药品中人血清白蛋白与3，5-二碘水杨酸的相互作用检测方案(圆二色光谱仪)

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Application Note 2/3 CDD SSpectroscopy Analyzing the interaction of human serum albumin and3，5-diiodosalicylic acid probed by circular dichroism Introduction Human serum albumin (HSA) is the most abundant protein inblood plasma. HSA binds with pharmaceutical compoundsand other in-vivo substances and plays an important role inthe transport of these substances to target organs. Severalstudies have reported on the binding affinity of HSA and itsinteractions with a variety of compounds. JASCO J-1500 CD spectrometer This application notes demonstrates the use of the J-1500CD spectrometer and ATS-530 automatic titrator to monitorthe interaction of HSA titrated with 3，5-diiodosalicylic acid.While 3，5-diiodosalicylic acid is achiral， its interaction withchiral HSA induces circular dichroism. View product information at www.jascoinc.com Keywords J-1500， circular dichroism， HSA， pharmaceuticals， ATS-530automatic titrator， biochemistry， pharmaceutical Experimental Measurement conditions Data acquisition interval 2 sec Scan speed 100 nm/min Spectral bandwidth 1nm Data pitch 0.1 nm Path length 10 mm Accumulations 2 Analyzing the interaction of human serumApplication Note albumin and 3，5-diiodosalicylic acid 50 mL aliquots of 0.025 mM of 3，5-diiodosalicylic acid was added to 2 mL of 0.0228 mM of HSA in 100 mM acetate buffer(pH 6.3) 20 times. The CD spectrum was scanned from 360 to 260 nm and also monitored at 320 nm. Results Chirality can be induced in an achiral substance interacting with a chiral substance. This interaction will exhibit circulardichroism. The resulting CD spectrum from the interaction of achiral 3，5-diiodosalicylic acid with chiral HSA shows apositive peak at 320 nm in Figure 1. While HSA does not show a CD signal at 320 nm， the induced CD from the interactionof HSA and 3，5-diiodosalicylic acid depicts this signal at 320 nm which increases with the increasing additions of3，5-diiodosalicylic acid and is shown in Figure 2. Figure 1. 3-D graphical representation of the CD spectrum of HSA titrated with 3，5-diiodosalicylic acid. Figure 2. CD spectra of HSA titrated with 3，5-diiodosalicylic acid. The arrows represent the increasingvolume of 3，5-diiodosalicylic acid. The increase in the induced CD signal at 320 nm was plotted in Figure 3 as a funtion of the 3，5-diiodosalicylic acidconcentration. Figure 3 is also known as a Hill plot which describes the cooperativity of the interaction between HSA and3，5-diiodosalicylic acid. A dissociation constant， which is the concentration at which 50% of a subtsance is bound， canalso be estimated from a Hill plot. Using Figure 3， the dissociation constant of 3，5-diiodosalicylic acid binding to HSA wasdetermined to be 0.023 mM. The Hill coefficient is approximately 3.1 which indicates a positive cooperative reaction. 28600 Mary's Court， Easton， MD 21601 USA Figure 3. CD signal monitored at 320 nm for the titration of 3，5-diiodosalicylic acid to HSA. Conclusion This application note demonstrates the use of the ATS-530 automatic titrator and J-1500 CD spectrometer to obtain CDspectra of the titration of 3，5-diiodosalicylic to HSA in order to determine a dissociation constant. References 1.R. Brodersen， J. Biol. Chem. (1977) 252，14，5067-5072. 2.U. Kragh-Hansen， Biochem. J. (1983) 209， 135-142. 3. Sinha， S. S.， Mitra， R. K.， and S. K. Pal， J. Phys. Chem.B. (2008) 112，4884-4891. 28600 Mary's Court， Easton， MD 21601 USA JASCO INC.Mary's Court， Easton， MD USATel：( Fax： ( pplication Library： http：//www.jascoinc.com/applications JASCO INC.Tel： ( Fax： ( pplication Library：http：//www.jascoinc.com/applicationsCopyright O JASCO Corporation This application notes demonstrates the use of the J-1500 CD spectrometer and ATS-530 automatic titrator to monitor the interaction of HSA titrated with 3,5-iiodosalicylic acid. While 3,5-diiodosalicylic acid is achiral, its interaction with chiral HSA induces circular dichroism.