荧光停流探测细胞色素c的酸展开

荧光光谱可以用来监测蛋白质芳香氨基酸残基周围环境的变化。细胞色素c是一种血红素蛋白，在血红素基团附近有一个色氨酸残基。在蛋白质的天然状态下，从色氨酸到血红素铁的非辐射能量转移使荧光猝灭。当蛋白质在变性条件下展开时，色氨酸和血红素之间的距离增加，同时荧光强度增加。将停流系统与荧光光谱仪相结合，研究人员不仅可以获得蛋白质结构变化程度的信息，还可以获得展开动力学的信息。Application Note Acid Unfolding of Cytochrome cProbed by Fluorescence Stopped-Flow2/3Application Note Fluorescence spectroscopy can be used to monitor changes in the environment su rr ounding a protein’s aroma t i c am i no acid r esidues. Cytochrome c i s a h eme protein tha t has one t r yptophan r esidue in close prox i mity to the heme grou p .I n the protein's native state ， the f l uorescence is quenched by the non-radiative energy t ransfer f rom the t ryptophan to the heme i ron. When t he protein unfolds under dena t uration conditions， t he distance between t he tryptopha n and heme in creases， concomitantly i n creasi n g the f luorescence i n tensity. Coupling a stopped -f low system wit h a spect r ofluorometer a l lows researche r s to acq u i r e in formation not o n ly o n the degree of structural cha n ges to t h e protein， but o n the u n f olding k in etics as well . SFS-852 Stopped-Flow Accessory This appl i cation note demonst r ates how stopped-flow fluo r escence ca n probe the unfolding kinetics of cytochrome c denatured by s u lfur i c ac i d. FP-8500， F l uorescence， Stopped-flow，SFS-852 Stopped-flow system， Kinetics 28600 Ma r y 's C ou r t， Easton， M D 21601 US A Excitation Wavelength 280nm Emission Wavelength 340 nm Excitation Bandwidth 5nm Emission Bandwidth 5nm Response Time 2 sec Sensitivity Manual Measurement Interval 25 msec Measurement Range 0-5000 msec Accumulations 4 Flow Time 35 msec Mixing Ratio 1：1 Flow Volume S1， S2： 100 uL 10 mL of 0.5 mg/mL horse heart cytochrome c was placed i n syringe 1 while 10 mL of 0.1 N sul f uric acid was placed in syringe 2. The data was acquired 35 mil li seconds befo r e the flow t ime ended . Re sults Figure 1 illu s trates t he measured (r ed) and calculated (blue) k inet i c traces of cytoch r ome c r eacting with sulfuric acid. The inc r ease in f l uorescence i nte n sity indicates that the protein begins to unfold upon its inte r action with sulfuric acid almost immed i ate l y and i s completely unfolded around 2000 msec. Figure 1. M e as u red (r ed) an d ca l culated (bl u e) f l uo r escen c e e m ission spectrum of cytochro m e c de n atured with s u lf u ric ac i d . 28600 Mary's Cour t ， E a s to n ， M D 21601US A UAGE O The react i on r ate a n d time constants were calcu l ated using t h e [Reaction Rate Calcu l a t i on] program and t h e r esults a r e shown in Table 1. The calculated r ange was 35 to 5000 msec and a 2-step reaction mechanism was appl i ed for t h e calcu l a ti on. T h e ca l culated results are shown to f i t the data. Table 1. Ki n e t ic p a ramete r s. Rate Equation Y(t)=-570.486·e(t/336.523)+-217.93·e(/3039.55) Step 1 Time Constant 336.523 msec Step 1 Rate Constant 0.00297157msec1 Step 2 Time Constant 3039.55 msec Step 2 Rate Constant 0.000329 msec C onclusion This application n ote demonstrates that the FP-8500 can be used to obta in thermal denatu r ation data for protein st r ucture studies. 28600 Mary's Cour t ， E a s to n ， M D 21601US A