细胞色素C的重折叠使用停流装置

CD光谱提供了关于蛋白质的二级和三级结构的信息。远紫外区域阐明了肽主链的结构信息，而光谱的近紫外区域突出了涉及芳香族氨基酸残基的变化。因此，将CD光谱仪与停流系统耦合被认为是分析蛋白质展开和重折叠的最佳方法之一。该系统现在不仅提供与相关蛋白质有关的结构信息，而且还以亚毫秒的时间尺度提供这些数据。用户现在可以获得每个蛋白质展开和重新折叠时的时间尺度的更详细的图片。本应用说明通过使用JASCO CD光谱仪和SFS-492停流系统探测变性细胞色素c的重折叠来监测中间状态的存在。Application NoteCDSSpectroscopy Refolding of cytochrome c2/3Application Note CD spe c t r a p r ov i d e info r ma t io n re g arding th e se c ond ar y an d t e r tia ry s tru c ture o f pro t e in s. Whil e the fa r -UV r eg i o n elu ci d at es st ru c tu ral i n f or mati o n of th e pepti de b ac k b o ne chain ， th e n ea r -UV region o f the spectr u m h ighlights chan ge s in vo l v in g the a r o mati c a mino a cid res i d u e s .There f o r e， c o upl i ng a CD spectro m e t e r w ith a st o p pe d-f l o w syste m i s con s ide r ed one of the best m et h o d s fo r a n a l ysing the u n fo l d in g an d r e f o l d in g of pr o tein s . T h i s s yst e m now prov i d e s n ot o n ly s tr u ctu ral i nfo r mati o n per ta in in g to th e pr otein in question ， bu t a ls o su p plies th is data on a s u b-mill iseco n d time sc al e. T h e u s e r c an n o w o b t ain a m ore de t aile d pi ctu r e of th e ti m e sca l e fo r w h e n ea c h p r o t e in u n f old s a n d r efo l ds . T h is application n o t es d emons tr ates t he use of t h e J -1500CD sp ectr o m et er an d SFS-492 S t o p pe d-F l ow s y s t em t o mon it o r t h e re fo l d in g pr o ces s o f cyto ch r o me c. Keywords J -1500， c ir cular d ich r o i sm， s t o p ped -f low， S F S-492， pr otein fo l d in g， bi oc h emis tr y， refo l d i n g J-1500 CD spectrometer View product information at w ww.j a s coi n c .com JASCO INC. 28600 M ary's C our t， Easton， M D 21601 US A Experimental Measurement conditions Wavelength 222nm 289nm Data acquisition interval 5 msec 10 msec Spectral bandwidth 4nm 2nm Response time 4 msec 8 msec Syringe 1 2 mg/mL cyt c： 4.3 M GuHCI 10 mg/mL cyt c： 4.3 M GuHCI Syringe 1 loading volume 30 uL Syringe 2 0.1 M acetic acid buffer (pH 6.3) Syringe 2 loading volume 270 uL Accumulations 36 times 24 times Flow rate 1.5 mL/sec A n aque o u s s ol ut i o n of cyto c h r o m e c de n atu r ed b y gu an id in e h ydroch l o ri d e (GuHCI) w a s dil u ted wi th 0.1 M acet i c ac id bu ff er sol u tion (1：9). T h e r e f o l d ing pro c es s w a s o b serv e d a t 222 an d 289 nm t o m on it o r th e s ec on dar y str u ctu r e ch a nge s a n d a r o mati c si d e c h ai n r esidue en v iron m e n t， res p ecti v e ly . Results The c h a ng e in th e CD va lu e a t 222 nm re f l ect s the f a st re f ol d i ng o f th e seco n d ar y s t r u ct ur e w i th in 200 m se c (Figure 1)The ch a nge a t 289 nm re f le c t i ng the a r omatic s i de c h ai n s residue e n viron m ent w as slo w er th a n the c h ange at 222 n m (Fi gure 2). T h i s s low e r chan ge app e a r s i n th e l a t t e r s tep o f th e re fo l d in g pro cess ， wh ich in di ca t e s the br i e f e x i ste n ce o f an i n ter me d i at e s tat e w it h a r efo ld ed secon d ary s tru c ture and un f o l d e d a r omati c s id e c h a in r e si d u es . Figure 1. Refolding measurement of cytochrome c monitored at 222nm Figure 2. Refolding measurement of cytochrome c monitored at 289 nm 28600 Mary's Cour t ， E asto n ， MD 21601US A Conclusion Th is appl i c a tion note m oni to r s th e e xi s t ence of an i n ter m ed ia te s t a te b y pr obin g the refold i ng of d e na tu red cy t ochrome c u sing a JA SCO CD s p ectr o meter an d t h e S FS -492 Sto pped -Fl ow s yst em . References 1. El ov e， G . A.， C h af fo t t e ， A. F.， Rod e r， H.， a n d M. E. Go l d b e r g ， Bi oc h emist r y (1992)， 31， 6876. 2. Cha ff otte， A. F.， Gu il lou ， Y.， and M . E. Gol d berg ， Bio c hem is try (1992)， 31，9694. 28600 Mary's Cour t ， E asto n ， MD 21601US A