刀豆蛋白A中蛋白质折叠与展开检测方案(圆二色光谱仪)

Application Note Unfolding of concanavalin A usingtrifluoroethanol2/3Application Note CDD SSpectroscopy Unfolding of concanavalin A using trifluoreoethanol probed by stopped-flow CD spectroscopy One way to study protein structure is by observing theprotein’s folding and unfolding processes. These events canspan from submillisecond time scales to hours. While circulardichroism measurements can provide information regardinga protein's secondary structure and the environmentsurrounding aromatic amino acids， stopped-flow techniquescan be used to measure kinetic reactions that occur overa span of several millisecond to several seconds. Bycombining these techniques， researchers can analyse thestructural kinetics of a protein's unfolding and refoldingprocesses. Concanavalin A in its natural form is abundant in B-sheetstructure. However， in the presence of trifluoroethanol (TFE)，the structure folds into predominantly a-helices. In this application note， the SFS-562T High-Speed Stopped-Flow System was used in conjunction with the J-1500CD spectrometer to measure the unfolding process ofconvanavalin A by TFE. Keywords J-1500， circular dichroism， stopped-flow， SFS-562T，secondary structure， concanavalin A， trifluoroethanol，kinetics， Reaction Speed Calculation program， biochemistry，material analysis， QA/QC， regulatory JASCO INC. 28600 Mary's Court， Easton， MD 21601 USA Experimental Measurement conditions Measurement wavelength 220 nm Path length 2mm Spectral bandwidth 1 nm Data pitch 2 msec Accumulations 4 Response time 1msec Syringe 1 (Concanavalin A) 0.2 mg/mL in HCI (pH 2) Syringe 2 TFE Mixing ratio 100 uL： 100 uL Total flow rate 10 mL/sec Results . Figure 1 illustrates the CD spectra of folded and unfolded concanavalin A. In its native form， concanavalin A exhibits apredominantly B-sheet structure (green)， while in a 50% TFE solution the protein is mostly a-helical. Figure 1. CD spectra of concanavalin A in HCl at pH 2 (green) and concanavalin A in solution with 50%TFE (blue). The CD spectra measurements were obtained with 0.1 mg/mL of concanavalin A in a 1 mmpathlength cell. Concanavalin A (0.2 mg/mL in pH 2 HCl) was mixed with TFE in a 1：1 ratio. Its unfolding process was measured usingstopped-flow CD at 220 nm， which is marker for a-helical structure. Figure 2 shows that the CD value at 220 nmdecreases with time， indicating a change from B-sheet-rich structure to a a-helical-rich structure as the protein unfoldsThis unfolding process was analyzed as a two-step reaction (A + B + C model)using the Reaction Speed Calculation(RSC) program. The results of the analysis are shown in Table 1. The black line in Figure 2 calculated from the RSCprogram demonstrates good fit of the stopped-flow data. 28600 Mary'’s Court， Easton， MD 21601 USA Figure 2. The unfolding process of concanavalin A in TFE measuremed by stopped-flow CD. Table 1. Reaction speed calculations of the unfolding of concanavalin A using trifluoroethanol. Analysis results Reaction speed equation Y(t)=20.5925 *e(-t/0.189295)+4.73648*e(-t/0.903939) Step 1 time constant (t) 0.189295 s Step 1 rate constant (k.) 5.28275 s1 Step 2 time constant (t) 0.903939 s Step 2 rate constant (k，) 1.10627 s1 Conclusion This application note demonstrates that stopped-flow CD measurements can be used to measure the protein unfoldingprocesses， analyze the secondary structure changes， and obtain reaction rate constants. References 28600 Mary’s Court， Easton， MD 21601 USA Tel：( Fax：(pplication Library： http：//www.jascoinc.com/applications This application note demonstrates that stopped-flow CD measurements can be used to measure the protein unfolding processes, analyze the secondary structure changes, and obtain reaction rate constants