真空紫外区蛋白质水溶液的圆二色性测量

       CD光谱学通常被认为是蛋白质二级结构分析的必要工具。二级结构估计的典型测量区域约为260纳米，并进入真空-紫外区域。然而，使用水作为溶剂由于其在真空-紫外区域的强吸光度而造成了显著的限制。使用水显著提高了175 nm左右的吸光度，而在D2O中取代只会使吸光度降低到170 nm。因此，为了分析真空-紫外区域的蛋白质二级结构，测量需要一个具有高信噪比（S/N）和宽动态范围的仪器来获得高质量的数据。       新型J-1500 CD光谱仪是专门为真空-紫外区高信噪比的测量而设计的。JASCO采用了几种新技术，包括具有数字锁定信号处理的新电子系统、宽动态范围探测器、高通量光学和高效低流量氮气吹扫系统。    结论：本应用说明表明，J-1500 CD光谱仪提供了良好的信噪比和较宽的动态范围，以获得真空-紫外区水中蛋白质样品的质量数据。从CD数据中计算出的二级结构估计与从x射线晶体学数据和CDSSTR程序中得到的计算结果很一致。Application Note CD measurement of protein water solution in thevacuum-UV region2/3Application Note CDS Spect r oscopy CD measurement of protein water solution in th e vacuum-UV regio n CD spect r oscopy is gene r a ll y consi d ere d an essent i al tool fo r the seco nd ary str u c t u re a n a lys is o f pr otei n s . The r egio n typ i c a lly mea s u r ed f o r se c on d a ry s tr u ctu re estimati o ns i s ar ound 260 n m a n d i n to th e vac u um-UV reg i on . H oweve r，the u s e o f w a ter a s a so l ven t cre a te s si g ni f i ca n t l i mit a t ion s du e t o i ts s tr o ng a b s o rb a nce i n th e v a cu u m -U V reg io n . Usin g H，O d r amatically in creases the absor b a n ce a r ound 175nm an d s u b s t it ut ing in D，O on ly d e c re a ses t h e a bso rb a n c e t o 170 nm. Th e r efo r e， i n o r de r t o a n a l y ze pr o t e in se c o n da ry st ru ct u re in the vacu u m-UV region ， measu r e m e n ts r eq u i r e an in s tru men t w ith a high si gnal -t o -no ise (S /N) r a tio and a w i d e d y n amic r a ng e to o b tai n qu a lit y dat a . Th e n e w J-1500 CD sp e c t rom eter h a s b ee n desi gned spe c ifically for measu r e m e n ts with high S/N i n the v a c u u m-UV re g i o n . JASCO h a s i n c o rp o r a t e d se ve ral new t ec h n o l og i e s， in clu di ng a n e w e le c t ron ic sys t em wi th d igital l oc k -in signal p r ocessing ， a wi d e dynamic range detector ，hi g h-th r oughpu t optic s ， an d a high l y e ffe c ti ve lo w -f low n itrog e n g a s pu rging syst e m. In th is appl i ca ti o n n ote ， th e vacu u m-UV CD spec tr um was o b tai n ed for a p r otein sample in order t o estimate the s eco n da ry s t r u ctu re u si n g th e J-1500 C D spe c t r o m ete r a n d n ew mu lti var i a t e SSE pr og r am (JWMVS-529). Keywords J-1500， ci r cular d ich r o i sm， vacuu m-UV r egion， seconda r y s t ru c tur e es t i m a t i on (SSE)， CD mult i v a ri a te SSE， b i o c h e mis try J ASCO IN C. 28600 Mary's Court ， Ea st on， MD 21601 U S A Experimental Measurement conditions Data acquisition interval 0.1nm Spectral bandwidth 1nm Accumulations 4 times Response time 2 sec Scan speed 50 nm/min Path length 0.1mm Results A CD spec tr um， each ref l ect i ng a specific secondary s tr uct u re was o b ta in ed for th r ee d iff erent pr o tein mo ti fs do w n t o 174n m. 0.1 mg /mL of h um a n s e ru m albu mi n (HSA) (gre e n)， co nca na va li n A (b