利用高通量圆二色性（HTCD）对多个样品进行热熔化分析

热变性CD的测量提供了关于蛋白质的热力学性质和二级结构特征的重要信息。JASCO的J-1500 HTCD系统不仅使研究人员不仅能够自动化CD光谱测量和二级结构估计，还可以实现多个样品的可变温度测量和热变性分析。本应用说明，J-1500 CD光谱仪结合HTCD系统可以为多个蛋白质样品提供高度精确的测量。此外，热力学参数可以很容易地计算使用热变性分析程序。Application NoteCDSSpectroscopy Thermal denaturation of multiple samples usinghigh-throughput circular dichroism (HTCD)2/3Application Note Thermal denaturation analysis of multiple samplesu sing high-throughput circular dichroism (HTCD) Introduction Th e r ma l de n atura ti o n CD m ea s uremen t s provide i m po r tan t in formation reg a rd in g a p rote in 's th ermodyn a mi c pro p e rt ie s a s w e ll a s se co n dary str uc tu r e c h ara ct e ri sti c s.JASC O 's J-1500 HTCD system enables r esea r che r s to au t oma t e not o n l y C D sp ectr a l me a s u r e men t s an d o btain s ec on dar y s t ru c tu r e es t i mat io n s ， but al s o var ia bl e t e mp e r a t ure m ea s uremen t s and th e r ma l de n atura ti on a n a l ysis o f mu l t i ple s a mple s. JASCO J-1500 high-throughput CD system View product information at w ww.j a sc oin c.c o m Th i s a ppl i c a ti o n no te de m ons t rate s th e u s e o f th e J -1500CD sp ectr o m e t er a n d Hig h -Thro u g h pu t s y s te m t o o b t a in va ri a bl e tempera tu re measu r ement data a n d seco n da r y stru c ture es t i m a t io n s us in g t h e Th e rm a l D en atu rati o n A n a l y s i s progra m. Keywords J -1500， ci r cular d ich r o i sm， protein s ， m el tin g temperatu r e an a l ysis， HTCD， AS U -800 Auto Sam p ler ， JFLC-498 Pe lti er f l ow ce ll ， phar ma ce u ti c a ls， bioc h e mi str y Experimental Measurement conditions Data i n terval 0.2℃Wave l e n gth 222 n m S p ectral b a n dwidt h 0.2nm Path l e n gt h 2nm 0.2 mg/mL of h u ma n se ru m a lbu m in (H S A )， l y s ozyme a n d rib o nec l ea s e A we r e p r e p a r ed in H，0 28600 M ary's C our t， Easton， M D 21601 US A Results The flo w cel l for the H T CD s y s t e m i s desi g ned to preven t the i n f lue n ce of tempe r atu r e in c r e a ses o n baseli n e m e a su remen t s . Fi g u r e 1 s h o ws th at th e C D si g n a l o f w a t e r u sed as a blank s o l v e n t is stabl e f r o m 20℃ t o 90℃. Figure 1. CD stability of t he HTCD system shown by using a water sample as a blank Th e ana ly s i s of th e CD results f rom the v ariable t emp e r atu r e me as u r e men t s of HS A ， l y soz ym e， an d r ib o n u cleas e A a r e s h own below (F i gur e 2). T h e th ermo d ynami c pro p er tie s w er e c a l c u la te d u sin g th e Th e r mal D e n a t urat i o n Pr o g ra m a n d a re s h o w n in Ta b le 1. Figure 2. Thermal Denaturation Analysis program displaying CD measurements f rom three protein samples 28600 Mary's Cour t ， E asto n ， MD 21601US A Table 1. Thermodynamic parameters o f three proteins measured using the HTCD system and calculated using the Thermal Denaturation Analysis program Sample name T(℃) AH (kcal/mol) AS (kcal/mol*K) Human serum albumin 73.5 46.4 0.134 Lysozyme 74.5 65.8 0.189 Ribonuclease A 58.4 135.4 0.408 Conclusion Th is appl i cation n ot e illustrate s that th e J-1500 C D s p ectromete r ， co u pl e d wi th t h e HT CD system can provide highly p r ecise measu r eme n ts fo r m u ltip l e p r otein samples. Add i ti ona ll y， thermod y na mi c pa r ame t e r s can b e easi l y calcu l a t ed u sing the Th ermal D e natu r ation A n al y sis pr og ram. 28600 Mary's Cour t ， E asto n ， MD 21601US A