 毫升, 在4摄氏度, 以下化合物和酶系统。<ol>
<li>混合100毫米磷酸盐缓冲液, pH 值 7.4, (0.375 毫升) 与10毫米的或生成系统 (10 毫米氯化镁2, 10 毫米 d-葡萄糖 6-磷酸盐, 10 毫米 NADP+, 1 U/毫升 d-葡萄糖 6-磷酸脱氢酶) (75 µL)。</li>
			<li>加入这种混合物微粒或纯重组 P450 在 Supersomes, 这是微粒分离的昆虫细胞与杆状病毒结构的基因重组 P450 酶, 50 pmol P450 酶在50µL 微粒体或 supersomal制剂. 在实验室或从商业来源分离的肝脏微粒体分数。</li>
			<li>添加1毫克小牛胸腺 DNA (0.3 毫升的库存溶液-3.3 毫克/毫升蒸馏水), 并在涡流振动筛上晃动5秒。</li>
			<li>最后, 添加7.5 µL 0.1 毫米测试 ellipticine 药物溶解在亚砜和42.5 µL 蒸馏水, 以达到一个数量的孵化混合0.75 毫升。根据检测 DNA 加合物的过程, 使用标记为3 H或14 C 或无标号化学物质的化学品。</li>
			<li>在涡流振动筛上摇动5秒, 在打开的管子上孵育37摄氏度, 30-60 分钟。</li>
			<li>也准备两个控制孵化相似, 但 (i) 没有活化系统 (微粒体样品) 或 (ii) 与它, 但没有测试化合物。</li>
		</ol>
</li>
	</ol></li>
	<li>孵化还原酶系统存在下的 DNA 检测化学物质 (致癌物/药物)
	<ol>
<li>准备孵化混合物, 其最终体积为0.75 毫升, 在4摄氏度, 以下化合物和酶系统。<ol>
<li>摄氏4摄氏度, 混合100毫米三 HCl 缓冲液, pH 7.4, 含0.2% 吐温 20, (0.375 毫升), 10 毫米溶液余子的 NQO1 还原酶 (µL), 胞浆分数 (肝胞浆分数-隔离在实验室或在使用胞浆的情况下从个人捐献者中分离出来的分数从含有1毫克蛋白质 (50 µL) 的商业来源获得。</li>
			<li>添加1毫克小牛胸腺 DNA (0.2 毫升的库存溶液-3.3 毫克/毫升蒸馏水), 并在一个摇摇器 5 s。</li>
			<li>最后, 添加7.5 µL 0.1 毫米测试化学物质 (溶解在蒸馏水, 甲醇, 乙醇或亚砜, 取决于化合物的溶解度) 和42.5 µL 蒸馏水达到0.75 毫升的容量为孵化混合物。根据检测 DNA 加合物的过程, 使用标记为3 H或14 C 或无标号化学物质的化学物质 (见下文)。</li>
			<li>用氩气将反应混合物清除1分钟, 在5秒的振动器上摇动, 在37摄氏度的封闭管中孵育 30-60 分钟。</li>
			<li>也准备两个控制孵化相似, 但 (i) 没有活化系统 (胞浆组分) 或 (ii) 与它, 但没有测试化学制品。</li>
		</ol>
</li>
	</ol></li>
	<li>用有机溶剂提取孵化混合物去除过量的化学试剂
	<ol>
<li>加入这些溶剂, 将孵化混合物与同一容积的醋酸乙酯 (或二乙基醚或己烷) 混合在试管中。在振动筛上摇动管子的内容, 直到乳液形成。</li>
		<li>在室温下离心机中, 在 1600 x g处旋转 (3 分钟)。如果有机和水相不正确分离, 旋转一次更长的时间或更高的离心速度。</li>
		<li>用吸管去除上部, 有机相收集。如果使用小体积 (< 400 µL), 则使用装有适当尖端的自动吸管。抛开这个有机的阶段。</li>
		<li>重复步骤 2.3.1, 2.3.2, 和2.3.3。通过氮气流 (至少需要 5-10 分钟) 去除残留的有机溶剂。</li>
	</ol></li>
	<li>孵化 DNA 的分离
	<ol>
<li>
苯酚/氯仿溶液中 DNA 的提取及其与乙醇的沉淀
		<ol>
