高通量圆二色性用于生物医学和pH依赖性分析

圆二色性（CD）的测量可以提供关于少量样品中蛋白质结构变化的信息。由于蛋白质的结构和活性密切相关，CD的测量现在被广泛接受于蛋白质的质量控制，其中包括生物医学。为了满足现代制药实验室对提高样品吞吐量的需求，JASCO开发了一种全自动高通量CD光谱测量系统。该系统由J-1500 CD光谱仪和自动进样装置组成。高通量CD系统实现了样品预处理、注射和清洗的自动化。Application NoteCDSSpectroscopy High-throughput CD spectral measurement：biomedicines and pH dependency2/3Application Note High-throughput CDsspectral measurement：biomedicines and pH dependenc y B i o m ed i c in e i s a med i c in a l r esponse to a physical o r bi o l og i c a l issue w ith the h u m a n bo d y. Bi o medi c in e s u se act iv e i ngr ed i en ts d er i v e d f r om pr o t e in s an d th e r esear ch a n d development be h ind these m ethod s i s i ncreas in g d a ily. Ho we v e r ， th e se bi o med i c in e s are more s e n si ti ve to en vi ro nm e n t a l c h a n ge s ， suc h as a c h ange i n te mperat u r e，pH， a n d salt con c e n tra ti o n ， than tr ad it io n al pharmaceu ti ca l s.Th i s e nv i ron men t a l s e n s i tiv i ty m a y be a poten t i al c au se o f bi o me d i c ines' deact iva ti o n d ur in g pro d u cti o n a nd st or a g e pr ocesses . Ci r c ul ar d i c h roism (CD) m ea s uremen t s can p r ov i de in f o r m ati o n r e g a rd in g c h an ges in pro t e in s t r u ctu re in s m all q u a nt ities of sampl e . S in c e pr o tein s tru c tu r e an d act i v i t y a re closely related ， CD measu r ements are now w idely accepted in t he qu al ity c o n tr ol o f pr o tein ， w h i c h i nc l u de s bi o me d i c in e s . To meet th e d e ma n d f o r inc rea s ed s a mpl e th r o u ghpu t in th e mo d er n pharmaceu t ical l a b o r a t ory， JASCO ha s devel o p e d a f u lly a u tomated high -th roughp u t CD s p ectral m easurement sy stem. Thi s sy s t e m is co mp osed of a J -1500 C D sp e c t r omet e r w i th a n a u tosa m p l er un it. The high-t h roughput CD sys t em e n a bl es the au t om a ti o n of sample pretreatmen t，in j e cti o n a n d w a s hin g. In th i s ap p l i ca ti o n no t e ， w e w il l d emon st rat e the u s e o f th e ASU-800 au t omated syste m to eva lu a t e t he pH depe n dency o n the s tru c tu r e o f h u ma n ser u m a lbumin (HS A ). Keywords B i om e d icines ， qu a lit y co nt ro l， au t o mate d me a s ure m e n t，h i g h-th r o u gh p u t s c re e n i n g， hu ma n s e ru m albu mi n ， c ir cular d i c h rois m ， J-1500，ASU-800，biochemistry， pha r maceut i ca l JASCO INC. 28600 M ary's C our t， Easton， M D 21601 US A Experimental The pH o f h u ma n ser um a lbumin (r e a g ent 1) was adj u s te d b y d il u te d su lf u ri c aci d or so d i u m h y d rox ide (r e ag en t 2) u sin g a 1：4 r a tio . Th e init ia l co n cen tr a ti o n of the 30 m g o f HSA u s ed w a s 0.05 m g/mL a n d the fi n al co nc en tr a t io n a fter mi x i n g w as 0.01 mg/mL. T he mi xed reagen t was in j ected into a 10 mm recta n gular cell i n the sa m pl e com p a r tme n t o f th e J-1500. The e nt i re s amp l ing pr o c e d ure， i n c l u d in g the mixi ng o f r e ag en t s ， CD s p e ctral m ea s u r e ment， a nd th e w ash i n g an d drying of cel l s w e re pre-pr og r a m m e d s o th at a f u ll y au t o mate d a n d u n a t ten d ed m e a s ur e m e n t c o ul d b e perfo rm ed . Measurement conditions Data acquisition interval 0.5nm Path length 10mm Spectral bandwidth 1nm Scan speed 100 nm/min Accumulations 2 Response time 1 sec Reagent 1 concentration (HSA) 0.5 mg/mL Reagent 1 used 30 mg Mixed reagent concentration 0.01 mg/mL Results F i g u r e 1 sh o ws th e CD spe c t r a of h u ma n ser u m albu m in for 10 diffe r e n t pH v a l u es (1.3，2.2， 3.1， 4.1，5.4，6.7，7.5，8.4，9.3，10.7). T h e pl ot illu st rat es t h at the CD de c rea s es as th e pH i n cr ea s es ， in dicatin g str u c t u ra l c h ang es to HS A. Figure 1. CD spectra of HSA for varying pHs. The arrow i ndicates the decrease in CD as the pH increases. 28600 Mary's Cour t ， E asto n ， MD 21601US A Figu r e 2 shows th ese st r uct u r a l ch a n g es i n mo r e det a il b y p lott in g the CD values a t 222 n m as a fun ct i o n of p H . 222 n m i s a C D m a r k er b a n d fo r a-helice s i n pr ot e i n s. By plo t t in g thi s b a nd， we can s h o w th e s t r uc tu r a l de v iati o n of th e pr otein's a-helical con t e n t . Bet w ee n p H 5 to 10， the a-h el i ca l stru c tu r e i s conse r ved . H owe v e r ， i n aci d i c cond iti o n s (<5) and bas i c con diti o n s (>10)， t he d ecreased CD i nte n si t y su g gests a sli g h t de n atura ti o n of t he HSA pr otein . Figure 2. The pH dependency of the CD intensity of the 222 nm band. Conclusion By mo n itor i ng th e pH de p e n dency o f h u ma n ser um a l bu m in u s i n g th e J -1500 CD s p ectrometer a n d sample-h a n dli n g A S U -800，n u me ro u s sam pl es c an b e au t o m a ti ca lly m e as u r e d t o d e ter mi n e th e s tru c tur a l in t e grity of the pr ot e i n . T h is appl i ca t ion n ot e d e mon st ra t e s t h at CD me as u remen t i s a n e ff e c t iv e t o ol f o r t h e qua lit y co n trol o f bi o me d i c ine s a n d that the JASCO J-1500 high -th roug h put CD system can assist pharmaceu ti ca l labora t o r i es i n the u n atte n ded screen i ng of l arge n u m b e r s of s a mpl es . 28600 Mary's Cour t ， E asto n ， MD 21601US A