Name: luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)
Uploaded: 2018-08-31T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

东南亚承认杜克捐赠特别倡议基金, 富国法戈基金, PhRMA 基金会的支持, 以及北卡罗来纳州大学夏洛特的启动资金。

1. 合成 BSA-Au (III.) 络合物
<ol><li>用1毫升高效液相色谱 (HPLC) 级水在5毫升反应瓶中溶解25毫克 BSA。 注意: 该解决方案应该看起来很清楚。</li>
	<li>将金 (III) 氯化三水 (四氯金酸) 溶解于 HPLC 级水中5毫米的浓度。 注意: 该解决方案应显示为黄色。在这种浓度下制备的四氯金酸溶液将导致 BSA 的金比为1:13。<ol>
<li>或者, 在 HPLC 级水中制备溶液, 其浓度介于0.38 毫米 (bsa: au = 1:1) 到20毫米 (bsa: au = 1:50) 之间的四氯金酸。 注: BSA 与金的不同比值将导致激发发射图的红色荧光模式截然不同。</li>
	</ol>
</li>
	<li>将 BSA 的反应瓶放在37摄氏度的水浴中, 用磁力搅拌器大力搅拌 750 rpm。</li>
	<li>搅拌开始后立即加入1毫升的四氯金酸溶液。解决方案的颜色应从透明转换为黄色。</li>
	<li>搅拌混合物2分钟在37°c 和 750 rpm 使用磁力搅拌器。</li>
	<li>进入反应瓶, 添加 100 ul 1 米氢氧化钠的解决方案, 使 pH 值为12。 注: 在添加氢氧化钠后, 溶液应略微变暗至黄褐色, 然后再转回黄色。</li>
	<li>继续搅拌在 750 rpm 2 小时和37摄氏度。解决方案应该慢慢地从黄色变成深黄色/褐色的颜色。这种颜色变化表明红荧光 BSA (III.) 复合物的形成。</li>
	<li>允许样品在室温下坐2天, 溶液将继续变暗到琥珀色的褐色, 荧光强度会增加。<ol>
<li>或者, 让样品继续搅拌37摄氏度12多 h, 因为溶液的颜色演变为琥珀色褐色。</li>
	</ol>
</li>
</ol>2. 合成 BSA-铜 (II)-Au (III)
<ol><li>在1毫升的 HPLC 级水中溶解25毫克 BSA。解决方案应该看起来很清楚。</li>
	<li>将氯化铜 (II) 二水合物溶解于 HPLC 级水中, 浓度为5毫米。该解决方案应显示为浅蓝色。</li>
	<li>添加1毫升的水 BSA 溶液到5毫升反应瓶和地方在水浴在37°c。搅拌 750 rpm 的混合物。</li>
	<li>立即在反应瓶中加入0.5 毫升氯化铜 (II) 溶液, 混合2分钟。溶液将保持浅蓝色。</li>
	<li>添加 75 ul 1 米氢氧化钠, 使 pH 值为 12, 并允许混合2小时。解决方案将变为紫色。</li>
	<li>将四氯金酸溶于 HPLC 级水中, 浓度为5毫米。</li>
	<li>在反应瓶中加入0.5 毫升的水四氯金酸, 用1米氢氧化钠将 pH 值调整回12。</li>
	<li>搅拌反应混合物2小时。 注意: 该解决方案应演变为褐色颜色。</li>
</ol>3. 合成 BSA 镍 (II)-Au (III)
<ol><li>在1毫升的 HPLC 级水中溶解25毫克 BSA。解决方案应该看起来很清楚。</li>
	<li>将氯化镍 (II) 六水合物溶于 HPLC 级水中, 浓度为5毫米。该解决方案应显示为浅绿色。</li>