<li>消除蛋白质从孵化混合物中提取的 dna 与苯酚, 苯酚/氯仿 (1:1) 的 dna 溶液, 和氯仿的步骤如下所示。</li>
			<li>将具有相同量的苯酚或苯酚/氯仿 (1:1) 的孵化混合物与一顶帽子的猎鹰或离心管结合在一起。搅拌混合物, 直到乳液形式。</li>
			<li>在室温下离心机中, 在 1600 x g处旋转 (3 分钟)。如果有机和水相没有适当的分离, 旋转一次更长的时间或更高的离心速度。</li>
			<li>用吸管将上水相转移到新的聚丙烯管上。如果使用小容量 (< 400 µL), 则使用装有适当尖端的自动吸管。与有机相一起去除蛋白质界面。</li>
			<li>将上水相与苯酚和氯仿混合物的相同量 (1:1) 结合起来。重现步骤2.4.1.2。-2.4.1.4。</li>
			<li>将上水相与相同量的氯仿结合, 重复步骤2.4.1.2。-2.4.1.4。用2卷的冷 (-20 °c) 乙醇回收 DNA。确定 DNA 溶液的体积。</li>
			<li>通过添加5米氯化钠, 对 0.1 m 的最终浓度进行强力搅拌, 以适应单价阳离子的浓度。结合2卷的冷 (-20 °c) 乙醇和适当的混合。冷却到-20°c.
</li>
			<li>储存在-20 摄氏度, 直到 DNA 沉淀。当 dna 在孵化 (e. g) 期间被碎片化时 (通过在酶活化反应过程中形成氧自由基) 或在隔离过程中的 dna 体积小 (< 1 kb) 或当 dna 小数量 (< 0.1 毫克/mL), 冷却时间必须增加, 温度降低到-70 摄氏度。</li>
			<li>旋转 (10 分钟) 在 1600 x g在0°c 在离心机。如果 DNA 以低浓度或小片段的形式存在, 则在更长的时间内旋转一次 (30 分钟)。放弃上清。</li>
			<li>要除去可能存在于沉淀 dna 中的任何溶质 (或残留的测试化学痕迹), 用70% 乙醇和二乙基醚冲洗 dna。用冷 (-20 °c) 70% 乙醇洗涤沉淀的 DNA。旋转 (10 分钟) 在 1600 x g在0°c 在离心机。放弃上清。</li>
			<li>重复步骤2.4.1.10。用冷 (-20 °c) 70% 乙醇洗涤沉淀的 DNA。旋转 (10 分钟) 在 1600 x g在0°c 在离心机。放弃上清。</li>
			<li>重复步骤2.4.1.10。将管子置于吸水纸的垂直状态, 以除去残留的上清。通过添加1毫升乙醚, 从分离的 dna 中去除试验化学物质的潜在残留物, 对 dna 颗粒进行清洗。旋转 (10 分钟) 在 1600 x g在0°c 在离心机。放弃上清。</li>
			<li>将 dna 颗粒溶于适当的体积 (通常在 100-400 µL 中, 以达到 0.5-1 µg/µL) 蒸馏水的 dna 浓度 (或在0.15 毫米柠檬酸钠和1.5 毫米氯化钠中)。dna 溶液可以在一夜之间站立4摄氏度, 或者可以加热到37摄氏度10-30 分钟, 以增加溶解 dna。</li>
			<li>贮存前, 将 dna 分离成小整除数 (10-20 µL), 因为 dna 溶液的反复冻结和解冻可能导致加成浓度下降。贮存在-80 摄氏度或更冷。 注: 分光光度法测定 dna: 一种简单而准确的方法, 它广泛用于测量制剂中 dna 的含量 (如果样品是纯的 (即), 而没有大量的污染物, 如蛋白质、苯酚或其他核酸), 是分光光度法测量碱基吸收的紫外线的 DNA 量 (见上文所述的过程)28。</li>
		</ol>
</li>
	</ol></li>
	<li>DNA 加合物形成的检测程序
	<ol><li>32对 postlabeling 的测定 注: DNA 水解利用本阶段制备的水解产物对加合物 (2.5.1) 以及正常 (未修改) deoxynucleotides (2.5.7) 进行分析。<ol>
<li>将 micrococcal 核酸酶 (锰) 溶于水中, 浓度为450单位 (U)/毫升。透析对蒸馏水和调整到 300 U/毫升。透析脾脏磷酸二酯酶 (SPD) 溶液和调整 4 U/毫升。</li>
