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The Stopped Flow method is used to analyze reactions occurred during the duration of several milliseconds
and several seconds meanwhile the CD measurement provides valuable information regarding the protein’s
secondary structures and environment of aromatic amino acids. Therefore, CD measurement with Stoppedflow
method provides one of the best techniques in analyzing the protein’s Unfolding and Refolding process.
Concanavalin A (derived from Jack bean) in natural state has abundant β-sheet structures, however, it is
known that its structure changes into the one with rich α-helix under trifluoroethanol (TFE) and its Unfolding
process has been reported also.
In this application, the model SFS-492 High-Speed Stopped-flow System was used to measure the Unfoldin
方案详情
s.crApplication Note CD-0004 Unfolding of Concanavalin A by the Trifluoroethanol The Stopped Flow method is used to analyze reactions occurred during the duration of several millisecondsand several seconds meanwhile the CD measurement provides valuable information regarding the protein’ssecondary structures and environment of aromatic amino acids. Therefore， CD measurement with Stopped-flow method provides one of the best techniques in analyzing the protein’s Unfolding and Refolding process. Concanavalin A (derived from Jack bean) in natural state has abundant B-sheet structures， however， it isknown that its structure changes into the one with rich o-helix under trifluoroethanol (TFE) and its Unfoldingprocess has been reported also. In this application， the model SFS-492 High-Speed Stopped-flow System was used to measure the Unfoldingprocess of Concanavalin A under TFE . The Concanavalin A’s in pH2 hydrochloric acid gives a CD spectrum specific to B-sheet structure. Incontrast， in a solution with 50% of TFE added it has a CD spectrum specific to o-helix structures. As seen inFigure 1， a big change from abundant B-sheet structure to abundant a-helix structure can be identified. Figure 1： CD Spectrum of Concanavalin A Green： CD spectrum of Concanavalin A in pH2 hydrochloric acid Blue： CD spectrum of Concanavalin A in solution with 50% TFE (Both are Concanavalin A： 0.1 mg/ml； cell path length： 1 mm) [Measurement Results1 Concanavalin A (0.2 mg/ml， in pH2 hydrochloric acid) was mixed with TFE with a ratio of 1：1 and itsUnfolding process was measured using the Stopped Flow method. The CD value at 220 nm showed anincrease in negative side， and change from abundant B-sheet structure to abundant a-helix structure wasobserved. By analyzing the reaction as a two-step reaction (A --> B --> C model) through the use of theReaction Speed Calculation Program， unsurpassed fitting of the spectra was obtained (Figure 2). [Measurement Conditions] Syringe2： TFE Mixing ratio：Total flow rate： 10 ml/secCell path length： 2 mm Wavelength for measurement：220 nm Data pitch： 2 msec Band width： 1 nm Accumulation： 4 times [Result of Analysis] Step 1 time constant： 0.189295 [s] Step 2 time constant： 0.903939[s] Figure 2： Unfolding process of Concanavalin A in TFE and analysis result Syringe 1： 0.2 mg/ml， Concanavalin A， in hydrochloric acid (pH2) 100 pl：100 p1 Reaction speed equation： Y(t)=20.5925*exp(-t/0.189295)+ 4.73648*exp(-t/0.903939) Step 1 speed constant (k1)： 5.28275 [s-1] Step2 speed constant (k2)： 1.10627 [s-1] (1) Qi Xu and Timothy A. Keiderling， (2005) Biochemistry， p.44， 7976-7987 copyrightOJASCO CorporationJASCO INTERNATIONAL CO.， LTD- Sennin-cho -chome， Hachioji， Tokyo JapanTel：+Fax： + The Stopped Flow method is used to analyze reactions occurred during the duration of several millisecondsand several seconds meanwhile the CD measurement provides valuable information regarding the protein’ssecondary structures and environment of aromatic amino
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