	<li>添加1毫升的水 BSA 溶液到5毫升反应瓶和地方在水浴在 37 oc. 搅拌混合物在 750 rpm。</li>
	<li>立即在反应瓶中加入0.5 毫升氯化镍 (II) 六水合物溶液, 混合2分钟。 注: 溶液将保持浅绿色。</li>
	<li>添加 75 ul 1 米氢氧化钠, 使 pH 值为 12, 并允许混合2小时。 注意: 溶液将变成深黄色。</li>
	<li>将四氯金酸溶于 HPLC 级水中, 浓度为5毫米。</li>
	<li>在反应瓶中加入0.5 毫升的水四氯金酸, 并将 pH 值调整回12。</li>
	<li>搅拌反应混合物2小时。 注意: 该解决方案应演变为褐色颜色。</li>
</ol>4. [Cys34]-Au (III.) 的合成
<ol><li>溶解2毫克的 n-ethylmaleimide (NEM) 在1毫升磷酸盐缓冲盐水 (PBS, pH 值 7.4)。</li>
	<li>将2毫克的 BSA 溶于1毫升的 PBS-NEM 溶液中。</li>
	<li>将溶液转移到5毫升反应瓶中, 在20摄氏度处搅拌500转1小时。</li>
	<li>透析的解决方案使用 12 kDa 透析油管在500毫升的 PBS, 搅拌 50 rpm 与磁力搅拌器过夜, 以消除反应 NEM。</li>
	<li>在 PBS 中溶解四氯金酸, 浓度为0.4 毫米。 注意: 解决方案将是一个微弱的黄色。</li>
	<li>将反应瓶转移到37摄氏度的水浴中。搅拌750转。</li>
	<li>立即加入1毫升的四氯金酸溶液的反应瓶, 并允许混合2分钟。</li>
	<li>添加 75 ul 1 米氢氧化钠的反应瓶, 使 pH 值为 12, 并允许混合2小时。</li>
</ol>5. 合成 [全硫醇上限-BSA]-Au (III.)
<ol><li>在 HPLC 级水中制备2米尿素和50毫米碳酸氢铵 (NH4HCO3, pH 8.0) 溶液。</li>
	<li>在上述溶液的1毫升中溶解3.3 毫克 BSA, 并转移到5毫升反应瓶。</li>
	<li>用0.25 米 (2-羧乙基) 膦 (TCEP) 的溶液, 将62.5 毫克的 TCEP 溶解在1毫升的 HPLC 水中。</li>
	<li>将 TCEP 的库存溶液添加到反应瓶中, 直到 TCEP 的最终浓度为8毫米。</li>
	<li>在50摄氏度的水浴中孵化溶液1小时。使用磁力搅拌器搅拌 500 rpm。</li>
	<li>让溶液完全冷却到室温。</li>
	<li>用1毫升的 HPLC 级水溶出12.5 毫克 NEM, 制备100毫米 NEM 的库存溶液。</li>
	<li>将 NEM 的库存溶液添加到反应瓶中, 直到 NEM 的最终浓度为16毫米。</li>
	<li>允许溶液结合2小时, 在20摄氏度。搅拌500转。</li>
	<li>透析的解决方案使用 12 kDa 透析油管, 搅拌解决方案在 50 rpm 与磁搅拌器隔夜500毫升50毫米 NH4HCO3 , 以消除过剩的 TCEP, NEM 和尿素。</li>
	<li>将反应瓶移动到37摄氏度的水浴中, 并在750转每分钟搅拌。</li>
	<li>将四氯金酸溶于 HPLC 级水中, 浓度为0.66 毫米。</li>
	<li>立即加入1毫升的四氯金酸溶液的反应瓶, 并允许混合2分钟。</li>
	<li>加入1米氢氧化钠, 直到溶液的 pH 值为 12, 并允许溶液继续混合2小时。</li>
</ol>