			<li>混合锰和 spd 到最终浓度150亩/µL 锰和2.5 亩/µL spd (锰/SPD 解决方案)。采取含有12.5 µg 的 DNA 溶液, 在蒸发器中蒸发至干燥。溶解在6.5 µL 蒸馏水。</li>
			<li>添加5.0 µL 锰/SPD 溶液 (最终浓度的锰是60亩/µL, 最终浓度的 SPD 是1亩/µL), 1.0 µL 消化缓冲 (最后浓度的琥珀酸钠是20毫米, 最后浓度 CaCl2是8毫米)。混合物的最终体积为12.5 µL. 混合并允许反应为3小时37摄氏度。</li>
			<li>去除2.5 µL (转移到另一管) 进一步稀释和分析未修改的 deoxynucleotides (2.5.7)。</li>
		</ol></li>
		<li>核酸酶 P1 浓缩程序
		<ol>
<li>对剩余的10.0 µL 水解物, 添加0.65 µL 醋酸钠缓冲液 (最终浓度, 40 毫米), 0.65 µL ZnCl2溶液 (最终浓度0.1 毫米), 1.25 µL NP1 溶液 (最终浓度, 0.385 µg/µL), 0.45 µL 蒸馏水。混合物的最后体积是13µL。</li>
			<li>允许混合物反应30分钟在37°c, 并且结束反应与增加3µL 的三溶液。</li>
		</ol></li>
		<li>n-丁醇浓缩过程
		<ol>
<li>对剩余的10.0 µL DNA 水解液, 添加215µL 11.6 毫米甲酸铵溶液, pH 值 3.5, 25 µL 10 毫米 TBA 氯溶液。提取与250µL n-丁醇 (饱和与水) 通过剧烈的混合。在 1600 x g处旋转 (3 分钟) 以分隔图层, 然后脱下上n-丁醇层。再提取250µL n-丁醇 (饱和与水), 旋转, 脱下上n丁醇层, 并与前萃取物结合。</li>
			<li>加入400µL 水 (饱和与n-丁醇) 对此萃取物, 并积极搅拌。旋转以分离层并删除底部的水层。用400µL 水 (用n-丁醇饱和) 重复此洗涤过程。将3µL 250 毫米的三 HCl 溶液, pH 9.5, 添加到n-丁醇层。蒸发n-丁醇在常温下在蒸发器中干燥。</li>
			<li>将残留物溶于100µL n丁醇, 再蒸发至干燥, 并在16.0 µL 水中溶解残渣。</li>
		</ol></li>
		<li>标签的加合物
		<ol>
<li>添加1µL bicine 缓冲液 (标记缓冲) 和4.5 µL 的混合包含100µCi [γ-32P] ATP, 45 pmol 的冷 ATP, 10.0 U T4-PNK (T4-phosphonucleotide 激酶) 到16.0 µL 溶液从 NP1 或丁醇浓缩混合。试剂的最终浓度将如下:20 毫米 bicine, 10 毫米氯化镁2, 10 毫米 dithiotreitol, 0.5 毫米胺, 0.5 U/µL T4-PNK, 3 µM ATP。混合物的总体积为20µL。</li>
			<li>允许混合物在室温下反应30分钟。将整个示例 (i. e20 µL) 应用到裴纤维素薄层板上 (2.5.6.)。</li>
		</ol></li>
		<li>NP1 或n-丁醇强化程序的疗效评价
		<ol>
<li>用50µL 水冲洗管子的底部。搅拌三十年代和旋转 (1 分钟) 在 1600 x g在离心机, 以确定没有污染的盖子。5µL 在裴纤维素薄层板上 (20 x 20 厘米)。色谱仪使用280毫米的解决方案 (NH4)2, 所以4和 50 mM NaH2PO4, pH 6.8。</li>
		</ol></li>
		<li>adducted deoxynucleotides 的薄层色谱分离
		<ol>
<li>用蒸馏水预洗薄层色板。特别推荐用于国产板材。 注: 此清洗应进行, 以消除黄色的板材, 一种颜色, 可以提高背景, 特别是在溶剂的前面。</li>