l u e)， and t r ypsin in hibit o r (re d ) in w a t er are s h o wn in F ig u r e 1. H SA i s r i c h i n a -h e l ic al str u ctu re， co n canavali n A shows a B -s h e e t str u ctu re， and tr ypsin in h i bi to r has a ran d o m coil seco nd ary st r ucture. Figure 1. CD spectra of human serum albumin， concanavalin A， and trypsin inhibitor in the vacuum-UV region. Secondary structure estimation The seco n da r y st ru c tu re es ti mat i on fo r eac h protein spectr u m was per f ormed using the m ul tiva ri a t e SSE p r ogram (J WMVS-529) a n d th e res u lts a re s h own in T abl e 1， a lon g wit h the X-r a y c ry s t a l s t ru c tu re an aly s i s a n d th e CDSSTR met h od fo r c ompari s o n . S econ d a ry st r u ctu re d eter mi n ati o n u si ng C D sp ec t r os copy is ge n er a lly c o ns idere d les s a c cu rat e f o r B-shee t m o t i fs tha n fo r helical st ru c tu res. H owever， the data i n Tab l e 1 c learly de m o n s tr ates th e st r ong cor r e l a ti on fo r the B-s hee t mo t if b et wee n th e mu ltiv ar i a t e SSE pr o gr a m ca l c u lati o n an d the res u lt s f r om the X -ra y c r yst all ogra p h y d ata. Th is i mp ro v e m en t w a s a chi eved by u s i ng a P LS /P C R mul ti v a r i at e an a lys i s met h o d w ith a c a l ibr atio n mo del tha t w as gen e rat ed using a r efe r e n ce d ata se t of CD spectra be t wee n 260-176 n m for 26 standard prote in s. Table 1. Secondary structure estimations using the JWMVS-529 multivariate program， X-ray crystallography data ，and the CDSSTR program. Helix (%) B-sheet (%) Turn (%) Random (%) Human serum X-ray' 71.8 0.0 8.2 20.0 albumin JASCO2 70.6 0.0 9.4 20.0 CDSSTR3 71.1 0.0 6.9 22.9 Concanavalin A X-ray 3.8 46.4 10.5 39.2 JASCO 9.7 42.7 10.4 37.2 CDSSTR 6.1 35.3 12.0 46.6 Trypsin inhibitor X-ray 1.7 33.1 10.5 54.7 JASCO 1.0 35.8 14.6 48.6 CDSSTR 5.1 17.5 16.2 59.5 iThe secondary structure estimation by X-ray crystal structure analysis referred to can be found in the RCSB PDB.The PDB IDs are as follows： HSA (1bm0)， concanavalin A (2ctv)， and t rypin inhibitor (1ba7). 2The JASCO mult i variate SSE prog r am (JWMVS-529) used the following PLS/PCR methods： HSA (PLS)， concanavalin A (PLS)， and trypsin inhibitor (PCR). 3The CDSSTR method used reference spectra SP22X[H，3/10， S， T ， P2，U]178-260 nm. Conclusion Th is applicat i o n n ote d em onstrates th at t h e J-1500 C D spec tr ometer provi d es good s i gna l -t o-n oise ratio and w i d e d y n a m i c r a n ge in o r der to ob t ain q u a l ity d a ta on p rote in s a mples in H，O in th e vac u u m-U V r eg i o n . T h e second ar y s tructu re e s ti ma ti o ns tha t we re c al c u l a t ed fr o m th e C D data w e re in g o o d agr e e m e n t w i th c al c u l a ti o ns made f ro m X -r a y c r ys t a l lography d a ta and t h e CDSSTR program . 28600 Mary's Cour t ， E asto n ， MD 21601US A