<ol>
	<li>Xie, J., Zheng, Y., Ying, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-Directed+Synthesis+of+Highly+Fluorescent+Gold+Nanoparticles.">Protein-Directed Synthesis of Highly Fluorescent Gold Nanoparticles.</a> Journal of the American Chemical Society. 131, 888-889 (2009).</li><li>Saha, K., Agasti, S. S., Kim, C., Li, X., Rotello, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles+in+Chemical+and+Biological+Sensing.">Gold Nanoparticles in Chemical and Biological Sensing.</a> Chemical Reviews. 112, 2739-2779 (2012).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Gold+Nanostructures+for+Sensor+Applications:+A+Review.">New Gold Nanostructures for Sensor Applications: A Review.</a> Materials. 7, 5169-5201 (2014).</li><li>Chen, L. -. Y., Wang, C. -. W., Yuan, Z., Chang, H. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Gold+Nanoclusters:+Recent+Advances+in+Sensing+and+Imaging.">Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging.</a> Analytical Chemistry. 87 (1), 216-229 (2015).</li><li>Cai, W., Gao, T., Hong, H., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Gold+Nanoparticles+in+Cancer+Nanotechnology.">Applications of Gold Nanoparticles in Cancer Nanotechnology.</a> Nanotechnology, Science and Applications. 1, 17-32 (2008).</li><li>Nune, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+for+Biomedical+Imaging.">Nanoparticles for Biomedical Imaging.</a> Expert Opinion on Drug Delivery. 6, 1175-1194 (2009).</li><li>Dorsey, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles+in+Radiation+Research:+Potential+Applications+for+Imaging+and+Radiosensitization.">Gold Nanoparticles in Radiation Research: Potential Applications for Imaging and Radiosensitization.</a> Translational Cancer Research. 2, 280-291 (2013).</li><li>Daniel, M. -. C., Astruc, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles:+Assembly,+Supramolecular+Chemistry,+Quantum-Size-Related+Properties,+and+Applications+toward+Biology,+Catalysis,+and+Nanotechnology.">Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology.</a> Chemical Reviews. 104 (1), 293-346 (2004).</li><li>Ferrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Nanotechnology:+Opportunities+and+Challenges.">Cancer Nanotechnology: Opportunities and Challenges.</a> Nature Reviews Cancer. 5, 161-171 (2005).</li><li>Huang, X., Jain, P. K., El-Sayed, I. H., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles:+Interesting+Optical+Properties+and+Recent+Applications+in+Cancer+Diagnostics+and+Therapy.">Gold Nanoparticles: Interesting Optical Properties and Recent Applications in Cancer Diagnostics and Therapy.</a> Nanomedicine. 2, 681 (2007).</li><li>Arvizo, R., Bhattacharya, R., Mukherjee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+Nanoparticles:+Opportunities+and+Challenges+in+Nanomedicine.">Gold Nanoparticles: Opportunities and Challenges in Nanomedicine.</a> Expert Opinion on Drug Delivery. 7, 753-763 (2010).</li><li>Doane, T. L., Burda, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Unique+Role+of+Nanoparticles+in+Nanomedicine:+Imaging,+Drug+Delivery+and+Therapy.">The Unique Role of Nanoparticles in Nanomedicine: Imaging, Drug Delivery and Therapy.</a> Chemical Society Reviews. 41, 2885 (2012).</li><li>Egusa, S., Ebrahem, Q., Mahfouz, R. Z., Saunthararajah, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+Exchange+on+Gold+Nanoparticles+for+Drug+Delivery+and+Enhanced+Therapeutic+Index+Evaluated+in+Acute+Myeloid+Leukemia+Models.">Ligand Exchange on Gold Nanoparticles for Drug Delivery and Enhanced Therapeutic Index Evaluated in Acute Myeloid Leukemia Models.</a> Experimental Biology and Medicine. 239, 853 (2014).</li><li>Qu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Gold+Nanoclusters:+Synthesis+and+Recent+Biological+Application.">Fluorescent Gold Nanoclusters: Synthesis and Recent Biological Application.</a> Journal of Nanomaterials. (784097), (2015).</li><li>Chakraborty, I., Pradeep, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomically+Precise+Clusters+of+Noble+Metals:+Emerging+Link+between+Atoms+and+Nanoparticles.">Atomically Precise Clusters of Noble Metals: Emerging Link between Atoms and Nanoparticles.