稀硫酸滴定聚L-谷氨酸钠的二级结构分析

智能文字提取功能测试中

由于蛋白质的功能与其结构密切相关，蛋白质和多肽的结构分析在帮助破译生物活性方面变得越来越重要。核磁共振和x射线结构分析是鉴定蛋白质结构的两种有效方法，但两者都需要大量的样本和较长的采集时间。另一方面，CD的测量值很容易获得，并且可以使用较小的样本体积。此外，还可以获得由pH、温度和外部配体引起的蛋白质和多肽的构象变化。该应用说明表明，JWSSE-513程序利用Reed的参考CD集，提供了良好的结果，可以描述肽环境中pH变化引起的二级结构的变化。Application NoteCDSSpectroscopy Secondary structure analysis of poly-L-glutamicsodium titration with dilute sulfuric acid2/3Application Note Secondary structure analysis of poly-L-glutamic sodium titration with dilute sulfuric acid Si n ce t he f u nc ti o n of a p rote in is close l y re l ated t o its s t ru c ture ， the stru c tu r a l an aly s is of prote in s a n d pepti d es i s b ecom ing in cre a s i n gly i m po r tan t t o a ss i s t i n d e c iph e r i n g bi oactivity. NM R a n d X-r ay structu r e an aly s i s are two effec ti ve m eth ods t o id e n tify p r otein st ru c ture ， howeve r， a l ar ge am o u n t of sa mpl e an d a len gth y a cquis iti o n t ime i s r eq u ired for bot h. On t h e other hand， CD measuremen t s ar e ea sy to o b t a in， a nd s m al l sampl e v o l u mes c an b e u s ed. Ad d iti o n a lly， co n f o rm a t io n al ch ange s of pr otei ns an c p e pti des due to pH， temperatu r e a n d exte r nal l i ga n ds can also be o b t aine d. Onc e CD m ea s u r e men ts are ob t a i ned ， th e a bu n d an c e r ati o of seco nd a r y st ru ct ure can b e calcu l a te d by a l e a s t s q uares method tha t uses a r efe r e n ce spectrum o f a-he l ix，B -s h ee t ， tu rn a n d r an dom s tr uc t ure. T he J WSS E-513 p rot ei n s eco n da ry str u ctu ra l a n a l y sis program u se s t he Cla ssi ca l Lea s t Squ a res (CLS) method ， wh ich i nc lu des t h e r efe r ence sp ectra of Y a ng an d R e ed². Yang’s r efe r e nc e s pe ct r a a re e x tr act e d f ro m th e CD sp ectr a of prot ein and are best suit ed for p r otein secondary stru c ture an a lys is 13 wh il e Reed's re f ere n c e spe ctr a a r e e xtr acte d f r o m CD measu r e m e n t s of p e p t ides an d are s u it a ble f or th e s e co n da r y st ru ct ure a n alysis o f peptides. Reed's r efe r ence s p ectra have less of an in flu e n c e fr om th e aro mati c a mi n o ac i d s id e c h ain res i d u es wh ich a re typ i cally s een i n prot e in s pec t ra. Th is app li catio n note d e mon s tra t es th e us e of the JWSSE-513 p r ogr a m i n ord e r to dete rmin e the seco n dary s t ructure c h a ng es o f po l y-L -gl u t a mi c sodi u m titra t ed w i th d ilu te su l f u ric aci d . Keywords J -1500， ci r cular dich r o i sm， p h armaceu ti ca l s， bioch e mistry，JWSSE -513， se c o nd ary stru c tur e an alysi s prog ram， ATS -429a u t o mati c titra ti o n u nit J ASCO IN C. 28600 Mary's Court ， Ea st on， MD 21601 U S A Experimental Measurement conditions Data acquisition interval 2 sec Spectral bandwidth 1nm Accumulations 2 times Data pitch 0.1nm Scan speed 100 nm/min Path length 2 mL o f a 0.02 mg/mL p oly -L -g lu ta m i c sod ium s ol u ti o n i s t itra t ed w it h 10-5 N s u l f u ri c a c id . T he t it rati o n i s ca r ried o u t 20ti mes fo r 50 p L a li q u ots u s ing the A T S-429 au t oma ti c titra ti o n u nit. Results The C D s p ectra of poly-L -gl u tamic sod iu m solu ti on ti trated w ith su l f u ri c aci d is s h own i n F igure 1. The figure ind i ca t es th at as d i lu te s ul furi c a c i d is add ed t o th e p e pti de sa mpl e ， t he CD sp e ctr um c h ang e s f r o m a r a n d om s tructu r e c o n f o rm a ti o n t o an a -heli ca l co n formati o n. Figure 1. CD spectra of poly-L-glutamic sodium solution titrated with sul f uric acid. A co m pa r ison of th e ex p er i ment a l a n d ca l cul a ted CD s p ec tra of p o l y-L-g lu tamic sod i u m so lu tion be f ore and a f te r add in g sul f u ric acid i s sho w n in F igur e 2 and u til izes Reed's r efe r e n ce s p ectra se t . These pl o t s i ll u st r ate th at t h e re si d u a l er r o r between t he expe r i me n tally d e te r mi n ed a n d calcu l ated spectra is sma ll. 28600 Mary's Cour t ， E asto n ， MD 21601US A 70000 60000卜 40000日 Mo l. Elp. 20000 0 -20000 -3000090 200 220 240 260 W a v eleng t h [nm] Figure 2. Comparison of the measured and calculated CD spectrum before (lef t ) and after (right) the titration of poly-L-glutamic sodium solution. The green l i ne represents the measured spectrium， the blue is the calculated spectrum， and the red line i s the residual error. Th e ra ti o of s e con d ary s tructur e s of th e me a su re d an d c a lculat ed da ta al so c an b e ca lcu la te d an d i s s hown in Fi gur e 3.As th e p e p t i de solut i on b ecomes more acidic due to th e add iti o n of su lf u r ic acid， the a -helical structure ra ti o in cr eases a n d 82.4% o f p o ly -L-glu tam i c ac id tran si ti o n s to a n a-he l i c al s tructu r e. U n d e r 6.6 uM su lfur i c aci d c on diti o n s ， the r a n do m s tructu r e r a ti o de c reases t o 3.2%. 一 +-h e lix T Bm 一 -f an d om Figure 3. The ratio of the measured to calculated secondary structure est i mations as a function of sulfuric acid concentration. The estimations were made using the JWSSE-513 software. Conclusion Th is app li c a tio n n o te demon s tra t es th at th e J WSSE-513 pro g ram， w h ich u tili zes Ree d 's r efe r ence CD set fo r pe p ti d es ，pro v ide s g ood results th at c an d e s c r i be c hange s in s econdar y st r u c ture d u e t o pH c h a nges in a pe p ti de's e nvir o n men t . References 1. Y an g， J. T .， Wu， C. C.， and H. M. Ma r tin e z ， Method s in En zy mol og y (1986)， 130，208-269. 2. J. Reed and T . A. R eed ， Anal.Biochem. (1997)，254，36-40. 3. Ch an g， C . T .， W u ， C. C.， a n d J . T . Y ang， An al. Bi oc h em. (1978)， 91，13-31.