圆二色光谱法研究核糖核酸酶A的微量热变性

圆二色性（CD）是一种测量和分析远紫外区域蛋白质和核酸的二级结构和热变性的有用技术。通常，1mm路径长度的矩形样品池用于这些测量。然而，这需要大约200µL的样品体积。对于只有非常小体积的样品，JASCO现在为小于10µL的样品提供了一种新的毛细管池。毛细管池也有一个用于热倾斜测量的护套。CD测量仍然易于执行，并且毛细管池是廉价且一次性的。该申请说明表明，变性温度值以及文档中图2所示的热变性曲线表明，使用毛细管池和护套进行核糖核酸酶A的微检测可以高精度进行。Application Note Microassay thermal denaturation ofribonuclease A probed by CD spectroscopy2/3Application Note CDS Spect r oscop y Microassay thermal denaturation of ribonuclease A probed by CD spectroscopy Introduction Cir c u l a r d i c h rois m (CD) i s a u se f ul t echn i que t o m eas u re and an a l y z e s eco n d a ry structu re a n d t h e th e rm a l d en atu rati on o f pro t e i n s a n d n uc le i c a c i ds in th e fa r -U V r egio n . Ty pi c all y，a 1 m m path l ength rec t a n gular cell is u s ed fo r these m e as uremen ts . Ho w ever， th i s req uir e s a s a mpl e vo lu me of ab o u t 200 p L. In t he c ase of sampl es w h e re on ly v er y s mall vo lu mes are ava il able ， J ASCO now offe r s a new ca p i l lary c e ll fo r sa mple vo l u mes s m a ll er th an 10 u L. The c ap ill a r y c e ll a l s o h a s a j a ck et for th er m al ramping m eas u remen t s ， s h ow n in Figure 1. C D measu r e m e n ts r e m a in s i mple to p e r form a n d the ca pi l l ar y c e ll s ar e in e xp e n s i ve a n d d is p o sabl e. JASCO J -1500 CD spectrometer View product i nformation at w ww.j a s c oi nc.cOm Th i s a pp li c at io n n ot e d e m o n st r a t e s th e u se of a c ap i lla ry c e l l an d ja c ket f or the rmal r amp in g stu di e s o f rib o n u c le a se A. Keywords Circu l a r d i c h rois m， ca p i l lary cell， mi croassay， t h e r ma l de n atu r a ti o n ， r ibo n u cl ea s e A， Denat ur ed P r otein An a ly s i s software， den atur ati o n t empera tur e，J -1500 Experimental Data acquisition interval 0.2℃ Path length 0.5 mm (capillary cell)， 1 mm (rectangular cell) Spectral bandwidth 1nm Rising temperature rate 1C/min Measurement wavelength 222 nm Response time 8 sec 1 mg/mL of r i b o n u cl ease A aque o u s so l u ti o n i s draw n u p i n to a capil l a r y cel l w ith a 0.5 mm o p tical p a t h le n gth ， a s seen in Fi g u re 1. T h e ce l l i s the n in ser t ed i n t o the capilla ry j ac k e t f or th ermal r a m p in g C D m easure me n t s. Results Fi g u re 1 s how s th e ther m al d en atur ati o n o f r i b o n u clease A. Th e J AS C O JWT D A -519 D en a t ur ed Pr otein A nal y sis s of t w a r e w as u sed to ca l c u late the denat u r ation temperat u re of r i bonuc l ease A i n bot h the capilla r y a n d r e c ta n g u la r cells . 59.4℃w a s c al c u l a t e d fo r the c ap i l l a ry cell w h i c h i s in g oo d a gre e ment w it h 59.7℃ o bt a in ed fo r th e dena tura tion t em p er atur e in th e re ct a ngu la r cell . Figure 2. Thermal denaturation curve of ribonuclease A measured at a wavelength of 222 nm. The green curve was obtained using the capi l lary cell while the blue curve was measured using the 1 mm rectangular cel l . 28600 Mary's Cour t ， E asto n ， MD 21601US A Conclusion This applic a ti o n note dem o n s tr ates th at the den a tu r a tion t empera tu re va lu es ， along w ith the t h e r ma l denaturat i on curves show n in Fi g u r e 2， i l lu s tra t e th at the m i c r oass ay o f ribon u clease A u sin g th e cap ill a ry c el l an d jacket can be c ar ri ed ou t with h igh acc ur acy . Note： Th e MSD-462 m ic r osampling disc ca n b e u sed f or s p ectral sca nn ing measu r e m e n ts o n s ample vol u mes b e twee n 2a nd 10 u L. T h e MSD-462 applic a tion s a r e sh o wn in the f ol l ow in g Application No t es ： 260-CD-0011 an d 260-CD-0019. 28600 Mary's Cour t ， E asto n ， MD 21601US A