</a> Chemical Reviews. 117, 8208-8271 (2017).</li><li>Raut, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+energy+transfer+from+tryptophan+to+BSA/HSA+protected+gold+nanoclusters.">Evidence of energy transfer from tryptophan to BSA/HSA protected gold nanoclusters.</a> Methods and Applications in Fluorescence. 2, (2014).</li><li>Le Gu&#233;vel, X., Daum, N., Schneider, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Characterization+of+Human+Transferrin-Stabilized+Gold+Nanoclusters.">Synthesis and Characterization of Human Transferrin-Stabilized Gold Nanoclusters.</a> Nanotechnology. 22 (27), (2011).</li><li>Kawasaki, H., Yoshimura, K., Hamaguchi, K., Arakawa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin-Stabilized+Fluorescent+Gold+Nanocluster+for+Sensitive+and+Selective+Hg2++Detection.">Trypsin-Stabilized Fluorescent Gold Nanocluster for Sensitive and Selective Hg2+ Detection.</a> Analytical Sciences. 27 (6), 591 (2011).</li><li>Lu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysozyme-Stabilized+Gold+Nanoclusters+as+a+Novel+Fluorescence+Probe+for+Cyanide+Recognition.">Lysozyme-Stabilized Gold Nanoclusters as a Novel Fluorescence Probe for Cyanide Recognition.</a> Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 121, 77-80 (2014).</li><li>Dixon, J. M., Egusa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Change-Induced+Fluorescence+of+Bovine+Serum+Albumin-Gold+Complexes.">Conformational Change-Induced Fluorescence of Bovine Serum Albumin-Gold Complexes.</a> Journal of the American Chemical Society. 140, 2265-2271 (2018).</li><li>Peters, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> All About Albumin. , (1996).</li><li>Masuoka, J., Saltman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc(II)+and+Copper(II)+Binding+to+Serum+Albumin.+A+Comparative+Study+of+Dog,+Bovine,+and+Human+Albumin.">Zinc(II) and Copper(II) Binding to Serum Albumin. A Comparative Study of Dog, Bovine, and Human Albumin.</a> Journal of Biological Chemistry. 269, 25557-25561 (1994).</li><li>Takeda, K., Wada, A., Yamamoto, K., Moriyama, Y., Aoki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Change+of+Bovine+Serum+Albumin+by+Heat+Treatment.">Conformational Change of Bovine Serum Albumin by Heat Treatment.</a> Journal of Protein Chemistry. 8 (5), 653-659 (1989).</li><li>Klotz, I. M., Curme, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Thermodynamics+of+Metallo-protein+Combinations.+Copper+with+Bovine+Serum+Albumin.">The Thermodynamics of Metallo-protein Combinations. Copper with Bovine Serum Albumin.</a> Journal of the American Chemical Society. 70, 939-943 (1948).</li><li>Fiess, H. A., Klotz, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Thermodynamics+of+Metallo-Protein+Combinations.+Comparison+of+Copper+Complexes+with+Natural+Proteins.">The Thermodynamics of Metallo-Protein Combinations. Comparison of Copper Complexes with Natural Proteins.</a> J. Am. Chem. Soc. 74, 887-891 (1952).</li><li>Rao, M. S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Study+of+the+Interaction+of+Nickel(II)+with+Bovine+Serum+Albumin.">A Study of the Interaction of Nickel(II) with Bovine Serum Albumin.</a> Journal of the American Chemical Society. 84, 1788-1790 (1962).</li><li>Peters, T., Blumenstock, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-Binding+Properties+of+Bovine+Serum+Albumin+and+Its+Amino-terminal+Peptide+Fragment.">Copper-Binding Properties of Bovine Serum Albumin and Its Amino-terminal Peptide Fragment.</a> Journal of Biological Chemistry. 242, 1574-1578 (1967).</li><li>Xue, Y., Li, X., Li, H., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Thiol-Gold+Interactions+towards+the+Efficient+Strength+Control.">Quantifying Thiol-Gold Interactions towards the Efficient Strength Control.</a> Nature Communications. 5, 4348 (2014).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Protein+Characteristics+in+the+Formation+and+Fluorescence+of+Au+Nanoclusters.">The Role of Protein Characteristics in the Formation and Fluorescence of Au Nanoclusters.</a> Nanoscale. 6 (3), 1515-1524 (2014).</li></ol>

