Development of Highly Sensitive Infrared Spectrometer with Femtosecond Time Resolution

飞秒时间分辨率高灵敏红外光谱仪的研制

基本信息

批准号：
11554028
负责人：
YOSHIHARA Keitaro
金额：
$ 6.85万
依托单位：
JAPAN ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The design and performance of a femtosecond time-resolved mid-infrared transient absorption spectrometer is illustrated. The spectrometer was developed from a set of commercially available Ti : sapphire femtosecond lasers utilizing difference frequency mixing technique. With this spectrometer, the transient absorption in the mid-infrared region from 800 to 2800 cm^<-1> was detected with a sensitivity of 1×10^<-4> for difference absorbance at a repetition rate of 1 kHz. With a sub-picosecond visible or ultraviolet pump, a time-resolution of 250fs has been obtained. A study on the binding and dissociation processes of carbon monoxide in myoglobin is presented as an example of its applications.CooA, which is a transcriptional regulator heme protein allosterially triggered by CO, is studied by femtosecond visible-pump mid-IR-probe spectroscopy. Transient bleaching upon excitation of the heme into Soret band is detected at around 1979cm^<-1>, which is the absorption region of the CO bound to the heme. The bleach signal shows a nonexponential decay with time constants of 56 ps and 290 ps, caused by the rebinding of the CO to the heme. About 97% of dissociated CO recombines geminately. The geminate recombination rate in CooA is significantly faster than that in myoglobin and hemoglobin. The angle of the bound CO with respect to the porphyrin plane is calculated to be about 78 degrees based on the anisotropy measurements. A shift of the bleach mid-IR spectrum of the bound CO is detected having characteristic time of 160 ps. It is suggested that the spectral shift is caused by a difference in the frequency of the bound CO in different protein conformations, particularly in active conformation and in intermediate one. Thus, the biologically relevant conformation change in CooA was traced. Possible assignment of the observed conformation change is discussed.

说明了飞秒时间分辨的中红外瞬态吸收光谱仪的设计和性能。光谱仪是由一套可使用差异频率混合技术的蓝宝石飞秒激光器开发的。使用该光谱仪，以1×10^<-4>的灵敏度为1 kHz的敏感性，以1×10^<-4>的灵敏度检测到中红外区域的瞬时吸收。使用亚皮秒可见或紫外泵，已经获得了250FS的时间分辨率。对肌红蛋白中碳一氧化碳的结合和解离过程的研究作为其应用的一个例子。在1979cm^<-1>左右检测到血红素兴奋后的瞬时漂白，这是CO与血红素结合的吸收区域。漂白信号显示由CO重新固定到血红素引起的时间常数为56 ps和290 ps的时间常数。大约97％的解离CO重组发出。 COOA中发射的重组率明显快于肌红蛋白和血红蛋白。基于各向异性测量值，计算出相对于卟啉平面的结合CO的角度约为78度。检测到具有160 ps的特征时间，检测到绑定CO的漂白中IR光谱的变化。据建议，光谱移位是由不同蛋白质构象中结合CO的频率的差异引起的，尤其是在活动构象和中间构型中。这是COOA中与生物学相关的构象变化的追踪。讨论了观察到的构象变化的可能分配。