			<li>将整个样品点到裴纤维素薄层板上, 开始色谱 (2.5.4);加合物的清理是通过在 D1 和 D2 方向上开发板块(图 2) 来完成的。</li>
			<li>以 D1 方向开发板 (图 2)。使用1.7 米磷酸钠缓冲液, pH 值为 6.8, 以确保 DNA 加合物在薄层色板开始时保持不动。类似, 使用此缓冲器在 D2 方向上开发板。有关过程的可能缓冲区的信息, 请参见为解决几种类型的加合物20、28而描述的解决方案. 注: 可省略 D2 方向的板的开发。</li>
			<li>在连续两个浴缸中, 用色谱法洗去去离子水中的盘子5分钟。然后把盘子擦干。</li>
			<li>D4 3.5 米甲酸锂缓冲液、ph 值为 D3 方向的8.5 米尿素、0.5 米三盐酸缓冲液、ph 8.0、含0.8 米 LiCl 和8.5 米尿素的 D4 方向 (图 2), 开发出 D3 和3.5 方向的板材。必须对溶剂进行调整, 以在薄层板上开发加合点。有关 D3 和 D4 开发过程可能的缓冲区的信息, 请参见为解决几种类型的加合物23、24、25、28而描述的解决方案.</li>
			<li>为避免任何问题在 D4, 在开发以后在 D4 方向和水洗涤, 开发板材 (沿 D4) 在 1.7 M 磷酸钠, pH 值 6.0 (通常被分配作为发展方向 D5), 到纸灯芯 (12 x 11.5 cm) 的顶部。 注意: D5 方向也可以省略。在这种情况下, 有必要打开薄层色谱池时, 溶剂已经达到了薄层色谱的顶部, 并允许运行高达60分钟。这种方法甚至比添加灯芯 (在许多实验室中经常使用的方法来删除 D4 中的任何问题) 要好。</li>
		</ol></li>
		<li>水解后正常 (未修改) deoxynucleotides 的量化
		<ol>
<li>稀释整除的水解物 (从2.5.1描述 DNA 水解) 与蒸馏水, (i. e, 2.5 µL 的消化混合物从2.5.1.调整为250µL, 而此解决方案的10µL 调整为150µL)。</li>
			<li>采取5µL (10 pmol 正常 deoxynucleotides) 整除本文摘, 在µL 中添加 2.5 2.5.4 10 毫米的三 HCl 缓冲液、pH 值9.0 和标签.(混合物的最后的容量是10µL)。允许在室温下反应30分钟。</li>
			<li>采取4µL 整除的混合物和稀释到750µL 10 毫米三 HCl, pH 9.0。混合和旋转, 以建立没有污染的盖子。在培纤维素薄层板上应用5µL。在280毫米 (NH4)2的解决方案中开发薄层色板, 所以4和 50 mM NaH2PO4, pH 6.5。允许干燥后薄层色谱。</li>
			<li>使用显影在室温下进行约45分钟, 以本地化四未修改的核苷酸双磷酸盐。通过液体闪烁或切伦科夫计数来进行量化的切割点。</li>
		</ol></li>
		<li>相对加合标记的计算 (RAL)
		<ol>
<li>确定加合点中的计数值和标记为未修改 (正常) deoxynucleotides 的整除中的计数。 注: 后者必须确定在18万计数比前者少的材料, 一个数字, 这是换算系数, 适用于未修改 (正常) deoxynucleotides 的计数, 以评估的 RAL 价值的加成水平。根据上面所示的方程式 (2) 计算出 DNA 加合物的 RAL。</li>
		</ol></li>
		<li>用放射性标记药物检测测试致癌物或药物对 DNA 的约束力
		<ol>
<li>用液体闪烁计数法评估用化学物质修饰的 DNA 的3H 或14C 的放射性。</li>
			<li>在闪烁瓶中添加 10-50 µL 的 DNA 溶液到3毫升的闪烁溶液中。混合好。使用闪烁计数器测量放射性。</li>
		</ol></li>
	</ol></li>
</ol>