作者没有什么可透露的。

在 pH 值为12的白蛋白 (III.) 化合物中制备的红色荧光在λem的发射波长 = 640 毫微米当激发与紫外 (紫外) 光λ前= 365 毫微米 (图 1A)。红色荧光的出现是一个缓慢的过程, 将需要几天在室温下增加到最大强度。在37摄氏度的反应将产生最佳的结果, 虽然更高的温度可以用来更快地产生红色荧光。蛋白质的不可逆转的退化可能发生在温度在45°c23</su...

一种白蛋白-金化合物, 呈紫外 (紫外) 兴奋的红色荧光, 具有显著的斯托克斯移位, 最初是由谢等 al合成的。1. 独特和稳定的红色荧光可以在诸如传感2、3、4、成像5、6、7或纳米医学8等领域找到各种应用. ,9,10,11,12,13。近年来,14、15、16等纳米科学领域的研究人员对此化合物进行了广泛的研究。BSA-au 化合物被解释为 au25簇。所提出方法的目的是对该化合物进行详细的研究, 了解红荧光的起源。通过采用所提出的方法, 可以说明多个 au 结合点的存在, 以及荧光的起源, 替代非盟25簇的单点核。同样的方法也可以用来研究其他蛋白质17,18,19与 Au (III) 的复合物如何改变它们的内在荧光特性。
红荧光 bsa-au 化合物的合成需要对 bsa 的摩尔比 (bsa: au) 进行狭义的控制, 以最大限度地提高荧光强度和激发发射图 (EEM)20中峰值的位置。可以证明, 有多个绑定站点存在于 Au (III) 上, 包括天门冬片段 (或 Asp 片段, 在 BSA N 终点的前四个氨基酸残留物)21,22。34次氨基酸的 BSA (Cys-34) 也显示, 以协调 Au (iii) 和参与的机制, 红色荧光 ([Cys34 上限-BSA]-Au (iii))20。在裂解所有的胱氨酸-胱氨酸二硫化物键和上限所有硫醇, 红色荧光不产生 ([全硫醇上限-BSA]-Au (III))。这表明了胱氨酸-胱氨酸二硫化物键作为非盟 (III.) 结合部位产生红色荧光的必要性。
蛋白质化学技术还没有被广泛用于研究纳米科学群落中的 BSA (III.) 配合物。然而, 使用这些技术来了解这些复合体的某些方面, 以及对 BSA 中的非盟 (III) 结合点有详细的了解是很有价值的。本文旨在展示其中的一些技术。

<table><tbody><tr><td>Bovine Serum Albumin (BSA), 96%</td><td>Sigma-Aldrich</td><td>A5611</td><td></td></tr><tr><td>gold (III) chloride trihydrate, 99.9%</td><td>Sigma-Aldrich</td><td>520918</td><td></td></tr><tr><td>Copper (II) chloride dihydrate, 99.999%</td><td>Sigma-Aldrich</td><td>459097</td><td></td></tr><tr><td>Nickel (II) chloride hexahydrate, 99.9%</td><td>Sigma-Aldrich</td><td>654507</td><td></td></tr><tr><td>N-Ethylmaleimide (NEM), &gt;99.0%</td><td>Sigma-Aldrich</td><td>4259</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &gt;98.0%</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Sodium hydroxide, &gt;98.0%</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Urea, 99.5%</td><td>Chem-Implex Int&#039;l</td><td>30142</td><td></td></tr><tr><td>Phospate buffered saline (PBS)</td><td>Corning</td><td>MT21040CV</td><td></td></tr><tr><td>Ammonium bicarbonate, 99.5%</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr></tbody></table>

luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)

所提出的协议的目的是研究金 (iii) 对 bsa 的结合过程, 产生构象变化诱导红荧光 (λem = 640 nm) 的 bsa-Au (iii) 配合物。该方法调整 ph 值, 表明红荧光的出现与 BSA 构象的 ph 诱导的平衡过渡相关。红色荧光 BSA-Au (III.) 配合物只能形成与 pH 值在9.7 或以上的调整, 这对应于 "A 形" 构象的 BSA。介绍了将 BSA 调整为金摩尔比的协议, 并对金 (III.) 结合过程的时间过程进行了监测。每 BSA (III) 的最小数目, 以产生红色荧光, 小于七。我们描述了该协议的步骤, 以说明在 BSA 中存在多个 Au (III) 绑定站点。首先, 通过添加铜 (二) 或镍 (镍 (ii)) 阳离子其次为 au (iii), 这种方法揭示了一个结合点的非盟 (iii), 这不是红色荧光。其次, 通过硫醇封堵剂对 BSA 进行改性, 揭示了另一种 nonfluorophore 形成的金 (III.) 结合部位。第三, 通过对二硫化物键的劈裂和封盖改变 BSA 构象, 说明可能的 Au (III.) 结合点。该协议描述, 以控制 BSA 构象和 Au (III) 结合, 可以广泛应用于研究其他蛋白质和金属阳离子的相互作用。

从 bsa-Au (III.) 络合物的荧光, 观察到, 白蛋白 (λem = 400 nm) 与红色荧光 (λem = 640 nm) 的内在蓝色荧光的转换发生在 pH 9.7 通过平衡转换 (图 1)。EEM (III.) 在不同的 bsa 到金摩尔比率如图 2所示, 这一数据表明, 改变摩尔比产生相同的发射波长在不同的激发波长。Cu (ii)、镍 (ii) 和 Au (III) 在 BSA (<strong class="xfi...

