真空-紫外区蛋白质膜样品的CD测量

CD光谱学通常被认为是蛋白质二级结构分析的必要工具。特定二级在2结构的标记带最典型的是60 nm以下的紫外区域，并尽可能进入远紫外区域。据广泛报道，使用同步辐射的CD测量已经成功地测量到140纳米左右的光谱。虽然同步辐射已被证明是测量真空-紫外区蛋白质膜样品的CD光谱的一种非常有效的方法，但它需要获得有限数量的昂贵设施。作为同步辐射的替代品，J-1500 CD光谱仪已经得到改进，用于真空-紫外区域的测量。J-1500的氮气净化效率最近通过计算机建模得到了优化，光学系统得到了增强，以获得更多的光吞吐量，新的系统电子学包括数字锁金技术。这些特征确保了CD光谱可以从强吸收和高信噪比（S/N）样品中获得横跨整个光谱并进入真空紫外区域。所获得的光谱数据的质量，包括在较短波长下获得的数据，大大提高了蛋白质二级结构分析的准确性。结论：该应用说明表明，在光谱的真空-紫外区域获得的CD测量为识别各种蛋白质样品中的二级结构标记条带提供了可靠的数据。Application Note CD measurement of protein film samples in thevacuum-UV region2/2Application Note CDS Spect r oscop y CD measurement of protein film samples in th e vacuum-UV regio n CD spect r oscopy is g e ne r a l ly co n si d ere d an essential tool fo r th e s ec on dary s t r u c ture a n a ly sis o f p rot e ins. M a rk e r b a n d s fo r sp e c i fic s ec o n d ar y s t ru c tu r es ar e mo st typic a l l y o b serve d i n t h e UV r egion below 260 n m a n d as fa r i n to th e f ar-U V regi o n a s p o s s ib le . It h a s be e n w ide l y repor t e d t hat CD meas ur e m e n ts u s i ng s y n c h ro t o n r a di a ti on h a v e b ee n u sed to successf u lly meas u re spect r a do w n t o a r o u nd 140n m. W h i l e s ync hr otro n ra d i a ti o n h as p roven t o b e a very e ff e cti v e me th od t o m easu r e th e C D spe ctr a of p ro t e in f i l m samples in the vac u um-UV reg i on ， it r eq u i r es access t o a limit ed n u mb e r o f cos tly f a c iliti es . A s an a lt er n a t iv e to s y n ch r otr o n radia t i o n ， t he J-1500 C D sp ectr om e t er ha s b een i mprove d f o r m e as u r e m en t s i n th e vac u um-UV r eg i on . T h e J-1500's n itrogen pu r ge eff i c i e n cy h a s r e ce n tly been op ti mi ze d th r o u g h c o m pu t e r mod e l li n g，the opti c al s y s t e m has b een e nh an ce d fo r m or e li g ht thro u ghpu t ， and n e w system e l ectron i cs inc lu de d i g it a l lo c k-in t ec h n o l ogy. Th e s e f eatu r es en s u re tha t CD sp e ctra c an b e o b tai n ed f rom str ongly a b s or bin g a n d h igh sign a l-to-no is e (S /N) samp l es across the spectr u m a n d in to th e vac u um-UV r egio n . The q ua l i t y o f s p ectral data o b tai ne d ， i n c lud in g dat a o b tai n e d a t s ho r t e r wa ve l e ng th s， s u b st a n tiall y im p rove s th e accu r acy of p rote in seco n da r y st ru c tu re a n alysis. T h is app li ca t io n note illu s tr a tes t he me a su r e m e n t and an alysi s o f a pr o t e in f i l m sa mpl e i n th e v a c uu m-UV r eg i o n u sing th e J -1500 C D spectrome t e r. Keywords J -1500， c ir cular d ich r o i sm， vacuu m-U V r eg i o n ， protein fi lm，bi och e mis tr y JASCO INC. 28600 Mary's Court ， Ea s t on， MD 21601 U S A Experimental Measurement conditions Data acquisition interval 1nm Spectral bandwidth 1nm Accumulations 4 times Response time 4 sec Scan speed A p r otein sam pl e in aqueous solu t io n was d r o pp ed onto a q u a r tz p l a t e a n d t hen evaporated to f or m a t hin fi l m . CD an d a b s o rbanc e s p ect ra we re me a s ure d in th e v acuu m-UV r e g i o n (250-163 n m ) fo r fo u r d iff e r e n t pr ot ein sa mple s . Results Th e C D spe c t r a of my g l obin (g r ee n)， ly soz yme (r ed)， con can a v a li n A (b l u e )， a nd tr y p s in i nh ib i t o r (pin k ) a r e sh ow n i n Fi g ur e 1. Mygl o bin s h ows a -h e li cal n ega t ive mark er band s a t 222 a n d 210 nm w h i le conc an ava li n A s h ows a positive b a n d a t 195n m and a nega ti ve band a t 220 n m i n dic a tive of B-shee t structu r e. Lysozyme illustr a tes a mi x tu r e of b oth a-h e lical and B -sh e e t co n ten t w ith a p o si tiv e ban d a t 195 n m and t w o n ega t i v e ma x es a r o u n d 175 a n d 222 nm . T ryp sin i n hibit o r sh ow s a ra n do m coi l str u ctu r e . Figure 1. Vacuum-UV CD (left) and absorbance (right) spectra of protein film samples. Conclusion Th is applicat i on n ote de m onstrates th at CD measu r e m e n ts o b ta in ed i n the vacu um -UV r egio n of the spectr u m p rov i de r e l i a bl e d a t a fo r i d e n ti f y i n g se co nda ry s t ructu r e mark er b a n ds in a va ri e t y of pro tein s a m p le s . Reference Mo de r n Te c h n iq u e s f or C ir cu la r Di c h r o i s m a n d S y nc h ro tr o n Radia t io n C irc u la r D i c h r o i sm s p ectrosc o p y， B. A . Wa l l a c e a nd R. W. J a n es (Eds.)， I OS Press ， 2009， p 43. JASCO INC. Jsc o