多角度激光光散射儀與凝膠滲透色譜聯(lián)用

技術(shù)及文摘

MALLS/GPC（SEC）

WYATT TECHNOLOGY CORPORTION

（BEIJING OFFICE）

地址：北京西直門北大街58號 金暉嘉園7-2302

      美國懷雅特技術(shù)公司北京代表處

：100082

：8610-82292806

8610-82290337

多角度激光光散射儀與凝膠滲透色譜聯(lián)用技術(shù)

Wyatt Technology Corp. 

一 前言

近十幾年來，光散射技術(shù)（Light scattering）在高分子特征分析領(lǐng)域的應(yīng)用得到了迅速的發(fā)展。將光散射技術(shù)和凝膠滲透色譜（Gel Permeation Chromatography, GPC）或尺寸排阻色譜（Size Exclusion Chromatography, SEC）分離技術(shù)相結(jié)合，不但可以測得大分子的分子量，分子旋轉(zhuǎn)半徑與第二維里系數(shù)，還可測得分子量分布，分辨分子量大小不同的族群，判斷溶液中大分子的構(gòu)象，分枝率及聚集態(tài)等。

作為多角度激光光散射儀的---Wyatt Technology Corp. 結(jié)合多個技術(shù)生產(chǎn)的DAWN系列產(chǎn)品在國內(nèi)外有著廣泛而的用戶基礎(chǔ)；的性能、可靠的質(zhì)量、穩(wěn)定的服務(wù)，Wyatt的產(chǎn)品一直是廣大科技工作者的產(chǎn)品。

二 光散射簡介

   早在十九世紀初，人們就開始對光散射原理進行研究。自六十年代激光被發(fā)明以來，光散射的原理與技術(shù)便得以迅速發(fā)展，至今已成為檢測微小粒子形狀，粒徑大小，分子量，界面電位及粒子間效應(yīng)的重要工具。隨著電腦技術(shù)的日新月異，許多過去需花費數(shù)小時甚至數(shù)日才能完成的實驗，如今只需數(shù)分鐘即可完成，而其準確性及重現(xiàn)性也大幅度提高了。

   光散射現(xiàn)象，如圖1 所示，當一束光通過一間充滿煙霧的房間就會產(chǎn)生散射。利用在不同角度，不同時間所測得的光散射強度，再借助各種光學(xué)理論及軟件，硬件設(shè)備，就可以測得微粒的許多特性。

入射光

散射光

圖1光散射現(xiàn)象

在光散射發(fā)展的歷程中，以下是一些具有代表性的人物：

▲James Clerk Maxwell （1833-1879）

解釋了光是一種電磁波，并正確地計算出光的速度。

• Lord Rayleigh（1842-1919）

研究了遠小于波長的微粒散射現(xiàn)象，發(fā)現(xiàn)了散射強度與波長的四次方成反比，并解釋了藍天被太陽光穿透大氣層所產(chǎn)生的散射現(xiàn)象。

• Abert Einstein（1879-1955）

研究了液體的光散射現(xiàn)象。

▲Chandrasekhara V.Raman （1888-1970）

印度籍物理大師，提出了Raman 效應(yīng)，其著作多次發(fā)表于印度文期刊，直至第二次世界大戰(zhàn)結(jié)束后才逐漸被人所知。

• Peter Debye（1884-1966）

延續(xù)了Einstein的理論，描述了分子溶解于溶劑中所產(chǎn)生的光散射現(xiàn)象，提出用Debye plot, 求得重量平均分子量Mw。

三 光散射理論

   激光照射到樣品時，會在各個方向產(chǎn)生散射光，于是我們可以在一個角度或多個角度收集散射光的強度。

1. 光散射所透露的信息

在任何方向的光散射強度與分子量和溶液的濃度成正比；散射光角度的變化與分子的尺寸大小有關(guān)。當分子小于10nm時，各個角度的散射強度都相同；當分子介于10至30nm時，散射強度則由低角度向高角度呈直線下降的趨勢；而當分子大于30nm 時，散射強度則隨角度增大呈曲線下降的趨勢。