作者没有什么可透露的。

本文展示了一种广泛应用的方法来研究化学物质对代谢中间体 bioactivated 的效力, 从而产生共价 DNA 加合物。这是一个关键的问题, 因为评价环境化学物质或药物对产生共价 DNA 加合物的代谢产物的作用, 是癌症及其治疗发展的一个重要领域。致癌物质对 DNA 的修饰被认为是肿瘤发展的原因, 现在被判定为致癌物质引起的致癌的中心教条。这一建议得到了各种结果的证实, 例如: (一) 各种致癌物的致癌?...

外来化合物 (环境化学物质或药物) 的新陈代谢发生在两个阶段 1.阶段 I 和 II 的目的是使最初疏水性 (不水溶性) 化合物更亲水性 (水溶性), 从而使它们容易 excretable 通过尿, 粪便或汗水。第一阶段 (功能化) 反应包括氧化、还原和羟化酶催化的酵素, 如细胞色素 P450s (P450s, CYPs), 过氧化物 (I. e, 环氧合酶, COX), 酮酮硝酸 (AKRs) 和微粒体黄素含 monooxygenases (FMOs)。第一阶段还包括减少反应, 介导的各种硝酸即,微粒体: 细胞色素 P450 还原酶 () 和胞浆 NAD (P) H: 醌氧化还原酶 (NQO1), 黄嘌呤氧化酶 (XO) 和醛氧化酶 (AO) 1.在第二阶段 (共轭) 中, 第一阶段所附的官能团用于共轭小极性分子, 进一步增加极性。考虑参与第二阶段反应的酶的例子包括 sulfotransferases (结果), N, O-乙酰转移酶 (NATs), 甲基转移酶如邻苯二酚-O-甲基 (COMT), 谷胱甘肽 S-转移酶 (禾采赞) 和苷二磷酸 glucuronosyltransferases (UGTs) 1。然而, I. 期和 II. 类酶的分类并不是硬性的, 有些酶可以被归类为任何一类。
P450 酶 (EC 1.14.14.1) 是含有多种生物的蛋白质的血红素, 参与多种化学物质的转化, 催化其转化为3,4。P450 酶催化许多基质的羟基化, 反应中, 氧气的一个原子被引入外来化合物分子中, 而第二个氧原子通过需要两个电子的反应而被还原形成水 [方程 (1)]3,4:
RH + O2 + NADP + h+ →卢武铉 + h2O + + (1)
哺乳动物细胞内质网膜中的 P450 酶 (微粒 P450 系统) 是酶甲烷系统的成员, 进一步包含了细胞色素 P450 还原酶 () 和细胞色素 b 5 , 该酶的基质称为 NADH: 细胞色素b5 还原酶。一个普遍接受的理论假设, P450 所需的两个电子的供体是系统。然而, 细胞色素b5 也可能充当 P450 电子的捐助者, 即作为电子减少 P450 的捐助者, 在其反应周期的第二次还原期间, 它与 NADH 一起行动: 细胞色素b5 还原酶2,3,4。
哺乳动物利用各种 P450 酶 (例如,家庭5、8、11、17、19、21、24、26和27的酶, 用于合成有价值的内源化合物, 如类固醇, 并用于天然产物的分解代谢2,3.其他 CYP 哺乳动物的酶, 如人类 CYP1A2, 2C9, 2C19, 2D6 和 3A4, 代谢外源化学药品作为药物。5,6最重要的酶催化代谢的药物是 CYPs 的3A 亚科, 特别是 CYP3A4。外来化合物的转换, 如亲致癌物和亲毒物, 由人类 CYP1A1、1A2、1B1、2a6 型坦克、2E1和3A4 2 5 介导.大多数这些 CYPs 都存在于肝脏中 (除了 CYP1A1 和 1B1)。然而, CYPs 也表达在一些肝外器官。这种 P450s 可能具有很大的意义, 主要是在参与它们在 bioactivation 代谢化学物质 (药物) 到反应中间体的这些器官7。各种 P450s 都是由其基底的几种化合物引起的, 但不一定如此。
许多 P450 酶在化学 (药物) 毒性中起着作用。它们不仅可以将外来化合物转化为解毒代谢物, 还能将它们激活到反应性物种, 从而改变另外表现出不同生物特性的内源大分子, 通常会引起它们的毒性。DNA、脂质和蛋白质可能是由活性化学物质产生的反应性 electrophiles 和自由基修饰的靶点。在 DNA 的情况下, 解决几个重要的基因应答及其机制已经被称为2,3,4,5。
DNA 的变化会导致细胞生长控制的减少, 这种现象被认为是导致致癌过程发展的主要因素。生成具有致癌效力的化学物质的共价 DNA 加合物被认为是致癌过程启动阶段最重要的步骤之一8, 9, 10, 11.结果表明, dna 加合物的形成与肿瘤发生之间存在关系, 而 dna 加合物的数量减少化学 8, 9, 10,11,12,13,14. 致癌/药物衍生的 dna 加合物的形成依赖于 dna 的个体基础, 并受 dna 中这些碱基序列的影响。dna 加合物的修复依赖于它们的位置 (在转录或非转录 dna 链上) 和类型的修改核苷酸序列8, 11, 12, 15, 16。
在本文中, 我们描述了使用酶催化转化的化学物质 (药物) 的过程, 以调查其效力, 以活化成代谢物, 改变 dna (生成 dna 加合物)。对于共价 DNA 结合, 测试化合物通常应由氧化或还原反应激活, 取决于个别药物。被测试的化学物质的氧化或还原活化由 P450-dependent 酶系统介导的微粒体亚细胞分数或减少与硝酸目前在微粒 (NADH: 细胞色素b5 还原酶, P450 酶) 和细胞胞浆亚组分 (NQO1, XO, AO, 过氧化物酶)。反应性代谢产物随后绑定到 dna 加合物 dna 的形成。由于氧化和还原反应对于激活几种药物对这些活性物种都很重要, 本文介绍了采用氧化还原酶体系的实验过程。此外, 还详细介绍了能够检测和量化这些 DNA 加合物的适当方法。
两个独立的程序, 以确定测试化学, 激活的酶系统, 是绑定到 DNA 的建议: 32p-postlabeling 技术和利用放射性标记化合物 (e. g, 3H 或14C)。对于第一个飞行员, 建议筛选32p-postlabeling 检测。精确评价的溶液中 DNA 含量的测定必须在两种方法之前。