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Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

本文介绍了研究金阳离子 (III.) 对牛血清白蛋白的各种构象和构象依赖性独特的 bsa-Au 荧光的表征的协议。

结合金 (III.) 对牛血清白蛋白不同构象的 Luminophore 形成

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

luminophore-formation-various-conformations-bovine-serum-albumin

Research

JoVE Journal

Chemistry

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

6.8K 次观看.  University of North Carolina. 本文介绍了研究金阳离子 (III.) 对牛血清白蛋白的各种构象和构象依赖性独特的 bsa-Au 荧光的表征的协议。

视频: Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

铱（III）荧光探针检测疟疾蛋白质生物标志物组氨酸丰富的蛋白质-Ⅱ

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

科研

化学

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

生物化学

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

光敏定位显微镜与双分子荧光互补（附设-PALM）

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

生物工程

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a ...

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: Au(III)-binding Luminescent Domain in Serum Albumins

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable red luminescence (&#955;em = 640 nm), with unusual Stokes shifts compared to the innate UV/blue fluorescence arising from the aromatic residues. Red-luminescent complexes are formed in highly alkaline conditions above pH 10 and require a conformation change within the protein to occur. In addition, preservation of Cys-Cys disulfide bonds in BSA is necessary to obtain this red luminescence. In order to understand the mechanism of this luminescence, elucidation of the luminophore-forming Au(III) binding site is essential. A facile way to assess the luminophore-forming site would be to (1) predictably fragment the protein by enzymatic digestion, (2) react the obtained fragments with Au(III), then (3) perform gel electrophoresis to observe the well-separated fragment bands and analyze the in-gel red luminescence. However, due to the alkaline conditions and the reaction with metal cations, new limited proteolysis techniques and gel electrophoresis conditions must be applied. Particularly, the presence of metal cations in gel electrophoresis can make the band separations technically difficult. We describe this new protocol in steps to identify the red-luminophore-forming metal binding domain in BSA. This protocol can thus be applied for analyzing protein fragments that must remain in a non-denatured or a partially denatured state, in the presence of metal cations. Because the majority of proteins need metal cations to function, analyses of metal-bound proteins are often desired, which have relied on x-ray crystallography in the literature. This method, on the other hand, could be used in supplement to study the interactions of proteins with metal cations without requiring the protein crystallization and at a desired pH condition.

Limited Proteolysis and Gel Electrophoresis in the Presence of Metal Cations: AuIII-binding Luminescent Domain in Serum Albumins

We present a protocol for studying the binding domain of Au(III) in bovine serum albumin (BSA).

The purpose of the presented protocols is to determine the domain of Au(III) binding in BSA. The BSA-Au(III) compound exhibits ultraviolet (UV)-excitable ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

生物应用近红外发光金纳米簇的合成

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Characterization of pH-Dependent Reversible Self-Assembly of Amyloid Beta 1-40-Coated Gold Colloids

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy and transmission electron microscopy (TEM). The observed pH-dependent shift of the SPR band of A&#946;1-40-coated 20 nm gold particles was correlated with alternation of aggregation and disaggregation observed in the TEM images. A pH of ~4&#160;induced an unfolded conformation, and a pH of ~10&#160;induced a folded conformation of A&#946;1-40 on the gold surface. This reversible aggregation process was observed by Raman imaging as the pH was gradually changed from pH 4 to pH 10. We observed a pH-dependent morphology change in A&#946;1-40 on the gold surface, where clear aggregates were observed at pH 4. However, we observed very subtle differences in the surface-enhanced Raman spectroscopy (SERS) spectrum between pH 4 and pH 10 conditions, with the most striking difference being spectral density in the region of 250 cm-1 and 1750 cm-1. Specifically, the mode analysis of the reversible aggregation indicated that the aggregates were formed by the unfolded conformation of A&#946;1-40 which involved the benzene ring section of Tyrosine and Phenylalanine. Conversely, the disassembly of the aggregates was associated with conformational changes in protein folding of A&#946;1-40 involving Histidine, Glutamine, Methionine, and Aspartic acid.

Here, we present a systematic method describing the quasi-reversible self-assembly of the amyloid beta 1-40 (A&#946;1-40)-coated 20 nm gold aggregates. The nano-size-dependent quasi-reversible networking between peptides was correlated with specific amino acids or sections of the A&#946;1-40 monomer.

β 淀粉样蛋白 1-40 包被的金胶体的 pH 依赖性可逆自组装的表征

The characterization of A&#946;1-40 coated over the nano-gold colloidal particle surfaces was conducted using surface plasmon resonance (SPR) spectroscopy ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

实时监控的配体 - 受体相互作用的荧光共振能量转移

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

生物学

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.