1. 基本理論

由Maxwell，Einstein，Debye及 Zimm 等人陸續(xù)發(fā)展起來，有關(guān)溶劑中分子量的光散射現(xiàn)象可由下列公式表達：

（1）

式中：

常數(shù)K^*=4π²（d_n/d_c）²n₀²（N_Aλ₀⁴）

n₀是溶劑的折光指數(shù)。

         N_A是阿佛加德羅常數(shù)。

λ₀是入射光的波長。

d_n/d_c是溶液折射率與濃度變化的比值，它說明了隨溶質(zhì)濃度變化的溶液折光指數(shù)變化。

C是溶質(zhì)分子的濃度（g/mol）。

R（θ）是單個角度的散射光（大于溶劑的散射光數(shù)量）除以入射光強度所得的分數(shù)即不同角度光散射強度。

Mw是重均分子量。

A₂ 是第二維里系數(shù)

P（θ） 是光散射強度的函數(shù)

將P（θ）代入式（1）展開得：

在上式中，R（θ）是測得值，K*c、λ_0、θ為輸入值，均為已知值；而Mw、A₂、r_g為未知值。

1. Zimm Plot

將K* C/ R_θ對sin2（θ/2）+kc作圖，可得到的Zimm曲線，如圖2所示，其中K為調(diào)整橫坐標的設(shè)定值。

                                      圖2 Zimm Plot

當θ→ O時，（2）式簡化為

斜率即是A₂

當C→ 0時，（2）式簡化為

斜率是r_g²

當θ→ O， C→ 0，（2）式簡化為

在縱座標上交點的倒數(shù)即為Mw。實驗的方法為配制一組不同濃度的溶液，依次在不同的角度測量其散射光強度，由計算機程序按照上列的公式繪出Zimm Plot，并求得Mw，<r_g²>及A₂值，這是極少數(shù)能直接測得分子量的方法之一。但由于結(jié)果僅為單一平均值，因此較適用于成分單一，分布較窄的分子，對于分布較寬或有不同族群分布的樣品，則較難看出全貌。

四 光散射與GPC/SEC

GPC/SEC可以將溶劑中的分子按重量或尺寸大小依次洗脫出來。利用此項技術(shù)將光散射儀器與GPC/SEC聯(lián)用，除了可以分出不同的族群，還可測得不同族群的分布，并且不需要另外標準樣品做標準曲線。由于光散射信號直接與分子量大小有關(guān)，因此可以直接測出重均分子量，并獲得其它許多有關(guān)的信息。

圖4 光散射強度與GPC層析圖

Chromatography with LS Set-up

圖5 光散射強度與GPC/SEC聯(lián)用

1 Debye plot

通常GPC/SEC 的樣品注射濃度就很低，再經(jīng)過色譜柱得到進一步的稀釋，圖4中光散射信號上的任何一點，其濃度都極低（趨近于零）。根據(jù)公式，當2 A₂C → 0，

將K* C/ R_θ對sin2（θ/2）+kc作圖6，其縱坐標交點即為1/Mw，由直線的斜率可得到<r_g²>，圖4光散射信號的每點都可以得到上述結(jié)果，由此可以求得分子量及旋轉(zhuǎn)半徑<r_g²>的分布，如圖8所示：