32p-postlabeling 技术利用非放射性化学物质 (致癌物/药物) 修饰的 DNA 的酶水解, 3´-phosphodeoxynucleosides, 在5´中加磷酸化与放射性磷 (32P)-OH 位置, 并通过色谱17 (图 1) 将化学核苷酸加合物从正常 (未修改) deoxynucleotides 分离出来。该化合物修饰的 DNA 由限制性、micrococcal 核酸酶和 exonuclease 的混合物水解而成, 称为脾脏磷酸二酯酶。含有正常 (未修改) 和修饰的 deoxyribonucleoside 3´ monophosphates 的水解 DNA 的混合物, 与 [γ-32P] atp 在 9.5 pH 值的载体 (非放射性) atp 和 T4-polynucleotide 激酶的存在下反应, 形成 5´-32P 标记的3´、5´ bisphosphates ("标准" 过程图 1)。所使用的碱性 pH 值能将 T4-polynucleotide 激酶的酶活性降到最低, dephosphorylate deoxyribonucleoside 3´ monophosphates。在聚乙烯亚胺 (PEI) 纤维素上采用多向阴离子交换薄层色谱 (TLC)对32 P 标记的加合物进行分离和解析, 其标记 deoxynucleotides 不经化学修饰 (图2).在第一和第二洗脱步骤 (在 D1 和 D2 方向), 标记正常 (未修改) deoxynucleotides 以及 [32P] 磷酸盐是洗脱从开始的薄层-裴-纤维素板使用电解液的水溶液到一个短的片断色谱纸应用于薄层板的顶部, 而含有束缚化学物质 (致癌物/药物-加合物) 的 deoxynucleotides 在培纤维素板的起始处保持, 以进一步解决在 D3 和 D4 方向有几个不同的溶剂系统 (图 2)。加合物的定位是使用屏幕增强显影进行的;分离的加合物被发现作为黑暗的可认识的斑点在 x 射线影片。斑点的区域从板材被切除并且用于定量放射性由液体闪烁或切伦科夫计数。目前还使用了一种存储荧光粉成像方法, 用于在32P-postlabeling 检测的图谱上对 DNA 加合物进行映射和量化。18快速成像机经常用于检测和量化 DNA 加合物。此方法比屏幕增强显影19技术提供的检测32P 的灵敏度高10倍。
DNA 加合物的数量被确定为相对加合标记 (RAL) 的值, 使用等式 (2) 计算如下:
cpm.在加合 deoxynucleotides RAL =-----------------------------------------------------------------------------------------(2) 32p-ATP (cpm/pmol) x pmol deoxynucleotides 的特定活动
RALs 的值是 adducted deoxynucleotides 的计数率与总计 [adducted 和正常 (未修改) deoxynucleotides] deoxynucleotides20、21的计数率的比率。但是, 此计算基于加合物和正常deoxynucleotides22 的相同标记效率。32postlabeling 技术的经典 ("标准") 过程适用于各种 dna 加合物 (笨重和/或非笨重的加合物), 然而, 它的灵敏度并不令人满意, 以检测在 DNA 中发现的低量加合物。使用此过程, 107中未修改的 deoxynucleotides dna (0.3 fmol 加合/µg dna) 中的加合量是可检测的。
对这种经典的32postlabeling 程序进行了多种修改, 提高了该技术的灵敏度。通过对 [γ-32P] ATP (强化过程) 的限制水平, 对32p 标记测定加合物的灵敏度达到了 10-100 倍以上。23,24进一步的过程, 增加了32p-postlabeling 方法的灵敏度, 利用含有加合物的消化 DNA 的孵化与核酸酶 P1 (从青霉马勃) 21(图 1)。这种酶倾向于 dephosphorylate 未修改的 deoxyribonucleoside 3´ monophosphates, 而 deoxynucleotides 与绑定化学品 (adducted 核苷酸) 本质上不是这种酶的基质。因此, 去磷酸化 deoxyribonucleoside 3´ monophosphates(即、deoxyribonucleosides) 不是由[32 p] 磷酸盐从γ-32 p] ATP 磷酸化而成的。然而, 某些化学物质被束缚的核苷酸 (adducted deoxynucleotides), 如 C8 脱氧的芳胺类加合物, 可以用这种酶 bedephosphorylated。与此相反, 大多数其他加合物(例如, 加合物在 N 2 脱氧中替换) 不是核酸酶 P1 去磷酸化的.对32postlabeling 的这种修改使此方法具有更高的灵敏度, 从而提高了三多个数量级的敏感性。此外, 此版本的32p-postlabeling 提供了一种方法, 即可以利用较高数量的 DNA (5-10 µg) 和过量的无载波 [γ- 32P] ATP。
加合物25 描述的另一种方法是利用笨重的核苷酸加合物的物理化学性质, 可以在相转移剂四丁基的存在下提取到 n 丁醇中。氯化物 (TBA) (图 1) 在 [32P] 磷酸盐标记之前, 而未修改的 deoxynucleotides 是由这种有机溶剂提取出来的。然而, 较少疏水性加合物, 例如 deoxynucleotides 改性与非芳香的笨重的基团或小烷基残渣, 没有有效地提取与n-丁醇。因此, 在对32postlabeling 方法进行此修改分析时, 它们基本上是无法检测到的。
前面提到的32postlabeling 的版本都增加了 DNA 加合物的灵敏度和量化能力 (多达三级), 能够检测到每109、10正常核苷酸 (0.3-3 amol/µg DNA)。这两种方法被推荐用于测试化学品的效率, 以共价键绑定到 DNA, 因此, 他们在这项工作中详细描述。

