低浓度水溶液中蛋白质二级结构分析

红外光谱技术是对蛋白质、糖、脂类等生物样品进行成分分析和相互作用分析的一种有效技术。FTIR光谱用于蛋白质二级结构分析，可用于测量固体和液体样品。JASCO之前已经报道了蛋白质水溶液的研究。在本申请说明中，我们报道了ATR PRO PENTA的结果，这是一种用于测量低浓度水溶液的最佳系统。ATR PRO PENTA的设计具有最佳的反射次数，因此吸水率不会饱和。此外，使用具有高灵敏度MCT检测器的FTIR光谱仪可以测量具有非常低的样品浓度的样品，这在以前是难以测量的。在这里，我们报告了三个低浓度蛋白质水溶液实例的测量结果。特点-ATR简易取样配件-独特的五角棱镜形状-Ge棱镜中的14次反射（图1）-与MCT检测器相结合的高灵敏度测量-样品体积只有几微升本应用中实验：低浓度蛋白水溶液的分析              制备了以下三种蛋白质水溶液（0.01% w / v），并使用ATR PRO PENTA测量了红外光谱。采用水的差分谱进行二级结构估计（SSE）                 1:溶菌酶（鸡蛋清）                  2：伴刀豆蛋白A                   3：胰蛋白酶抑制剂KunitzApplication Note140-AT-0263 Protein secondary structure analysis2/3Application Notein low concentration aqueous solutions Protein secondary structure analysis in low concentration aqueous solutions I nt r oduction Inf r ared spect r osco p y is an e f fect i ve tec h nique for component analysis and in t eract i on analys i s of biological sa m ples suc h as proteins， sugars and l i pids. F T IR spectrosco p y can b e used to m easure solid and liquid samples when appl i ed to seco n da r y st r ucture analysis of prote i ns. JASCO has previously reported th e study of aqueous p r ote i n sol u t ions". I n t his applicat i o n note， we r eport r esu l ts fr om the ATR PRO PENTA an optimal syste m for meas u ring l ow concentration a qu eous sol uti ons . The ATR PRO PENTA h as bee n designed w i th an optimum nu m ber of reflec ti o n s so th a t wa t e r a bsorpt i o n i s n o t saturated. Furt h e r more， th e use of an FT IR spect r ometer wit h highly sensitive MCT detec t or al l ows measureme n t of samples wit h very low sample concentration s ， whic h were previou s ly diff i cult to mea s ure. Here we report th e res u lts of m easurement of three examples o f low co n ce n t r at i o n protein aqueous solu t ion. Figure 1. F T/IR-6800 and ATR PR O PENTA (Ove r v i ew a n d Pr i s m i m a g e ) F ea t u r es - ATR easy samp l ing accessory -Unique pe n tago n al pr i sm shape -14 r ef l ect i ons i n a Ge p ris m (Figure 1) - Hig h se n sitivity measureme n t i n combinat i on wit h a n MCT detector - Sampl e vo l um e of o nl y a f e w m icroliters *1FT/I R Ap p licat i o n Note： 210-TR-0127 K eywords S e c ond ar y s truct ur e e s t im ation，l o w c on c e nt ra tion， AT R， mul t ip l e bo unce s， MC T detec tor， p r oteins Exper i me n tal Analysis of low concentration protein aqueous solution T h e f ollowi n g t h r ee p rote in aqueou s solut i o n s (0.01% w / v) were pre p ared and IR spectr a meas u red usi n g a n ATR PRO P ENTA. Secondary s tr ucture es t imatio n (SSE) was pe r for m ed by taki n g t h e diffe r e n ce spectrum wit h wate r. 1： Lysozyme (C hi cke n Egg White) 2： Co n canavali n A 3： Tryps in in hi b itor Kunitz Measurement Conditions Main Unit FT/IR-6800 Accumulation 90 times Accessory ATR PRO PENTA Resolution 4cm² Detector MCT (PV type) F igur e 2 shows t h e I R spect ru m of 0.01% L ysozym e aqueou s solution a n d t h e diff e r en c e s pectr u m wit h wat e r. In t h e di f f erence spec t ru m， protein amide l and amide Il b ands were clear l y obse r ved. Figure 2. 2 IR S pectr u m of 0.01% aqu eo u s Lysozy me (le ft )， di ff e re n c e s p e ct ru m (b e f o re ATR c o r r ect io n ) (rig ht) T h e SSE r esult of t h e amid e l ba n d of the AT R -corrected d i ffe re nce spectrum and the r eference value² by X-r ay diffraction are s h own . Each samp l e was measured i n tripl i cate and a ll were i n good cor r elation with t h e re f erence value. From this resu l t， it was shown t h at ATR PRO PENTA is effect i ve fo r a n alysis o f low concentration prote i n aqueous sol u t i ons. *2 Sarve r ， R. W.， Krueger， W. C.， 1991. Anal . Biochem.， 194，89-100. *3 De p e n ding on th e type of prot e i n ， it may adsor b on t h e prism and affect th e r e sults Figure 3. Re s u l t o f S SE a n aly s is o f t h ree pr ot ein aq u e o u s so lutio n s (0.01% w/v) System Configuration Model Description Part Number Main Unit FT/IR-6800 FT/IR Spectrometer 7085-J006A*1 Detector MCT-6000PV MCT(PV) detector 6584-J345A2 Attachment ATR PRO PENTA Multi reflection ATR 6584-J343A Software IR SSE-4000 Secondary structure estimation program 4880-7234A *1 FT/IR-4600/4700 a n d FT/IR-6600/6700 are a l so ava i lab l e. *2 When using FT/IR-4600/4700， MCT-4000PV should b e used.