圖6 Debye plot

圖7 積分分子量

圖8 分子量對洗脫體積作圖

2 分子形狀  

  不論是分布較寬或是多峰分布的樣品，皆可通過測量分子量及分子旋轉(zhuǎn)半徑得到分子形狀的數(shù)據(jù)。

  球形分子

  r_i3 ∞ M_i→log r_i = k + 1/3 log M_i

無規(guī)則線團狀分子

  r_i2 ∞ M_i→log r_i = k + 1/2 log M_i

棒狀分子

r_i1 ∞ M_i→log r_i = k + 1/1 log M_i

 將log r_i， log M_i作圖，有直線的斜率可以獲知分子的形狀，如圖9所示：

                              棒狀（斜率=1）

                                               無規(guī)線團（斜率0.5-0.6）

                logr_g

                                               球型（斜率=1/3）

 logM

圖9-1  M、r_g與分子形狀的關(guān)系

圖9-2 構(gòu)型判斷, r_g對M_w作圖。斜率為0.54 ± 0.01。表明分子是具有無規(guī)則線團構(gòu)象的線性聚合物

圖9-3 構(gòu)型判斷，r_g對M_w作圖。斜率大于0.6，表明分子具有伸展結(jié)構(gòu)。斜率為1.0，表明是棒狀結(jié)構(gòu)。

圖9-4 構(gòu)型判斷，r_g對M_w作圖。其U型曲線表明為典型的高支化度結(jié)構(gòu)。

3 需要量多少個角度

   在低角度的時候，有雜質(zhì)所產(chǎn)生的噪音信號干擾會很大，如圖10所示，所以只取低角度，加上九十度兩個角度，其誤差就會相對很大；若只取九十度則只能求得分子量，無法測得旋轉(zhuǎn)半徑，所以zui起碼要加上一組高角度來修正，則誤差會減少很多。

圖10 雜質(zhì)較多的GPC層析圖

4 光散射儀器

zui基本的儀器mini DAWN TREOS如圖11所示，在與入射光成45度、90度、135度角配置三組光電二極管檢測器，同時檢測不同角度的光散射強度，而激光經(jīng)由樣品槽的毛細管通道，樣品槽為石英材質(zhì)。

如果要增加測量角度，可以如DAWN HELEOS

在樣品槽的兩側(cè)以不對稱得方式增加檢測器的數(shù)目，

如圖12所示可高達18個角度之多。

圖11 三個檢測角度

五 應(yīng)用

   光散射強度與分子的大小及分子量有直接的關(guān)系，而SEC/GPC能分離不同尺寸及分子量的分子，結(jié)合此兩種特性，可以得到許多有用的信息，并廣泛地應(yīng)用于高分子，生化及動力學(xué)等研究領(lǐng)域。

圖12 十八角度檢測器

1 高分子聚合物和天然高分子的特性研究

利用多角度激光光散射系統(tǒng)（Multi-Angle Laser Light Scattering_MALLS） 結(jié)合SEC/GPC，不必依賴泵的流速，校正曲線及其它任何的假設(shè)，即可直接求得重均分子量及分子量分布等數(shù)據(jù)。

圖13 光散射強度，洗脫體積與角度作圖

MALLS利用色譜柱分離出的樣品在各個角度的光散射量（如圖13），由RI 檢測器得到的洗脫液濃度及dn/dc值，即可計算出各個切片的分子量。MALLS測得分子量所需的各種物性均可由實驗直接求得，無需作任何假設(shè)。而GPC的色譜柱又有分離雜質(zhì)的功能，可以避免傳統(tǒng)的光散射需極小心準備樣品的麻煩。圖14顯示高分子混合物經(jīng)SEC分離后MALLS及RI的洗脫體積對照圖。由此圖看出RI 對大分子量濃度低的物質(zhì)較不敏感，而對低分子量高濃度者較敏感。

圖14 這是由ASTRA軟件得到的miniDAWN（上）和Optilab示差檢測器（下）信號。

1   BSA     67,000   64,300±700     1%    2   溶解酵素 14,300   14,600±300     1%

3   緩激肽   1,060    1,090±10      2%    4   亮氨酸腦啡肽 556  592±6          3%

圖15分子量對洗脫體積圖，有四個明顯的峰

2． 蛋白質(zhì)及其聚合體

在各種工業(yè)應(yīng)用中，決定蛋白質(zhì)的特性不僅嚴格而且必要，例如在生化工程應(yīng)用上，以蛋白質(zhì)為基質(zhì)的產(chǎn)品必須很純而且無任何聚集存在。而測定蛋白質(zhì)的分子量和是否有聚集態(tài)存在，光散射法是的工具之一。

