DEVELOPMENT OF NIR FOR MONITORING ENZYME-SUBSTRATE REACTIONS

开发用于监测酶-底物反应的近红外光谱

• 批准号: 3843283
• 负责人: R L BERGER
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3843283
• 关键词: adenosine diphosphate; adenosine triphosphate; adenosinetriphosphatase; bioengineering /biomedical engineering; biomedical equipment development; conformation; enzyme activity; enzyme substrate complex; infrared spectrometry; ions; protein structure; solutions; temperature

Much progress has been made in the quantification of ATP, ADP, and P(1) in a biological reaction, namely Na(+)K(+)ATPase hydrolyzing ATP. Several features of the reactions must be accounted for in order to fully deconvolute the spectra. The water spectrum is the largest hurdle as it is a strong absorber in this region. In addition, the water spectrum is very dependent upon temperature and is affected by the presence of dissolved ions. By obtaining a spectrum of the enzyme within the solution, a effective background is achieved and observation of the hydrolysis of ATP can be accomplished. An error of approximately 10% was found, quite high as compared to the calibration curves of the separate components. Possible contributors to the overall error include dissolved ions, intermediates in the reaction, and protein conformational changes, where it is known that changes in the secondary structure of the protein can alter the vibrational spectrum and thus the near infrared spectrum.

在ATP、ADP和P(1)的量化方面取得了很大进展 一种生物反应，即Na(+)K(+)ATPase水解三磷酸腺苷。几个 必须考虑反应的特点，以便充分地 去卷曲光谱。事实上，水的光谱是最大的障碍 在这一地区具有很强的吸波性。此外，水的光谱非常 取决于温度，并受溶解物质存在的影响 离子。通过获得溶液中酶的光谱， 获得了有效的背景并观察到了ATP的水解 是可以实现的。误差约为10%，相当高 与各单独组分的校准曲线进行比较。可能的 总误差的贡献者包括溶解的离子、中间体 这个反应和蛋白质的构象变化，在那里是已知的 蛋白质二级结构的变化可以改变振动 光谱，因此近红外光谱。