<table>
	<tbody>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>252859</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Roth</td>
			<td>0032.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol/Chloroform/Isoamylalcohol</td>
			<td>Roth</td>
			<td>A156.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Penta</td>
			<td>70390-11000</td>
			<td></td>
		</tr>
		<tr>
			<td>Calf thymus DNA</td>
			<td>Sigma-Aldrich</td>
			<td>D4522</td>
			<td></td>
		</tr>
		<tr>
			<td>NADH</td>
			<td>Sigma-Aldrich</td>
			<td>N7004</td>
			<td></td>
		</tr>
		<tr>
			<td>NADP+</td>
			<td>Sigma-Aldrich</td>
			<td>N5755</td>
			<td></td>
		</tr>
		<tr>
			<td>NADPH</td>
			<td>Sigma-Aldrich</td>
			<td>N7505</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose 6-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>7647001</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose 6-phosphate dehydrogenase</td>
			<td>Sigma-Aldrich</td>
			<td>G6378</td>
			<td></td>
		</tr>
		<tr>
			<td>Supersomes</td>
			<td>Corning Gentest</td>
			<td>456211, 456203, 456220, 456204, 456210, 456222, 456219, 456212, 456206, 456207, 456202</td>
			<td></td>
		</tr>
		<tr>
			<td>Human liver microsomes</td>
			<td>Corning Gentest</td>
			<td>452172</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>77662</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Hydroxypyrimidine</td>
			<td>Sigma-Aldrich</td>
			<td>H56800</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>437549</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>179272</td>
			<td></td>
		</tr>
		<tr>
			<td>Micrococcal nuclease from Staphylococcus aureus</td>
			<td>Sigma-Aldrich</td>
			<td>N3755</td>
			<td></td>
		</tr>
		<tr>
			<td>Spleen phosphodiesterase from calf spleen, Type II</td>
			<td>Calbiochem</td>
			<td>524711</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease P1 from Penicillium citrinum</td>
			<td>Sigma-Aldrich</td>
			<td>N8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Bicine</td>
			<td>Sigma-Aldrich</td>
			<td>163791</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiotreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma-Aldrich</td>
			<td>S2626</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrabutylammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>86870</td>
			<td></td>
		</tr>
		<tr>
			<td>n-Butanol</td>
			<td>Sigma-Aldrich</td>
			<td>437603</td>
			<td></td>
		</tr>
		<tr>
			<td>T4-polynucleotide kinase</td>
			<td>USB Corp</td>
			<td>70031Y</td>
			<td></td>
		</tr>
		<tr>
			<td>[&#947;-32P]ATP</td>
			<td>Hartman Analytic GmbH</td>
			<td>FP-201</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI-impregnated cellulose TLC plates</td>
			<td>Macherey-Nagel</td>
			<td>801053</td>
			<td></td>
		</tr>
		<tr>
			<td>Packard Instant Imager A202400</td>
			<td>Packard</td>
			<td>G120337</td>
			<td></td>
		</tr>
		<tr>
			<td>Ellipticine</td>
			<td>Sigma-Aldrich</td>
			<td>285730</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Nitrobenzanthrone</td>
			<td colspan="2">prepared (synthesized) as shown in ref. 40</td>
			<td></td>
		</tr>
	</tbody>
</table>