以往，在水相中用低角度光散射測量法（LALLS）受到溶劑中不純的物質(zhì)干擾相當大。而MALLS的多角度測量大大降低了背景噪音的干擾，并能提供完整的信息和良好的重現(xiàn)性結(jié)果。圖16顯示蛋白質(zhì)混合物的MALLS 和RI的信號。樣品在0.1M NaCl 中含0.05M的磷酸鹽緩沖液中進行RI為Wyatt Optilab 903，流速為0.1mL/min ,色譜柱為Shodex KW-803 和KW-804。雖然此樣品為標準樣品，但MALLS仍很清楚地檢測到聚集現(xiàn)象，此現(xiàn)象在RI幾乎無法辨認。

圖16蛋白質(zhì)洗脫體積及聚集體信號圖    

3．分枝

高分子聚合物的分枝程度和分布是影響其物理和化學(xué)性質(zhì)的一個重要因素。采用多角度激光光散射系統(tǒng)（MALLS）與GPC/SEC系統(tǒng)聯(lián)用是*決定分枝系數(shù)g_M的方法。雖然也有其他確定分枝的方法，但都不能直接且需要眾多假設(shè)及“虛擬因子”。

由傳統(tǒng)的RI或Viscometer （粘度檢測器） 測定的高枝化分子的分子量與值有很大的差別，若欲做有效的色譜柱校正，則需以一系列與待測物成分相同的標準品作校正。若標準樣品與待測物的成分或組分不同，則會產(chǎn)生很大的誤差。例如分子量相同的球形高分子的洗脫時間比無規(guī)則線團狀分子要長。

因為MALLS所求得分子量和大小為值，因此計算分枝系數(shù)g_M不需要任何假設(shè)。由MALLS直接所求得的分子大小會直接影響分枝率。對分子量相同的長鏈狀分子而言，其值越小，則分枝程度越大。分枝比的定義為分枝分子的旋轉(zhuǎn)半徑與長鏈分子的旋轉(zhuǎn)半徑之比，即g_M=<r²>_b/<r²>_l由MALLS測得。

圖17為由MALLS測得的分枝狀和長鏈形的高分子（PS）的旋轉(zhuǎn)半徑和分子量對照圖。由圖中可看出，雖然其分子量相同，但分布明顯不同。圖18 為r_g對 Mw做圖。

圖17 PS線形與枝化分子的對照圖

圖18旋轉(zhuǎn)半徑對分子量圖（分子構(gòu)型圖），可以看出樣品（藻酸鈉）在輻射后分子構(gòu)型的變化。

6. 動力學(xué)/反應(yīng)速率

MALLS還可以用于如抗原、抗體等反應(yīng)迅速的溶液系統(tǒng)，粒子和蛋白質(zhì)聚集現(xiàn)象的檢測。因為MALLS 內(nèi)部同時裝有數(shù)個固定的檢測器，所以不需移動任何儀器硬件來掃描樣品，即可及時多方位同時捕捉反應(yīng)速率的現(xiàn)象。使用MALLS，可研究抗原-抗體反應(yīng)，反應(yīng)發(fā)生時就可決定聚集粒子的大小。當改變溫度，濃度或催化劑時，MALLS可記錄下反應(yīng)發(fā)生時，分子的特殊變化。

圖19 濃度變化對蛋白聚集的影響

使用DAWN 檢測器研究濃度對分子量為75KD單分子蛋白質(zhì)聚集作用的影響。圖19描述了這種特殊蛋白質(zhì)從30ug/ml到1mg/ml范圍內(nèi)得到的濃度相關(guān)性。如圖所示，該蛋白質(zhì)在低濃度作為單一分子而在濃度大于700ug/ml時聚集為六聚體。該結(jié)果與由gluteraldehyde高度交聯(lián)技術(shù)所得結(jié)合*相符。

