α-乳清蛋白变性引起的荧光各向异性光谱变化

CD光谱技术是蛋白质结构分析的领先技术之一，荧光光谱技术和荧光各向异性技术提供了互补的信息。CD光谱提供了关于蛋白质二级结构的信息，而荧光和各向异性光谱提供了关于荧光团局部环境的信息。特别是，荧光各向异性提供了关于这些荧光团的旋转运动的见解，这是不能单独通过荧光光谱获得的。JASCO J-1500 CD光谱仪可用于测量CD、吸收、激发和发射以及荧光各向异性。这种技术不仅允许二级结构估计，还允许分析蛋白质配体结合和旋转运动。结论：本应用说明表明，J-1500 CD光谱仪可用于各种光谱技术的样品测量。在J-1500光谱仪上添加FPA-580偏光器，用户可以在同一台仪器上获得CD和荧光各向异性数据。Application NoteCDSSpectroscopy Change in the fluorescence anisotropy spec-trum by the denaturation of a-lactalbumin2/2Application Note Change in the fluorescence anisotropy spectrum by the denaturation of a-lactalbumin CD spect r oscopy is o n e of th e lead i ng techn i q u es i n pro t e in s t r uct u re a n a ly s i s， with fl uo r e s c enc e s pe c tr o s c o p y an d fluo r es c en c e a ni s otr o py p rov i d i n g c o mple men t a ry in formation . While CD spec tr a pro vi de info r mation regarding the sec o n dary s t ru c tu re o f pr o te i n s， fl uo re s cen c e a nd a n i sotr o p y sp ec tr a provi de in fo r m ati o n a b o u t th e l o cal e n v i r onmen t of f lu o r ophores. I n pa rt icu l a r ， f lu o r esce n ce an is otr opy offe rs in si ght a b ou t th e ro t a ti o n al mo v e me n t of th ese fl u oropho r es w hi c h c an n ot be o b tai n e d b y t lu oresce n ce spectrosco p y a l one . T h e JASCO J-1500 CD spectrome t e r ca n be u sed t o me a s ure CD， ab s o rpti o n， e x citati o n an d em i ssi o n ， a n d f l uor es c enc e a n iso t r opy. T h is v a r iety of t ec h ni q ue s no t only a ll ows f or seco n dary stru c ture estimat i o n but also th e an a ly s i s of pro t e in-l igan d bin din g a n d ro t a ti o n a l mov e me n t in pr o t e ins . Th is appl i ca t ion n o te d e scr i b es th e change s in f lu ore s cenc e a n isot r opy meas u remen t s of the den atu ration o f a-lac t a l b umin by g u a n adi n iu m h ydr oc h l or i d e (GuHC I ). Keywords J-1500， cir cul a r d i c h r o is m ， C DF -426， fl uo r es c en c e，a n isot r opy， a -lac t albu min， GuHC l ， seco n dary st r ucture ，d e na tu rati o n ， bi ochemi str y JASCO INC. 28600 Mary's Court ， Ea s t on， MD 21601 U S A Experimental Measurement conditions Data acquisition interval 0.1nm Excitation bandwidth 7nm Response time 2 sec Scan speed 100 nm Results Th e f l u oresce n ce a ni s o t ro p y spe c tra of b o t h the na ti ve-st a te a -l a ct a lbu mi n in H 2O a n d the u n fo l ded a -l a c t albu mi n i n 3.4M GuHCl a r e sho wn i n Fi g u r e 1. B o th spe ctr a show a p eak ma xima at 267 nm an d pea k min i ma at 283 a n d 291 n m. A ll three peaks r esu lt f ro m th e t r y p to ph an res i d u e of the protein . Fi g u re 1 i llu s tr ates th at the denat ur ation o f a-lac t a l bu min cle arl y dec r e a se s th e f l u ore s ce n ce anisotr o p y， i n d i c a tin g a mo r e fr eely r ot a tin g tr y p t o phan res i d u e. Figure 1. Fluorescence anisotropy spectra o f the denaturation of a-lactalbumin by GuHCl . The green line indicates 0.02 mg/mL a-lactalbumin ， 0.1 mM EDTA in the absence of GuHCl， and the blue line is 0.02 mg/mL a-lactalbumin， 0.1 mM EDTA in 3.4 M GuHCI. Conclusion T h i s app l i ca ti o n n o te demon s trates that th e J-1500 CD sp ectr o meter can b e us e d to mea s u r e sam ples fo r a v a r iet y o f sp e c t rosco pi c te c h ni qu e s . The a dd ition o f th e FP A -580 p olar i z e r to the J-1500 spec t rometer allows u sers to obtain C D and fluorescence an i sotropy data al l o n the sa m e in s tru m e n t. References 1. C a n e t ， D.， D oe rin g， K .， Do b so n ， C. M.， an d Y. Dup o n t ， B i oph ys i c a l J o ur nal (2001)， 80，1996-2003 2. J . R. Lakow i cz， Pr i nciple s of F l u ore s c en ce Spe ctrosc o p y， Ne w Yor k， 446-487. JASCO INC. Jsc o T el ： (800) 333-5272， Fa x ： (410) 822-7526 A ppli c at io n Li brary： htt p ：//www.j as c o i n c .co m/applica t ion s