以纯猪心肌高铁肌红蛋白(pMetMb)为对象,建立荧光光谱条件并经同步荧光指认其主要功能域. 研究表明,以600 nm/min扫描速度和中等响应时间为基本条件,pMetMb的适宜荧光光谱条件分别是20 mmol/L pMetMb、10 nm狭缝宽度和270 nm或430 nm为激发光波长. 经同步荧光光谱解析,酪氨酸(Tyr-)、色氨酸(Trp-)和铁卟啉环(heme-Fe3+)荧光特征峰分别在312、355和630 nm处. 270 nm是Trp-和Tyr-适宜激发光波长,而 430 nm 是heme-Fe3+适宜激发光波长. 与马肌红蛋白(hMb)比较,其Trp-和heme-Fe3+荧光光谱特征峰出现红移. 430 nm可激发630 nm处heme-Fe3+,发生电子迁移和能量跃迁. 随着Trp-和heme-Fe3+特征峰的红移,推测pMetMb中heme-Fe由五配位高自旋(Fe2+)转变为六配位低自旋(Fe3+).Purified pig cardiac metmyoglobin(pMetMb)was used to study its functional domains under the suitable fluorescence conditions. When 600 nm/min scanning speed and the medium response time were set up,the optimal fluorescence conditions for pMetMb spontaneous(emission)fluorescence were 20 mmol/L pMetMb,10 nm slit width with 270 nm or 430 nm excitation wavelength,respectively. By the resolve of the synchronous fluorescence spectra,the domains of tyrosine(Tyr),tryptophan(Trp)residue and heme-Fe3+ group were located at 312 nm,355 nm and 630 nm,respectively. Those were their special emission peaks of fluorescence spectra. 270 nm was the optimal excitation wavelength for Tyr and Trp residues,while 430 nm was optimal for heme-Fe3+ group. At those locations their powerful spontaneous fluorescence spectra were found. Compared with horse myoglobin(hMb-heme-Fe2+),the Trp residue and heme-Fe3+fluorescence spectra of pMetMb appeared as redshift from 595 nm to 630 nm. The results also showed that if excitation wavelength was far away from another molecule(as 270 nm for heme-Fe3+ group),spontaneous fluorescence was quenched without inducing clearly electron transfer and energy transition. However in a short-distance,a 430 nm excitation wavelength could induce the fluorescence emission of heme-Fe3+ group at 630 nm mostly. In a summary,it was suggested that high-spin pentacoordination of Mb-heme-Fe2+ was changed into low-spin hexacoordination of MetMb-heme-Fe3+