圖

圖20 溫度變化對PMMA分子量和大小的影響

7. 低分子量的測定

DAWN HELEOS或mini DAWN TREOS的固定光電二極管檢測器可以捕捉到很微弱的光散射信號，使得低分子量的測定成為可能。使用DAWN 系列標準配制的任何一款激光器，都可以輕易地測量分子量低于2000D的聚合物，并具有相當?shù)臏蚀_性。  

由于DAWN HELEOS 或mini DAWN TREOS具有三個以上的多角度同時捕捉散射信號的能力，即使極微弱的信號，如只比背景值略高的低分子量樣品所散射出的信號也可以從不同的角度去捕捉，累計在一起就可以計算出相當準確的結(jié)果。這是單角度或者兩角度檢測器所無法做到的。

圖21為分子量分別為580、1400及2000D 的聚苯乙烯樣品分子量對洗脫體積的對應(yīng)圖。樣品量濃度分別為7.1mg/ml，2.9mg/ml及2.2mg/ml。經(jīng)ASTRA  軟件分析得出如表2的平均分子量。

圖21 低分子量樣品與洗脫體積圖

圖22低分子量樣品分子量圖分布圖

表2樣品平均分子量

一般傳統(tǒng)光散射儀給人們的印象是不易測得分子量較低的樣品，甚至低于10000D就比較困難了。但是采用的多角度激光光散射儀就可以輕易且相當準確的測量幾百D分子量的樣品。

六 代表性文獻

1. Design and Testing for a Nontagged F1-V Fusion Protein as Vaccine Antigen against Bubonic and Pneumonic Plague   

B. S. Powell, G. P. Andrews, J. T. Enama, S. Jendrek, C. Bolt, P. Worsham, J. K. Pullen, W. Ribot, H. Hines, L. Smith, D. G. Heath, J. J. Adamovicz, "Design and testing for a nontagged F1-V fusion protein as vaccine antigen against bubonic and pneumonic plague", Biotechnology Progress 21（5）, 1490-1510 （2005）.

Summary: During discovery and testing of the F1-V fusion protein, proposed for development as the new plague vaccine antigen, the purified protein was observed to form soluble aggregates under certain conditions. Dr. Powell and colleagues used the DAWN EOS and Optilab to characterize the molecular structures of pure F1-V and its constituent subunits, the Yersinia pestis F1 and V proteins. Their investigation showed that aggregation was caused by the F1 subcomponent which forms soluble aggregates 10-times larger than the fusion protein under physiological temperature and salt. SEC-MALS studies also showed that the F1-V fusion protein structure is more stable to cold temperature, high salt, or reducing conditions than the individual subcomponent proteins.

1. Structure of nerve growth factor complexed with the shared neurotrophin receptor

        X.-L. He, K. C. Garcia, "Structure of nerve growth factor complexed with the shared neurotrophin receptor p75," Science 304 870-875 （2004）.

Summary: The Garcia team at Stanford University put their DAWN instrument to work in order to help confirm that NGF homodimers and other NT dimers bind to only one neurotrophin receptor p75 in solution. In order to ensure that the 2:1 NGF/p75 stoichiometry isn't an artifact of crystallization, they measured the ligand:receptor stoichiometry and the assembly thermodynamics of p75 complexes in solution with NGF, NT-3, and NT-4/5. MALS and SEC of p75 alone revealed it to be a mixture of a higher and lower molar mass species determined dimer and monomer of 55.1 and 27.0 kDa respectively..

1. Oxidized mono-, di-, tri-, and polysaccharides as potential hemoglobin cross-linking reagents for the synthesis of high oxygen affinity artificial blood substitutes

   J. H. Eike, A. F. Palmer, "Oxidized mono-, di-, tri-, and polysaccharides as potential hemoglobin cross-linking reagents for the synthesis of high oxygen affinity artificial blood substitutes," Biotechnol. Prog. 20 953-962 （2004）

Summary: Professor Palmer's team at the University of Notre Dame used their DAWN, Optilab and Eclipse systems to to measure the absolute molecular weight distribution of PolyHb dispersions in their study of oxidized mono/di/tri/poly saccharides as potential hemoglobin cross-linkers in order to produce oxygen carriers with high oxygen affinities and high molar masses.

1. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein

     R. Ando, H. Hama, M. Yamamoto-Hino, H. Mizuno, A. Miyawaki, "An optical marker based on the 

UV-induced green-to-red photoconversion of a fluorescent protein," PNAS 99（20） 12651-12656 （2002）

Summary: The RIKEN group has cloned a fluorescent protein which emits green, yellow and red light, which they have named Kaede. The protein includes a tripeptide, His-Tyr-Gly. Various techniques used to test the transition from one color to another.The team used their DAWN instrument in conjunction with a Shodex HPLC system in order to characterize their Kaede protein. C-terminal His-tagged protein was found to have a mass of 115.0 kDa which is 4.33 times larger than that deduced from the primary structure of the protein. It was therefore concluded that Kaede forms a homotetrameric complex.

1. Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A

    M. G. Bertero, R. A. Rothery, M. Palak, C. Hou, D. Lim, F. Blasco, J. H. Weiner, N. C. J. Strynadka, "Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A," Nature Structural Biology 10 681-687 （2003）.

Summary: The team at the University of British Columbia has provided fundamental molecular details for understanding the mechanism of proton-motive force generation by a redox loop by studying the crystal structure of NarGHI at a resolution of 1.9 Angstroms. The group used their miniDAWN and Optilab DSP instruments to analyze the oligomerization of NarGHI in the presence of 0.7 mM Thesit.

1. Rational design of low-molecular weight heparins with improved in vivo activity

    M. Sundaram, Y. Qi, Z. Shriver, D. Liu, G. Zhao, G. Venkataraman, R. Langer, R. Sasisekharan, "Rational design of low-molecular weight heparins with improved in vivo activity," PNAS 100（2） 651-656 （2003）

Summary: Robert Langer's group at MIT demonstrates a practical analytical method enabling measurement of a structural correlate to in vivo anticoagulant function.They have used this information to to develop low-molecular weight heparins （LMWHs） with increased anticoagulant activity and decreased polydispersity. Desirable in vivo pharmacokinetic properties and the ability to cause release of tissue factor pathway inhibitor from the endothelium are among several beneficial aspects of these LMWHs. The findings demonstrate a simple approach for the creation of designer LMWHs. The group used their miniDAWN in conjunction with a Shodex HPLC system to determine the molar mass and polydispersity of their heparins.

1. Chaperonin-mediated stabilization and ATP-triggered release of semiconductor nanoparticles

    D. Ishii, K. Kinbara, Y. Ishida, N. Ishii, M. Okochi, M. Yohda, T. Aida, "Chaperonin-mediated stabilization and ATP-triggered release of semiconductor nanoparticles," Nature 423 628-632 （2003）.

Summary: The team at the University of Tokyo used their DAWN detector in conjunction with their Shodex HPLC system to find the molar mass of their T.th cpn/CdS nanoparticle inclusion complex. These results demostrate that T.th cpn within the protein-nanoparticle complex preserves its own structural identity, without formation of higher aggregates or dissociation into protein subunts. They further report that GroEL and T.th cpn can also enfold CdS semiconductor nanopartilces, giving them high thermal and chemical stability in aqueous media. Such biological mechanisms integrated into materials science may open a door to conceptually new bioresponsive devices. For information on specifics on their MALS data, please see the Supplementary Information for this publication.

1. Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp 130 complex

    M. J. Boulanger, D.Chow, E. E. Brevnova, K. C. Garcia, "Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp 130 complex," Science 300 2101-2104 （2003）.

Summary: Professor Garcia's group at Stanford characterized the structure and assembly of the immunoregulatory cytokine, interleukin-6 （IL-6）, which activates a cell-surface signalling assembly. A structural model of the complex is presented. The complex forms a hexamer containing 2 IL-6, 2 IL6R-alpha and two gp 130 which assemble sequentially and cooperatively. This structure reveals a conserved architectural blueprint for assembly of all gp130-cytokine signalling complexes. For details on how the DAWN EOS was used, please see the Supporting Online Material for this paper.

