用圆二色性探测溶菌酶和N-乙酰葡萄糖胺的相互作用

蛋白质CD光谱的变化直接反映了其结构的变化。在CD光谱的240nm以下可以看到源自蛋白质肽键的吸收。因此，通过测量远紫外线波长区域的CD光谱，可以获得关于蛋白质二级结构的信息。另一方面，在光谱的210-220nm波长区域中可以看到由于芳香族氨基酸侧链残基引起的吸收。在超过240nm的波长处存在额外的吸收带，其不与肽键的吸收带重叠。出于这个原因，为了研究侧链残基，CD光谱通常在大于240nm的波长区域测量，这被称为近UV。本应用说明演示了J-1500 CD谱仪和ATS-530自动滴定仪在探测CD谱的近紫外区域方面的应用，以阐明蛋白质特定芳香侧链残基的构象变化。Application Note Lysozyme and N-acetyl glucosamine interactionprobed by circular dichroism2/3Application Note Lysozyme and N-acetyl glucosamine interactionp robed by circular dichroism Changes in a p rotei n 's CD spect ru m di r ectly re f le c t th e chan ge s in it s s tru c ture. The a b s or pt i o n d e r ived f ro m the pro t e i n 's p ep ti d e b o n d s c an b e se e n bel ow 240 n m of the CD spect ru m. T h ere fo re， by measur i ng t h e CD spec tr um in th e fa r -U V w a ve l en gth re g i o n ， in f or mati o n r e g ard ing a pro t e in's s ec o nd ary s t ruc t ure c an b e o bt a in ed . On t he o th er h and， absor p t i o n due to aromatic amino acid s i de c h ai n r e s idu es ar e se en in th e 210-220 n m w a vel e n gt h r eg i o n of the sp ectr u m. Th e r e ar e ad diti o n a l a b so r p t i o n b a n ds at w ave l ength s beyo n d 240 n m w h ich d o not ove r la p w ith the a b s orpt i o n b an d of the pepti d e b o n ds . F or t hi s r e a s o n ，in o rd er to st udy side ch a in res i dues th e CD sp ec t ru m i s u s u ally measu r ed in th e wavelengt h region longer t h a n 240n m w h i c h i s k n o w n as the n ea r -UV. I n th is appl i cati o n no t e， th e in t e ra c t i o n b et w een ly soz ym e an d its in hibit or ， N-ac e tyl-(D +)-gl u co samine (NAG) was m ea s ured in th e n ea r -UV regi o n u s ing a J-1500 CD sp ectr om e t er an d a ATS -530 a u t om ati c t i t r at or . Keywords J -1500， cir c u l a r d i c h rois m ， n e a r -UV， side ch a in res i d u es ar omatics ， A T S-530， automat i c t itrator， bi oc h emis tr y JASCO INC. 28600 Mary's Court ， Ea s t on， MD 21601 U S A Experimental Data acquisition interval 1 sec Data pitch 0.1nm Spectral bandwidth 1nm Scan speed 50 nm/min Accumulations 4 Path length Th e t i t r a ti o n o f 225 mM NA G to 0.07 mM l y s o z ym e solu ti o n w a s me a s u re d between 260-320 n m.NAG wa s sl owly t i tr a t ed in 50 u L i nc r eme n ts at a co n s t a n t tempe r atu r e of 20℃. Results The CD spec tr a of l ysozy m e， lysozyme +NAG ， and NAG a r e sho w n i n Fig ur e 1. T h e l ysozyme CD peaks a r e p os i tive a n d o b se r ve d at 293.5，289， a n d 283.2 nm . Th e a d diti o n of NAG t o ly s o z ym e i n cr ease s th e C D in ten s it y b e tween 300-270nm w h i l e th e pe a ks in the CD sp e ctr um o f NAG o n ly ar e b a r e l y obs e r vabl e. Figure 1. CD spectra of lysozyme only， NAG only， and NAG t i trated into the lysozyme solution i n the near-UV region. Figu re 2 s h o ws a 3-D grap h i c a l repr es e n t a ti o n o f t he c h ang es i n t he C D sp e c t r a re fl e ct e d by th e titr ation o f NAG t o th e lysozyme so l u ti o n . F igure 3 i l lu s trates the c h a n ge in the CD inte n s i ty at 293.5 nm d u ring the t i tration o f l ysozyme. X-ra y c ry s t a llo g rap hy d at a i llu s trates th at ther e i s a t ryp t o ph a n r esi d u es i n th e s ubstr a te b o n d in g reg i o n o f l yso z ym e . Th e X -ray d at a sugge st s t h a t the C D d ata c an b e in ter p re t ed as inc r e a s i n g in C D in ten sity d ue t o th e i n t e ract i o n of NAG w ith lysozyme's t r yptophan residue . 28600 Mary's Cour t ， E asto n ， MD 21601US A Figure 2. 3-D graphical representation of the CD spectra changes due to the ti t ration of NAG to lysozyme. Figure 3. CD spectrum a t 293.5 nm as a function of the NAG concentration. By co n du c ti n g th i s CD m eas u remen t ， th e ch ange i n th e c o n f o r m ati o n of the en zy me's side ch a in s c an be o b ser v e d during in teract i ons w ith th e s u bs tr ate a n d whe n the s u bs tr ate b o n ding region i s k now n ， th e t ype o f s i d e c h a in can be e s ti m a t ed u sin g th e CD s p ectru m o b t a i n e d . Conclusion T h i s app l i ca t i o n n o t e d e mon st ra t es the u se of t h e J -1500 CD s p e c tro met er a nd ATS -530 au t o mati c t i tra t or i n probin g t he near -U V reg i o n of th e CD spe c tr um in ord e r t o e lu cidat e con f or mati o n a l c h ange s i n specific aromatic s i d e c h ai n re s i du es of proteins . JASCO INC. 28600 Mary's Cour t ， E asto n ， MD 21601US A