超耐热纤维素酶的圆二色性热变性分析

蛋白质的热力学性质和二级结构揭示了有关其结构和功能的重要信息。虽然 Differential scanning calorimetry（DSC）和CD光谱法都经常用于测量蛋白质的热变性，但CD光谱法比DSC有几个优点。例如，CD光谱可以在各种pH和更宽范围的溶剂条件下使用较低蛋白质浓度的蛋白质来测量。然而，由于水溶液的沸点极限，CD光谱通常不能在100°C以上的温度下测量。因此，JASCO开发了TC-700/700PC高压/高温测量样品室，用于100°C以上的高温测量。TC- 700允许在使用市售的N2气体将样品溶液加压到1 MPa，在高达170°C的温度下测量CD光谱。探测超耐热蛋白的热变性的CD测量现在可以用TC-700来测量。本应用说明表明，通过使用TC-700/700PC和JC-1500CD光谱仪，可以在100°C以上的温度下测量热变性曲线，以测试高熔化温度下的耐热蛋白。Application NoteCDSSpectroscopy Thermal denaturation analysis of superthermostable cellulase using a TC-700PC2/3Application Note Thermal denaturation analysis of super thermostable cellulase using a TC-700PC Th e thermod y n a m i c p rope r ties and seco n dary s tr uct u re o f pr ote i ns r eve al i mp o rtan t in formati o n reg a rd in g t he i r s t ructure and th ere fo re， their fun c ti o n. W h il e d i f f eren ti a l scan n ing calorimetry (DSC ) a n d CD spe c tr osco p y are bo t h o ft e n u s ed t o m e a s u r e the th e rm a l d e n atura ti o n of pro t e i n s ，CD s p ectr o s c opy h as se v e r a l a d van t a ge s o v er DS C . F o r example ， CD spe c t r a ca n be measured u s i n g lower pro t e in c o nce n tr a ti o n s o f p ro teins at v ari o u s pHs a n d in a w id e r ra ng e o f s o lv e n t c on diti o n s. Ho w eve r ， CD spe c tr a ca nnot typ i cally b e meas ur ed at tempe r atu r es above 100℃ because of t he b o i l in g p o i n t lim i t o f a q u eo u s-b as ed s o l u t i on s . JA SCO h a s t he r efo re de v e l o p e d th e TC -700/700PC h i g h pres s u r e/h ig h t e mp e ratu r e me as u r e men t sampl e c h am ber fo r high t empera tu re measu r eme n ts above 100℃. The TC-700 all ows fo r C D spectr a t o b e me as u re d at tem pe ra tur e s up t o 170℃ by pre s s u ri z i n g t h e s a m pl e s o lu ti o n t o 1 MP a u sing comme r cially a v aila bl e N， gas . CD measu r e m e n ts pr o b ing th e ther ma l d e n at u r a ti o n of s up e r th e r mo stable pr ot e i ns ca n now b e mea su r ed u s in g the TC -700. Th is appl i ca t io n no t e d e scrib e s th e ther ma l denat ur ati o n a n a l ysis of s u pe r therm o stable cellulose u s ing the TC-700PC an d th e De n atu ra t ion A n a lysi s prog ram. Keywords Ther ma l d en atur ati o n ， therm a l st a bil i t y， ci r cu l ar d i c h r o i sm，J -1500， TC-700/700PC ， Dena tu ration Analysis， super therm o s table c ellu l a s e Temperature controller TC-700 sam p le holder View product information at www.j as coi nc.co m Experimental Measurement conditions Data acquisition interval 0.1nm Spectral bandwidth 1nm Response time 2 sec Temperature interval* 0.1℃ Path length 10mm Scan speed 100nm/min Pressure 0.8 MPa Temperature slope* 1C/min *For CD thermal denaturation curve monitored at 220 nm. 1.2 mg /mL of s up er th ermost a b l e cel lul ose i n 20 mM T r i s-HCI bu ff er (p H 8.0) wa s diluted w ith d isti l led wa t e r t o the desirec co n cen tr ati o n s a n d m easured u s in g t he J-1500 a n d T C -700P C . Results T h e CD sp ectr a of 0.01 mg /mL s up e r th er mos t able c e l lulase wa s m ea s ur e d at 45， 80， 100 and 120℃ an d i s sh o w n in F i gu r e 1. S u pe r thermostable cell u lase mai n tain s its act i vity a t t e m pe r a t ures u p to 90℃， wh i c h i s con s is t e n t wit h o u r d ata il lustra t i ng th at the th ermal de n atu r a ti o n o f th e p ro t e in ca n o nl y b e s e e n f or t em per a tu re s a b o v e 100℃. Figure 1. Far-UV (195-260 nm) CD spectra of 0.01 mg/mL of super thermostable cellulose at 45℃ (red)， 80℃ (blue)，100℃ (yellow) and 120℃(green). 28600 Mary's C o u r t， Easto n， MD 21601 USA CD measu r eme n ts we r e a l so obtained at 220 n m t o p r o b e th e therma l d e na tu ration of supe r thermostable cellulase. The r esu lt a n d a na ly s i s u sing th e D enatu r atio n A n al y sis pr og r a m a r e sh own in Fi gur e 2. Con sisten t w ith Fi g ur e 1， there is a sh ar p dec re a s e i n t he CD va l u e at t e m pera tu re s ov e r 100℃， i n di ca tin g the l os s of s ec o ndary s tru c tur e an d d e n atu r a ti o n of the pro t e in a b ove 100℃. T h e m el ti ng tempe r atu r e (T) of super th ermo s ta bl e cel l u l ase was calc u lated by the D ena tu r a ti o n An a l y s i s software a n d fo u n d t o b e 106.3℃. Figure 2. Thermal denaturation results for 0.025 mg/mL super thermostable cellu l ase. Conclusion Th is ap p li ca t i o n no t e ill u st ra te s that by u si n g th e T C-700/700PC wi th th e J -1500 CD s p e c t r om eter， th e r mal de n atu r a ti o n c u rv es c a n b e m e a su r ed a t t e mp e r a t ures a b o v e 100℃ t o te s t t her mo stab l e pr oteins wit h h igh m elting temperat ures . 28600 Mary's C o u r t， Easto n， MD 21601 USA