热变性溶菌酶的荧光测量

荧光光谱可用于监测蛋白质的溶剂环境和芳香族氨基酸残基的变化。提高溶液温度可以在蛋白质展开时诱导蛋白质的结构变化，这可以反映在发射光谱中。然后可以获得热力学性质以提供关于蛋白质的热稳定性的信息。Application NoteFP-O001 Fluorescence Measurement of2/3Application NoteHeat-Denatured Lysozyme Fluorescence Measurement of Heat-Denatured Lysozyme Introdu c tio n Fluorescence spectroscopy can be used to monitor changes i n the solvent environmen t and a r omatic amino acid residues of a prote i n. Inc r easing the sol u tion temperature can i nduce struct u ral c h anges in a protein as i t u nfolds， which can be reflected in the emiss i on spect ru m. The thermodynam i c proper t ies ca n then be o b tained to provide informat i on regard in g the prote i n 's th ermal stabi l ity. T h is application notes i ll ustrates the temperature-dependence of lysozyme by mo n itoring the protein's ter t iary structure using a FP-8500 and the r mostatted cell holde r . FP-8500 Spectrof l u o rom e ter Keyword s FP-8500， F l uorescence， Temperature-dependence，ETC-272 Pe l tier thermostatted cell holder Exp e rimental Excitation Wavelength 280nm Wavelength Range 290-450nm Excitation Bandwidth 3nm Emission Bandwidth 5nm Response Time 0.5 sec Gain Medium Temperature Range 15-90°℃ Temperature Interval 5℃ A buffered aqueous so l ution of 0.1 mg/mL of lysozyme was st i r r ed with a magnetic stirrer to ensure even sample temperatu r es in the cell . A tempe r ature ram p i n g r ate of 1C/m in was controlled by the Peltier cel l h older and spectra were measured within 60 seco n ds after r eaching t h e in divid u al set temperature points. R esul t s Figure 1 i l lustrates the change in the emission in tensity at 340 nm as a function of tempe r atu r e. As a protein is denatu r ed and begins to u nfold， the f l u o r escence in tens i ty is expected to decrease. While l ysozyme is known to den a t u re around 70℃， Figure 1 s h ows a consisten t decrease in the fluorescence i n tensity as the tem p e r ature inc r eases. However ， the con t o u r plot o f t he emission spectr u m (Figure 2) i llustrates a slight s h ift in the spec t ra at 70℃. Int Figure 1. The t e m per a ture-de p en d en t f lorescence i n tensity o f lys o z yme at 340 nm . Figure 2. Counto u r plot of t he e mi s s ion spectra from 40 to 90℃. The emission spect r a of lysozyme at room temperature has an emission ma x imum at 340 nm whi l e at 90℃ the maximum is at 348 n m. Figu r e 3 de p icts a plot of the intensity ratios f or the two wavelengt h s as a f u n ct i on of tempe r ature. Figu r e 3clear l y shows a t ra n sition a t 70℃， cor r esponding to the dena tu ration tem p erat u re of lysozyme. Figure 3. Maximum fluorescence i nte n si ty ratio (340/348 n m) a s a fu n ctio n of temperature. Conclu s ion T h is application n ote demonst r ates t hat t he FP-8500 can b e used to obta in th erm a l de n atu r atio n data for protein s tr ucture st u dies. JASCO INC. 28600 Mary's Cour t ， E a s to n ， M D 21601US A