1. Crystal structure of a tetradecameric assembly of the association domain of Ca²⁺/Calmodulin-dependent kinase II

     A. Hoelz, A. C. Narin, J. Kuriyan, "Crystal strucutre of a tetradecameric assembly of the association domain of Ca²⁺/calmodulin-dependent kinase II," Molecular Cell 11 1241-1251 （2003）.

Summary: The National Academy of Sciences team led by Professor Kuriyan used their DAWN EOS and Optilab instruments to confirm the crystal structure of the 143 residue association domain of Ca²⁺/Calmodulin-dependent kinase II （CaMKII）. This association domain forms a hub-like assembly which is held together by extensive interfaces. The group has determined an atomic model for the hub and spoke architecture of the association domain, possibly the most complex of the hundreds of pritein kinases in animal cells. This architecture is a critical aspect of CaMKII's to respond to calcium signals and retain a molecular memory of activation events.

1. An "endless" route to cyclic polymers

       C. W. Bielawski, D. Benitez, R. H. Grubbs, "An 'endless' route to cyclic polymers," Science 297 2041-2044 （2002）,  California Institute of Technology>

Summary: The team lead by Professor Robert Grubbs at Cal Tech developed a novel synthetic route in which the ends of growing polymer chains remain attached to a metal complex throughout the entire polymerization process, which eliminates the need for linear polymeric precursors and high dilution. A GPC system consisting DAWN EOS and Optilab detectors played crucial roles in providing strong physical evidence for circularity of the polymers synthesized. The DAWN EOS detector was used not only for molar mass determination, but also to found that the ratio of mean square radii of cyclic and linear polymers to be approximay 0.5 over a wide range of molar masses, as predicted by theory.

1. Structural evidence for feedback activation by Ras-GTP of the Ras-specific   nucleotide exchange factor SOS

      S. M. Margarit, H. Sondermann, B. E. Hall, B. Nagar, A. Hoelz, M. Pirruccello, D. Bar-Sagi, J. Kuriyan, "Structural evidence for feedback activation by Ras-GTP of the Ras-specific nucleotide exchange factor SOS," Cell 112 685-695 （2003）.

Summary: This National Academy of Sciences team led by Professor Kuriyan discovered a highly conserved Ras binding site on SOS. It is also shown that Ras-GTP forms ternary complexes with SOS^cat in solution. A DAWN EOS and  an Optilab were used extensively to characterize their purified SOS complexes as well as verify the theoretically calculated values. Their research indicates the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras.

1. Light Scattering to Detect Compound Aggregation in Screening Assays

      Dr. Bingyi Yao, Bob Collins, Dr. Michelle Chen "Light scattering to detect compound aggregation in screening assays," DPI 4,1 +32-2-240-26-11

Summary: During the high-throughput screening of compound libraries, false positive results can be generated because some compounds form aggregates that nonspecifically inhibit receptors. These compound aggregates are readily detectable by optical methods such as light scattering. This article describes a system that measures dynamic light scattering, using the same conditions as for HTS, allowing the identification of false positives very early in the screening process.

1. Characterization of Hyaluronic Acid with On-Line Differential Viscometry, Multiangle Light Scattering, and Differential Refractometry

    Jason Waters, Danielle Leiske, "Characterization of Hyaluronic Acid with On-Line Differential Viscometry, Multiangle Light Scattering, and Differential Refractometry," LCGC 23,3 （732） 225-9500.

Summary: Wyatt Technology in collaboration with Professor Skip Rochefort's group at Oregon State University used a DAWN EOS, an Optilab rEX and a ViscoStar to measure the intrinsic viscosity and Mark-Houwink-Sakurada （MHS） behavior of Hyaluronic Acid （HA）. Unusual MHS behavior was demonstrated, perhaps helping explain the large variation in HA MHS coeffiecents that have been reported in the literature.