formation of covalent dna adducts by enzymatically activated carcinogens and drugs in vitro and their determination by 32p-postlabeling

由化学物质或具有致癌效力的药物形成的共价 DNA 加合物被认为是致癌过程起始阶段最重要的因素之一。这种共价键被认为是肿瘤发生的原因, 现在被评估为化学致癌的中心教条。在这里, 使用细胞色素 P450 和其他生物转化酶催化反应的方法来研究化学物质或药物对形成这些 DNA 加合物的代谢物的活性的效力。介绍了用细胞色素 P450 或其他生物转化酶 (微粒体和胞浆样品) 分离具有生物转化酶的细胞组分的方法, 即, 过氧化物, 细胞色素 P450 氧化还原酶 (P) H: 醌氧化还原酶, 或黄嘌呤氧化酶)。此外, 描述的方法可以用于代谢活化的分析化学物质的这些酶, 以及那些分离的 DNA。此外, 能够检测和量化化学/药物衍生 DNA 加合物的适当方法,即,对32 P postlabeling 技术的不同修改和使用放射性标记分析的化学品, 是详细显示。

使用这里描述的协议, 利用酶催化活化 (即, P450, 过氧化物酶, 还原酶), 以调查化学物质 (致癌物/药物) 的效力, 将代谢成中间体, 导致其共价绑定到 dna (生成 dna 加合物), 我们能够解决 (i) 抗癌剂 ellipticine 药理作用的新机制 (查看,29, 30, 31), (二) 两种病因学肾病与上尿路癌相关的植物生物碱马兜铃酸 ...

Watch this Scientific Journal Video about Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling at JoVE.com

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

酶活化致癌物和药物对共价 DNA 加合物的生成研究体外及其对32 P-postlabeling 的测定

评价环境化学物质和药物的效力, 将其酶 bioactivated 到产生共价 DNA 加合物的中间体, 是癌症及其治疗发展的一个重要领域。方法描述了复合活化形成 DNA 加合物, 以及检测和定量的技术。

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

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Research

JoVE Journal

Biochemistry

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

13.8K 次观看.  Charles University. 评价环境化学物质和药物的效力, 将其酶 bioactivated 到产生共价 DNA 加合物的中间体, 是癌症及其治疗发展的一个重要领域。方法描述了复合活化形成 DNA 加合物, 以及检测和定量的技术。

视频: Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

In this manuscript, periodate oxidation and glutaraldehyde cross-linking methods for covalent immobilization of antibodies on paper discs are presented. The binding activity of immobilized antibodies was evaluated. Based on these results, a glutaraldehyde cross-linking method was used to develop a novel paper-based immunoassay for immunoglobulin G (IgG) detection.

共价结合的抗体，以纤维素纸光盘及其裸眼比色法免疫分析应用

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) ...

科研

生物化学

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

通过智能手机实时跟踪DNA片段分离

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

毛细管电聚焦法对蛋白质及其亚型的高灵敏度和定量检测

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA序列识别利用高通量原酶分析的DNAPrimase

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

通过质量光度法快速测定抗体抗原亲和力

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

酶的结晶和结构确定:多功能微流体芯片中串行晶体学的基板复合物

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...